Hua Xu
- Associate Professor
- Associate Professor, Physiological Sciences - GIDP
Contact
- (520) 626-7050
- Arizona Health Sciences Center, Rm. 006338
- Tucson, AZ 85724
- hxu@arizona.edu
Bio
No activities entered.
Interests
No activities entered.
Courses
2019-20 Courses
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Senior Capstone
MCB 498 (Spring 2020) -
Senior Capstone
MCB 498 (Fall 2019)
2018-19 Courses
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Independent Study
MCB 499 (Spring 2019) -
Independent Study
MCB 399 (Fall 2018)
Scholarly Contributions
Chapters
- Ghishan, F. K., Xu, H., & Kiela, P. R. (2021). Na+/H+ Exchangers in Epithelia. In Studies of Epithelial Transporters and Ion Channels: Ion Channels and Transporters of Epithelia in Health and Disease(pp 125-209). Springer.
- Xu, H., Ghishan, F., & Kiela, P. (2018). SLC9 Gene Family: Function, Expression, and Regulation.. In Compr Physiol.
- Xu, H., Xu, H., Ghishan, F., & Ghishan, F. (2018). Molecular Physiology of Gastrointestinal Function during Development. In Physiology of the Gastrointestinal Tract.
- Xu, H., Xu, H., Collins, J. F., & Ghishan, F. K. (2012). Molecular Physiology of Gastrointestinal Function during Development. In Physiology of the Gastrointestinal Tract. Elsevier Inc. doi:10.1016/B978-0-12-382026-6.00014-2More infoThe mammalian intestinal tract undergoes dramatic changes during the first few weeks of postnatal life. Genetic and neurohormonal regulators influence the changes in digestive and transport function that mediate the development of the gut. In mammals, the most significant changes occur during the suckling/weaning transition. At birth, the intestine’s digestive and secretory function are not fully developed, and thus nutrient absorption occurs throughout the small and large intestines and along the entire length of the crypt–villus axis. During the suckling period, factors from the mother’s milk affect intestinal function and regulate the expression of genes involved in nutrient transport and immune function. After weaning, a different set of physiologic regulators and dietary factors direct rapid functional maturation of the gut epithelium. This chapter reviews the key discoveries that have led to the current understanding of ontogenic maturation of the gastrointestinal function (secretory, digestive, absorptive, and regulation).
- Collins, J. F., Bai, L., Xu, H., Xu, H., & Ghishan, F. K. (2006). CHAPTER 13 – Molecular Aspects and Regulation of Gastrointestinal Function during Postnatal Development. In Physiology of the Gastrointestinal Tract(pp 375-404). doi:10.1016/B978-012088394-3/50016-7
Journals/Publications
- Tong, H., de Miranda Azevedo, Claudio -, C., Curiel, L., Xu, H., & Ghishan, F. (2023). The expression of NHE8 in liver and its role in carbon tetrachloride induced liver injury.. Gastro Hep Advances, 2(2), 199-208.
- Bernardazzi, C., Sheikh, I., Xu, H., & Ghishan, F. (2022). The Physiological Function and Potential Role of the Ubiquitous Na/H Exchanger Isoform 8 (NHE8): An Overview Data. . Int J Mol Sci.
- Guo, W., Jamwal, D., Wang, J., & Xu, H. (2022). Single Cell Analysis – Discovery, Development and Implications to Study Cell-Cell and Cell-Pathogen Interactions. Front. Cell Dev. Biol, 951506.
- Xu, H., Zhou, K., Amiri, M., Salari, A., Yu, Y., Seidler, U., & Nikolovska, K. (2021). Functional characterization of the sodium/hydrogen exchanger 8 and its role in proliferation of colonic epithelial cells. American Journal of Physiology-Cell Physiology, 321(3), C471-C488. doi:10.1152/ajpcell.00582.2020
- Zhou, K., Amiri, M., Salari, A., Yu, Y., Xu, H., Seidler, U., & Nikolovska, K. (2021). Functional characterization of the sodium/hydrogen exchanger 8 and its role in proliferation of colonic epithelial cells. Am J Physiol Cell Physiol, 321(3), C471-C488.
- Bernardazzi, C., Xu, H., TOng, H., Laubitz, D., Figliuolo, d. P., Curiel, L., & Ghishan, F. (2020). An indisputable role of NHE8 in mucosal protection. Am J Physiol Gastrointest Liver Physiol., 319(4), G421-G431.
- Ghishan, F. K., Laubitz, D., Xu, H., Bernardazzi, C., Tong, H., Figliuolo da Paz, V., & Curiel, L. (2020). An indisputable role of NHE8 in mucosal protection. American Journal of Physiology-Gastrointestinal and Liver Physiology, 319(4), G421-G431. doi:10.1152/ajpgi.00246.2020
- Xu, H., Xu, H., Li, J., Ghishan, F. K., & Chen, H. (2019). NHE8 Deficiency Promotes Colitis-Associated Cancer in Mice via Expansion of Lgr5-Expressing Cells.. Cellular and molecular gastroenterology and hepatology, 7(1), 19-31. doi:10.1016/j.jcmgh.2018.08.005More infoLgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal Na+/H+ exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in an ulcerative colitis-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors..We assessed NHE8 expression in human CRCs by immunohistochemistry and studied tumor burden in NHE8 knockout (KO) mice using an azoxymethane/dextran sodium sulfate colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NOD scid gamma (NSG) mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by polymerase chain reaction and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of β-catenin and c-Myc also were analyzed to evaluate Wnt/β-catenin activation..NHE8 was undetectable in human CRC tissues. Although only 9% of NHE8 wild-type mice showed tumorigenesis in the azoxymethane/dextran sodium sulfate colon cancer model, almost 10 times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared with HT29NHE8 wild type cells. Furthermore, NHE8 deficiency resulted in increased Lgr5 expression in the colon, in HT29-derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/β-catenin activation..NHE8 might be an intrinsic factor that regulates Wnt/β-catenin in the intestine.
- Xu, H. (2018). Up-regulation of NHE8 by somatostatin ameliorates the diarrhea symptom in infectious colitis mice model. Korean J Physiol Pharmacol.
- Xu, H., Li, J., Chen, H., & Ghishan, F. (2018). NHE8 Deficiency Promotes Colitis-Associated Cancer in Mice via Expansion of Lgr5-Expressing Cells.. Cell Mol Gastroenterol Hepatol..
- Xu, H., Li, X., Lei, X., Cai, L., Geng, C., & Wang, C. (2018). Up-regulation of NHE8 by somatostatin ameliorates the diarrhea symptom in infectious colitis mice model. The Korean Journal of Physiology & Pharmacology, 22(3), 269. doi:10.4196/kjpp.2018.22.3.269
- Xu, H., Xu, H., Li, J., & Ghishan, F. K. (2017). Loss of NHE8 Expression Impairs Crypt Function in the Colon. Gastroenterology, 152(5), S646. doi:10.1016/s0016-5085(17)32284-9
- Li, X., Cai, L., Xu, H., Geng, C., Lu, J., Tao, L., Sun, D., Ghishan, F. K., & Wang, C. (2016). Somatostatin regulates NHE8 protein expression via the ERK1/2 MAPK pathway in DSS-induced colitis mice. American journal of physiology. Gastrointestinal and liver physiology, 311(5), G954-G963.More infoPrevious studies reported that administration of somatostatin (SST) to human patients mitigated their diarrheal symptoms. Octreotide (an analog of SST) treatment in animals resulted in upregulation of sodium/hydrogen exchanger 8 (NHE8). NHE8 is important for water/sodium absorption in the intestine, and loss of NHE8 function results in mucosal injury. Thus we hypothesized that NHE8 expression is inhibited during colitis and that SST treatment during pathological conditions can restore NHE8 expression. Our data showed for the first time that NHE8 is expressed in the human colonic tissue and that NHE8 expression is decreased in ulcerative colitis (UC) patients. We also found that octreotide could stimulate colonic NHE8 expression in colitic mice. Furthermore, the somatostatin receptor 2 (SSTR2) agonist seglitide and the somatostatin receptor 5 (SSTR5) agonist L-817,818 could restore NHE8 expression via its role in suppressing ERK1/2 phosphorylation. Our study uncovered a novel mechanism of SST stimulation of NHE8 expression in colitis.
- Xu, H., Li, Q., Zhao, Y., Li, J., & Ghishan, F. K. (2016). Intestinal NHE8 is highly expressed in goblet cells and its expression is subject to TNF-α regulation. American journal of physiology. Gastrointestinal and liver physiology, 310(2), G64-9.More infoWhile the intestine plays an important role in digestion and absorption, the mucus lining the epithelium represents a pivotal function in mucosal protection. Goblet cells are scattered in both the crypts and among enterocytes, and they secrete an important component of mucus, mucin. We have reported that sodium/hydrogen exchanger (NHE) 8 is a novel player in mucosal protection, since loss of NHE8 function resulted in reduced mucin production and increased bacterial adhesion. While NHE8 has been shown to be expressed in enterocytes and its expression is reduced during intestinal inflammation, nothing is known about the role of NHE8 in goblet cells. This current study is designed to define the expression of NHE8 and the role of TNF-α in the regulation of NHE8 in goblet cells. Using HT29-MTX cells as an in vitro model, we detected abundant NHE8 mRNA in goblet cells. Immunohistochemical staining localized NHE8 protein on the plasma membrane and in the intracellular compartments in goblet cells. Furthermore, NHE8 expression in goblet cells is regulated by the proinflammatory cytokine TNF-α. The expression of NHE8 in HT29-MTX cells was significantly reduced at both mRNA and protein levels in the presence of TNF-α. This inhibition of NHE8 mRNA expression could be blocked by the transcriptional inhibitor actinomycin D. Promoter reporter assay showed that NHE8 promoter activity was indeed reduced by TNF-α. Mechanistically, TNF-α reduced Sp3 protein binding to the human NHE8 basal promoter region. Therefore, NHE8 is expressed in goblet cells, and the inflammatory cytokine TNF-α downregulates NHE8 expression by a transcriptional mechanism.
- Xu, H., Li, X., Cai, L., Geng, C., Lu, J., Tao, L., Sun, D., Ghishan, F. K., & Wang, C. (2016). Somatostatin regulates NHE8 protein expression via the ERK1/2 MAPK pathway in DSS-induced colitis mice. American Journal of Physiology-Gastrointestinal and Liver Physiology, 311(5), G954-G963. doi:10.1152/ajpgi.00239.2016
- Xu, H., Wang, C., Tang, C., Li, X., Gao, J., & Cai, L. (2016). Sa1421 Somatostatin Maintains Intestinal Epithelial Barrier Function by Modulation of Tight Junctions in DSS-Induced Colitis Mice. Gastroenterology, 150(4), S311. doi:10.1016/s0016-5085(16)31090-3
- Li, J., McCoy, A., Xu, H., Zhao, Y., & Ghishan, F. K. (2015). Sodium butyrate stimulates NHE8 expression via its role on activating NHE8 basal promoter activity. American Journal of Physiology-Gastrointestinal and Liver Physiology, 309(6), G500-G505. doi:10.1152/ajpgi.00194.2015
- Xu, H., Wang, C., Tang, C., Li, X., & Cai, L. (2015). Tu1397 Somatostatin Regulates NHE8 Protein Expression via the MAPK -ERK1/2 Pathway in Colitis Mice. Gastroenterology, 148(4), S-879. doi:10.1016/s0016-5085(15)32984-xMore infoBackground and aim:diarrhea is a common disease in gastroenterology which is caused by various affects such as intestinal infection, non-infectious inflammation, tumors and so on. The imbalance of fluid/sodium absorption and secretion is considered playing a crucial role in diarrhea. Sodium, as an important electrolyte, involves three mechanisms to transport. Of note, sodium/hydrogen exchanger (NHE) may occupy a crucial position in modulating intestinal water and sodium absorption. Previous study had shown that somatostatin (SST) could stimulate NHE8 expression in physiological intestine. To determine the benefical effect of SST on NHE8 protein expression in colitis mice and its mechanisms, experimental colitis was induced in mice utilizing dextran sulfate sodium (DSS), models of Caco-2 cells intervened by TNF-α were also established to further explore the mechanism of somatostatin modulating NHE8 expression. Methods: To induce diarrhea via intestinal inflammation, mice were fed with 3% Dextran sulfate sodium (DSS) water for seven days. On the eighth day, treatment groups were administrated with octreotide at dose of 50 μg/ kg body weight three times a day for three days. On the eleventh day, mice were euthanized and colonic tissues were collected. Diarrheal symptoms were assessed every other day. Diarrheal score was recorded based on fecal shape, color and hardness. For the TNF-α study, Caco-2 cells were incubated with TNF-α for 18 hours before adding somatostatin. Cells were exposed to somatostatin for 1 hour before harvest. To further explore the mechanism of somatostatin modulating NHE8 expression, Caco-2 cells were incubated with TNF-α for 18 hours. Subsequently, Caco-2 cells pretreated with MAPKK inhibitor (PD98059) were administratedwith SST for 1 hour before harvest.Results: For DSS colitis mice, the expression of somatostatin in colon were decreased in DSS colitis mice compared with the control mice. Moreover, SST could not only ameliorate diarrhea in inflammatory colitis (diarrheal score: 1.7 ± 0.78 vs. 3.6 ± 0.16, n = 3, P
- Wang, M., Xu, H., Zhao, Y., Xu, H., Wang, M., Li, J., & Ghishan, F. K. (2014). NHE8 in human conjunctival epithelial cells: expression and regulation (1122.3). The FASEB Journal, 28.
- Wang, M., Xu, H., Zhao, Y., Xu, H., Wang, M., Li, J., & Ghishan, F. K. (2014). TNF-α Inhibits NHE8 Expression in Conjunctival Epithelial Cells. Investigative Ophthalmology & Visual Science, 55(13), 2761-2761.
- Xu, H., Wang, Q., Wang, C., Tao, L., Lu, J., Li, X. X., & Cai, L. (2014). Somatostatin regulates tight junction proteins expression in colitis mice.. International journal of clinical and experimental pathology, 7(5), 2153-62.More infoTight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P
- Xu, H., Xu, H., Li, J., Ghishan, F. K., & Chen, H. (2014). NHE8’s role in male fertility involves LHR protein trafficking (1097.8). The FASEB Journal, 28.More infoWe reported last year that NHE8, the newest member of the sodium/hydrogen exchanger family, plays an important role in male fertility. NHE8 has abundant expression in the male reproductive organs, particularly in Leydig cells and epididymal duct cells. Male mice lacking NHE8 are infertile. Loss of NHE8 expression also resulted in reduced luteinizing hormone receptor (LHR) expression in male mice. Furthermore, luteinizing hormone (LH)-stimulated cAMP production was reduced in isolated NHE8KO Leydig cells. After NHE8 expression was silenced by NHE8 siRNA in Leydig cells, reduced LHR expression and dramatically reduced cAMP production after LH stimulation were noted. Confocal microscopic imaging showed that LHR protein was targeted to early endosome in Leydig cells in the absence of NHE8 function instead of the plasma membrane. Therefore, male infertility in NHE8KO mice is due to the disruption of LHR protein trafficking, which results in an interruption in the precise hormone regulation that governs normal ...
- Xu, H., Xu, H., Xia, L., Sjovall, H., Johansson, M. E., Jabbar, K. S., Holmen-larsson, J., Hansson, G. C., Gustafsson, J. K., Ghishan, F. K., Gewirtz, A. T., & Carvalho, F. A. (2014). Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis.. Gut, 63(2), 281-91. doi:10.1136/gutjnl-2012-303207More infoThe inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis (UC)?.The colon mucus layer from mice deficient in Muc2 mucin, Core 1 O-glycans, Tlr5, interleukin 10 (IL-10) and Slc9a3 (Nhe3) together with that from dextran sodium sulfate-treated mice was immunostained for Muc2, and bacterial localisation in the mucus was analysed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10(-/-) mice revealed a thicker mucus layer than wild-type, but the properties were different, as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsy samples. In contrast, mucus on colon biopsy specimens from patients with UC with acute inflammation was highly penetrable. Most patients with UC in remission had an impenetrable mucus layer similar to that of controls..Normal human sigmoid colon has an inner mucus layer that is impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in patients with active UC allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated, and this suggests a novel model of UC pathophysiology.
- Xu, H., Zhao, Y., Xu, H., Wang, M., Stevenson, W., Li, J., & Gishan, F. (2014). Hyperosmolar Stress Downregulates NHE8 Expression in Human Conjunctival Epithelial Cells. Investigative Ophthalmology & Visual Science, 55(13), 2764-2764.
- Wang, M., Xu, H., Xu, H., Wang, M., Li, J., & Ghishan, F. K. (2013). NHE8 Participates in Electrolyte and Water Secretion in Conjunctival and Lacrimal Gland Epithelia. Investigative Ophthalmology & Visual Science, 54(15), 2109-2109.
- Xu, H., Chen, H., Li, J., & Ghishan, F. K. (2013). NHE8 plays important roles in male fertility. The FASEB Journal, 27(S1). doi:10.1096/fasebj.27.1_supplement.736.8
- Xu, H., Xu, H., Liu, C., Li, J., & Ghishan, F. K. (2013). 891 NHE8 Participates Intestinal Mucosal Protection Against Bacterial Infection. Gastroenterology, 144(5), S-157. doi:10.1016/s0016-5085(13)60566-1
- Xu, H., Xu, H., Wang, C., Li, J., Ghishan, F. K., & Chen, H. (2013). NHE8 plays important roles in gastric mucosal protection.. American journal of physiology. Gastrointestinal and liver physiology, 304(3), G257-61. doi:10.1152/ajpgi.00433.2012More infoSodium/hydrogen exchanger (NHE) 8 is an apically expressed membrane protein in the intestinal epithelial cells. It plays important roles in sodium absorption and bicarbonate secretion in the intestine. Although NHE8 mRNA has been detected in the stomach, the precise location and physiological role of NHE8 in the gastric glands remain unclear. In the current study, we successfully detected the expression of NHE8 in the glandular region of the stomach by Western blotting and located NHE8 protein at the apical membrane in the surface mucous cells by a confocal microscopic method. We also identified the expression of downregulated-in-adenoma (DRA) in the surface mucous cells in the stomach. Using NHE8(-/-) mice, we found that NHE8 plays little or no role in basal gastric acid production, yet NHE8(-/-) mice have reduced gastric mucosal surface pH and higher incidence of developing gastric ulcer. DRA expression was reduced significantly in the stomach in NHE8(-/-) mice. The propensity for gastric ulcer, reduced mucosal surface pH, and low DRA expression suggest that NHE8 is indirectly involved in gastric bicarbonate secretion and gastric mucosal protection.
- Xu, H., Zhang, B., Xu, H., Liu, C., Li, J., Johansson, M. E., Hansson, G. C., & Ghishan, F. K. (2013). NHE8 plays an important role in mucosal protection via its effect on bacterial adhesion.. American journal of physiology. Cell physiology, 305(1), C121-8. doi:10.1152/ajpcell.00101.2013More infoThe Na⁺/H⁺ exchanger NHE8 is expressed on the apical membrane of intestinal epithelial cells and is particularly abundant in the colon. Our previous study showed that Muc2 expression was significantly reduced in NHE8-knockout (NHE8-/-) mice, suggesting that NHE8 plays a role in mucosal protection in the colon. The current study confirms and extends our studies on the role of NHE8 in mucosal protection. The number of bacteria attached on the distal colon was significantly increased in NHE8-/- mice compared with their wild-type littermates. As expected, IL-4 expression was markedly increased in NHE8-/- mice compared with wild-type mice. Immunohistochemistry showed disorganization in the mucin layer of NHE8-/- mice, suggesting a possible direct bacteria-epithelia interaction. Furthermore, NHE8-/- mice were susceptible to dextran sodium sulfate-induced mucosal injury. In wild-type mice, dextran sodium sulfate treatment inhibited colonic NHE8 expression. In Caco-2 cells, the absence of NHE8 expression resulted in higher adhesion rates of Salmonella typhimurium but not Lactobacillus plantarum. Similarly, in vivo, S. typhimurium adhesion rate was increased in NHE8-/- mice compared with wild-type mice. Our study suggests that NHE8 plays important roles in protecting intestinal epithelia from infectious bacterial adherence.
- Xu, H., Xu, H., Li, J., Ghishan, F. K., & Chen, H. (2012). 1 Loss of NHE8 Expression Results in Gastric Ulcers in Mice. Gastroenterology, 142(5), S-1. doi:10.1016/s0016-5085(12)60001-8More infoNHE8 is a member of the SLC9 family. This Na+/H+ exchanger is expressed on the apical membrane of intestinal epithelial cells. Although NHE8 has been shown to be important for intestinal sodium absorption early in development, its physiological role in the gastrointestinal tract remains unknown. By using NHE8 knockout (NHE8KO) mouse model, we discovered that NHE8 plays an important role in protecting the gastric mucosa against gastric acid. Stomach tissue was isolated from NHE8KO mice and their wild-type littermates. Localization of NHE8 expression in the stomach was detected using Immunohistochemistry. Mucosal surface pH was measured using a surface pH meter. Histology of the stomach of NHE8KOmice was also analyzed. RNA was isolated and used for PCR to detect the expression of PAT1 and DRA. Total tissue lysate was prepared for Western detection of NHE8, PAT1 and DRA. Immunohistochemistry indicated that NHE8 is expressed on the apical membrane of gastric epithelial cells. Histological evaluation showed no abnormality of the stomach in NHE8KO mice. Interestingly, higher incidence of gastric bleeding was observed in NHE8KO mice. The appearance of ulcers was seen in the glandular stomach. Mucosal surface pH measurement indicated that NHE8KO mice had decreased mucosal surface pH compared to wild-type mice (3.72 ± 0.13 in NHE8KO vs. 4.26 ± 0.09 in wild-type). Lower surface pH of NHE8KO gastric mucosa suggests that HCO3secretion may be altered. Therefore, we studied the expression of PAT1 and DRA in NHE8KO mice. Indeed, PAT1 and DRA mRNA expression in the stomach of NHE8KO mice were significantly reduced by ~30% and ~90%, respectively, in NHE8KO mice compared to their wild-type littermates. At the protein expression level, PAT1 protein abundance was reduced by ~70% and DRA protein abundance was nearly abolished in NHE8KO mice. Thus NHE8 may play an important role in mucosal protection by participating bicarbonate secretion.
- Xu, H., Zhang, B., Xu, H., Wang, C., Li, J., Ghishan, F. K., & Chen, H. (2012). Impaired mucin synthesis and bicarbonate secretion in the colon of NHE8 knockout mice.. American journal of physiology. Gastrointestinal and liver physiology, 303(3), G335-43. doi:10.1152/ajpgi.00146.2012More infoSodium/hydrogen exchanger 8 (NHE8), the newest member of the SLC9 family, is expressed at the apical membrane of the epithelial cells in the intestine and the kidney. Although NHE8 has been shown to be an important player for intestinal sodium absorption early in development, its physiological role in the intestine remains unclear. Here, we successfully created a NHE8 knockout (NHE8(-/-)) mouse model to study the function of this transporter in the intestinal tract. Embryonic stem cells containing interrupted NHE8 gene were injected into mouse blastocyst to produce NHE8(+/-) chimeras. NHE8(-/-) mice showed no lethality during embryonic and fetal development. These mice had normal serum sodium levels and no signs of diarrhea. Apically expressed NHE2 and NHE3 were increased in the small intestine of the NHE8(-/-) mice in compensation. The number of goblet cells and mucin (MUC)-positive cells in the colon was reduced in NHE8(-/-) mice along with mucosal pH, MUC2 expression as well as downregulated in adenoma (DRA) expression. Therefore, the role of NHE8 in the intestine involves both sodium absorption and bicarbonate secretion.
- Xu, H., Zhang, B., Xu, H., Wang, C., Li, J., Ghishan, F. K., & Chen, H. (2011). Impaired Intestinal Mucin Synthesis and Bicarbonate Secretion in NHE8KO Mice. Gastroenterology, 140(5), S-660. doi:10.1016/s0016-5085(11)62737-6
- Xu, H., Zhang, B., Xu, H., Wang, C., Li, J., Ghishan, F. K., & Chen, H. (2011). Tryptophan Affects Bone Density by Regulating Phosphate Homeostasis. Gastroenterology, 140(5), S-358. doi:10.1016/s0016-5085(11)61463-7
- Xu, H., Zhang, B., Xu, H., Wang, C., Li, J., King, N., Ghishan, F. K., Chen, R., & Chen, H. (2011). NHE2X3 DKO mice exhibit gender-specific NHE8 compensation.. American journal of physiology. Gastrointestinal and liver physiology, 300(4), G647-53. doi:10.1152/ajpgi.00546.2010More infoNHE8, the newest member of the sodium/hydrogen exchanger family, is expressed in the epithelial cells of the intestine and the kidney. Intestinal expression of NHE8 is significantly higher than that of NHE2 and NHE3 at a young age, suggesting that NHE8 is an important player for intestinal sodium absorption during early development. The current study was designed to explore if NHE8 plays a compensatory role for the loss of NHE2 and NHE3 function in NHE2X3 double-knockout (NHE2X3 DKO) mice. We further explored the regulatory mechanism(s) responsible for the change in NHE8 expression in NHE2X3 DKO mice. We found that >95% of NHE2X3 DKO mice survived through weanling. However, only 60% of male NHE2X3 DKO mice and 88% of female NHE2X3 DKO mice survived to 6 wk of life. We also found that the expression of NHE8 in wild-type female mice was higher compared with wild-type male mice after puberty. In NHE2X3 KDO mice, NHE8 expression was increased in females but not in males. Using Caco-2 cells as a model of the small intestine, we showed that testosterone inhibited endogenous NHE8 expression by reducing NHE8 mRNA synthesis, whereas estrogen had no effect on NHE8 expression. Thus our data show for the first time that intestinal NHE8 has a compensatory role in NHE2X3 DKO mice and this regulation is gender-dependent.
- Xu, H., Zhang, B., Xu, H., Wang, C., Tang, C., Li, J., Ghishan, F. K., & Chen, H. (2011). Somatostatin stimulates intestinal NHE8 expression via p38 MAPK pathway.. American journal of physiology. Cell physiology, 300(2), C375-82. doi:10.1152/ajpcell.00421.2010More infoDiarrhea is a common manifestation of gastrointestinal disorders. Diarrhea-induced losses of fluid and electrolyte could lead to dehydration and electrolyte imbalances, resulting in significant morbidity and mortality, especially in children living in developing countries. Somatostatin, a peptide hormone secreted by D-cells, plays an important role in regulating motility and intestinal Na(+) absorption. Although octreotide, a somatostatin analog, is used to treat diarrhea, its mechanisms of action are unclear. Here we showed that octreotide increased brush-border membrane Na(+)/H(+) exchanger 8 (NHE8) expression in the small intestine to the exclusion of other NHEs that participate in Na(+) absorption. The same effect also occurred in human intestinal cells (Caco-2). We found that the increase of NHE8 expression by somatostatin required p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, the somatostatin receptor SSTR2 antagonist CYN154806 could abolish somatostatin-induced NHE8 expression and p38 MAPK phosphorylation. Thus our data provided the first concrete evidence indicating that somatostatin stimulates intestinal Na(+) absorption by increasing intestinal NHE8 expression through the SSTR2-p38 MAPK pathway.
- Xu, H., Xu, H., Li, J., Ghishan, F. K., & Chen, H. (2010). T1858 TNF-α Inhibits Intestinal NaPi-IIB Expression Through Competitive Binding With EGFR, Independent of TNFR1. Gastroenterology, 138(5), S-593. doi:10.1016/s0016-5085(10)62736-9
- Xu, H., Xu, H., Wang, C., Ghishan, F. K., & Chen, H. (2010). M2036 Modulation of NHE8 by Somatostatin in Human Intestinal Epithelial Cells. Gastroenterology, 138(5), S-463. doi:10.1016/s0016-5085(10)62144-0
- Xu, H., Zhang, B., Xu, H., Li, J., King, N. M., Ghishan, F. K., Chen, R., & Chen, H. (2010). W1776 Gender Differences in NHE8 Response to NHE2/NHE3 Double Knockout (Nhe2×3 Dko) Mice. Gastroenterology, 138(5), S-737. doi:10.1016/s0016-5085(10)63396-3
- Xu, H., Zhang, B., Xu, H., Tong, W., Li, J., King, N., Ghishan, F. K., & Chen, H. (2010). Testosterone down-regulates sodium-hydrogen exchanger isoform 8 (NHE8) expression. The FASEB Journal, 24.
- Xu, H., Zhang, B., Xu, H., Tooley, J. E., Li, J., Ghishan, F. K., & Chen, H. (2010). Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription.. American journal of physiology. Cell physiology, 299(1), C51-7. doi:10.1152/ajpcell.00081.2010More infoSodium/hydrogen exchangers (NHEs) play a major role in Na(+) absorption, cell volume regulation, and intracellular pH regulation. Of the nine identified mammalian NHEs, three (NHE2, NHE3, and NHE8) are localized on the apical membrane of epithelial cells in the small intestine and the kidney. Although the regulation of NHE2 and NHE3 expression has been extensively studied in the past decade, little is known about the regulation of NHE8 gene expression under physiological conditions. The current studies were performed to explore the role of epidermal growth factor (EGF) on NHE8 expression during intestinal maturation. Brush-border membrane vesicles (BBMV) were isolated from intestinal epithelia, and Western blot analysis was performed to determine NHE8 protein expression of sucking male rats treated with EGF. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcriptional factors involved in EGF-mediated regulation. Our results showed that intestinal NHE8 mRNA expression was decreased in EGF-treated rats and Caco-2 cells, and NHE8 protein abundance was also decreased in EGF-treated rats. The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by EGF treatment. This could be explained by reduced binding of transcription factor Sp3 on the NHE8 basal promoter region in the presence of EGF. Pretreatment with MEK1/2 inhibitor UO-126 could prevent EGF-mediated inhibition of NHE8 gene expression. In conclusion, this study showed that EGF inhibits NHE8 gene expression through reducing its basal transcription, suggesting an important role of EGF in regulating NHE expression during intestinal maturation.
- Xu, H., Zhang, B., Xu, H., Tooley, J. E., Li, J., Ghishan, F. K., & Chen, H. (2010). T1732 Inhibition of NHE8 Expression by Dexamethasone. Gastroenterology, 138(5), S-567. doi:10.1016/s0016-5085(10)62610-8More infowith this activity. A combination of four subunits reached highest glucogenesis indicating a broader spectrum of glucosidase activities than any individual subunit alone. Some alphaLDx was not completely available for enzyme hydrolysis, and residues are considered either slowly digestible or resistant to human enzyme digestion. This study for the first time shows the direct role of individual mucosal glucosidase subunits in starch digestion and reveals the potential of producing slow glucose release dextrins.
- Xu, H., Zhang, B., Xu, H., Tooley, J. E., Li, J., Ghishan, F. K., & Chen, H. (2010). W1778 Epidermal Growth Factor Effect on NHE8 in the Intestine. Gastroenterology, 138(5), S-738. doi:10.1016/s0016-5085(10)63398-7More infoG A A b st ra ct s acetylcholine (ACh)-induced vasodilation in pressurized mesenteric arteries (diameter: 170250 microns).Methods: Isobaric preparations of 4th-order mesenteric arteries from SpragueDawley rats were used. The arterial diameter was continuously recorded using video microscopy. The effect of Chrm1-4, COX, NOS and potassium channel inhibitors on ACh-induced vasodilation was investigated. The role of Chrm on pressure-induced vascular tone (20-100 mm Hg) and phenylephrine sensitivity was also determined. Only arteries that developed myogenic tone were used. Results: Arteries spontaneously developed >20% reduction in passive diameter. ACh-induced dose-dependent (0.001-10 μM) vasodilation (ANOVA: P
- Xu, H., Zhang, B., Xu, H., Wang, C., Li, J., Ghishan, F. K., & Chen, H. (2010). Transcriptional inhibition of intestinal NHE8 expression by glucocorticoids involves Pax5.. American journal of physiology. Gastrointestinal and liver physiology, 299(4), G921-7. doi:10.1152/ajpgi.00227.2010More infoSodium/hydrogen exchangers (NHEs) are a family of proteins that transport sodium ions into the cells by moving protons out of the cells. They play a major role in sodium absorption, cell volume regulation, and intracellular pH regulation. Three out of nine identified NHEs (NHE2, NHE3, and NHE8) are expressed on the apical membrane of intestinal epithelial cells. Glucocorticoids have been found to regulate NHE3 function in the intestine, but it is unknown if they have a similar function on NHE8 expression. Interestingly, high glucocorticoid levels in the intestine coincide chronologically with the change from high expression of NHE8 to high expression of NHE3. Studies were performed to explore the role of glucocorticoids on NHE8 expression during intestinal maturation. Brush-border membrane vesicles were isolated from intestinal epithelia, and Western blotting was performed to determine NHE8 protein expression of suckling male rats treated with methylpredisolone. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcription factors involved in glucocorticoid-mediated regulation. Our results showed that the expression of NHE8 mRNA and protein was decreased in glucocorticoid-treated rats and human intestinal epithelial cells (Caco-2). The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by glucocorticoid treatment. GMSAs suggested that the reduction in promoter activity in the presence of glucocorticoids was due to enhanced transcription factor Pax5 binding on the NHE8 proximal promoter region. In conclusion, this study showed that glucocorticoids inhibit NHE8 gene expression by increasing Pax5 binding on NHE8 gene promoter, suggesting an important role for Pax5 during intestinal maturation.
- Xu, H., Xu, H., Li, J., Ghishan, F. K., Dong, J., & Chen, H. (2009). Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis.. American journal of physiology. Gastrointestinal and liver physiology, 296(4), G775-81. doi:10.1152/ajpgi.90722.2008More infoPhosphate homeostasis is critical for many physiological functions. Up to 85% of phosphate is stored in bone and teeth. The remaining 15% is distributed in cells. Phosphate absorption across the brush-border membrane (BBM) of enterocytes occurs mainly via a sodium-dependent pathway, which is mediated by type IIb sodium-phosphate cotransporters (NaPi-IIb). Patients of inflammatory bowel diseases (IBDs) suffer not only from diarrhea and nutrient malabsorption but also from bone loss. About 31-59% of patients with IBD develop bone disorders. Since the intestine is a primary location for dietary phosphate absorption, it is logical to postulate that there is an inverse relationship between gastrointestinal disorders and phosphate transport, which, in turn, contributes to bone disorders observed in patients with IBD. Phosphate absorption and NaPi-IIb expression was studied with BBM vesicles isolated from trinitrobenzene sulphonic acid (TNBS) animals as well as in Caco-2 cells. The mechanism of TNF-alpha downregulation of NaPi-IIb expression was investigated by luciferase assay, gel mobility shift assay (GMSA), and coimmunoprecipitation. Intestinal phosphate absorption mediated by NaPi-IIb was reduced both in TNBS colitis and in TNF-alpha-treated cells. Transient transfection indicated that TNF-alpha inhibits NaPi-IIb expression by reducing NaPi-IIb basal promoter activity. GMSAs identified NF1 protein as an important factor in TNF-alpha-mediated NaPi-IIb downregulation. Signaling transduction study and coimmunoprecipitation suggested that TNF-alpha interacts with EGF receptor to activate ERK1/2 pathway. Intestinal phosphate absorption mediated by NaPi-IIb protein is reduced in colitis. This inhibition is mediated by the proinflammatory cytokine TNF-alpha through a novel molecular mechanism involving TNF-alpha/EGF receptor interaction.
- Xu, H., Xu, H., Tong, W., Li, J., & Ghishan, F. K. (2009). T1756 The Effect of Interleukin-1β On Intestinal NHE8 Expression. Gastroenterology, 136(5), A-573. doi:10.1016/s0016-5085(09)62637-8
- Xu, H., Xu, H., Uno, J. K., Li, J., Ghishan, F. K., Dong, J., Chen, R., & Chen, H. (2009). Tumor necrosis factor-{alpha} downregulates intestinal NHE8 expression by reducing basal promoter activity.. American journal of physiology. Cell physiology, 296(3), C489-97. doi:10.1152/ajpcell.00482.2008More infoNHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial Na(+) transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of Na(+)/H(+) exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-alpha-mediated NHE8 expression downregulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-alpha produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-alpha could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-alpha inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-alpha decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation.
- Xu, H., Lynch, R. M., Xu, H., Lynch, R. M., Ghishan, F. K., Dong, J., & Chen, H. (2008). Gastrointestinal distribution and kinetic characterization of the sodium-hydrogen exchanger isoform 8 (NHE8).. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 21(1-3), 109-16. doi:10.1159/000113752More infoNHE8 is a newly identified NHE isoform expressed in rat intestine. To date, the kinetic characteristics and the intestinal segmental distribution of this NHE isoform have not been studied. This current work was performed to determine the gene expression pattern of the NHE8 transporter along the gastrointestinal tract, as well as its affinity for Na(+), H(+), and sensitivity to known NHE inhibitors HOE694 and S3226. NHE8 was differentially expressed along the GI tract. Higher NHE8 expression was seen in stomach, duodenum, and ascending colon in human, while higher NHE8 expression was seen in jejunum, ileum and colon in adult mouse. Moreover, the expression level of NHE8 is much higher in the stomach and jejunum in young mice compared with adult mice. To evaluate the functional characterictics of NHE8, the pH indicator SNARF-4 was used to monitor the rate of intra-cellular pH (pH(i)) recovery after an NH(4)Cl induced acid load in NHE8 cDNA transfected PS120 cells. The NHE8 cDNA transfected cells exhibited a sodium-dependent proton exchanger activity having a Km for pH(i) of approximately pH 6.5, and a Km for sodium of approximately 23 mM. Low concentration of HOE694 (1 microM) had no effect on NHE8 activity, while high concentration (10 microM) significantly reduced NHE8 activity. In the presence of 80 microM S3226, the NHE8 activity was also inhibited significantly. In conclusion, our work suggests that NHE8 is expressed along the gastrointestinal tract and NHE8 is a functional Na(+)/H(+) exchanger with kinetic characteristics that differ from other apically expressed NHE isoforms.
- Xu, H., Xu, H., Ghishan, F. K., Dong, J., & Chen, H. (2008). 408 TNF-α Inhibits Human Intestinal NHE8 Gene Expression in Caco2 Cells Via Tyrosine Kinase Pathway. Gastroenterology, 134(4), A-57. doi:10.1016/s0016-5085(08)60271-1
- Xu, H., Xu, H., Li, J., Ghishan, F. K., Dong, J., & Chen, H. (2008). S1659 TNF-α Inhibits Human Intestinal NaPi-Iib Gene Expression in Caco2 Cells Via Epidermal Growth Factor Receptor Activation. Gastroenterology, 134(4), A-244. doi:10.1016/s0016-5085(08)61130-0
- Xu, H., Xu, H., Uno, J. K., Lipko, M. A., Kiela, P. R., Hua, P., Ghishan, F. K., & Dong, J. (2007). Sp1 and Sp3 mediate NHE2 gene transcription in the intestinal epithelial cells.. American journal of physiology. Gastrointestinal and liver physiology, 293(1), G146-53. doi:10.1152/ajpgi.00443.2006More infoOur previous studies have identified a minimal Sp1-driven promoter region (nt -36/+116) directing NHE2 expression in mouse renal epithelial cells. However, this minimal promoter region was not sufficient to support active transcription of NHE2 gene in the intestinal epithelial cells, suggesting the need for additional upstream regulatory elements. In the present study, we used nontransformed rat intestinal epithelial (RIE) cells as a model to identify the minimal promoter region and transcription factors necessary for the basal transcription of rat NHE2 gene in the intestinal epithelial cells. We identified a region within the rat NHE2 gene promoter located within nt -67/-43 upstream of transcription initiation site as indispensable for the promoter function in intestinal epithelial cells. Mutations at nt -56/-51 not only abolished the DNA-protein interaction in this region, but also completely abolished NHE2 gene promoter activity in RIE cells. Supershift assays revealed that Sp1 and Sp3 interact with this promoter region, but, contrary to the minimal promoter indispensable for renal expression of NHE2, both transcription factors expressed individually in Drosophila SL2 cells activated rat NHE2 gene promoter. Moreover, Sp1 was a weaker transactivator and when coexpressed in SL2 cells it reduced Sp3-mediated NHE2 basal promoter activity. Furthermore, DNase I footprinting confirmed that nt -58/-51 is protected by nuclear protein from RIE cells. We conclude that the mechanism of basal control of rat NHE2 gene promoter activity is different in the renal and intestinal epithelium, with Sp3 being the major transcriptional activator of NHE2 gene transcription in the intestinal epithelial cells.
- Xu, H., Xu, H., Uno, J. K., Tooley, J. E., Mah, A. T., Kiela, P. R., & Ghishan, F. K. (2007). Altered Expression of Renal Sodium-Phosphate Cotransporter in Mice with Chemically Induced Colitis. The FASEB Journal, 21(6).
- Ghishan, F. K., Xu, H., Yang, C., Yin, Y., Wang, Z., Shen, Y., Squires, J. E., Li, J., Collins, J. F., & Fan, M. Z. (2006). Visceral distribution of the type II sodium‐dependent phosphate cotransporter (NaPi‐II) isomer mRNA and the expression of NaPi‐IIc mRNA along the intestinal longitudinal axis in the post‐weaned pig. The FASEB Journal, 20(5). doi:10.1096/fasebj.20.5.a1064-c
- Xu, H., Lynch, R. M., Xu, H., Uno, J. K., Stewart, M., Lynch, R. M., Ghishan, F. K., & Chen, H. (2006). Functional Characterization of a Novel Intestinal NHE Isoform (NHE8). The FASEB Journal, 20(5).
- Xu, H., Lynch, R. M., Xu, L., Xu, H., Nullmeyer, K. D., Lynch, R. M., Kiela, P. R., Ghishan, F. K., & Dixit, M. P. (2006). Regulation of Na+/H+ exchanger-NHE3 by angiotensin-II in OKP cells.. Biochimica et biophysica acta, 1758(4), 519-26. doi:10.1016/j.bbamem.2006.02.023More infoPrevious studies have shown that circulating Angiotensin II (A-II) increases renal Na+ reabsorption via elevated Na+/H+ exchanger isoform 3 (NHE3) activity. We hypothesized that prolonged exposure to A-II leads to an increased expression of renal NHE3 by a transcriptionally mediated mechanism. To test this hypothesis, we utilized the proximal tubule-like OKP cell line to evaluate the effects of 16-h treatment with A-II on NHE3 activity and gene expression. A-II significantly stimulated NHE3-mediated, S-3226-sensitive Na+/H+ exchange. Inhibition of transcription with actinomycin D abolished the stimulatory effect of A-II on NHE3-mediated pH recovery in acid-loaded OKP cells. This prolonged exposure to A-II was also found to elevate endogenous NHE3 mRNA (by 40%)-an effect also abolished by inhibition of gene transcription. To evaluate the molecular mechanism by which A-II regulates NHE3 expression, the activity of NHE3 promoter driven reporter gene was analyzed in transient transfection assays. In transfected OKP cells, rat NHE3 promoter activity was significantly stimulated by A-II treatment, and preliminary mapping indicated that the A-II responsive element(s) is present between 149 and 548 bp upstream of the transcription initiation site in the NHE3 gene promoter. We conclude that a transcriptional mechanism is at least partially responsible for the chronic effects of A-II treatment on renal NHE3 activity.
- Xu, H., Xu, H., Uno, J. K., Kolek, O. I., Kiela, P. R., Ghishan, F. K., & Feferman, H. R. (2006). The Role of TNF{alpha} in Down-Regulation of Osteoblast Phex Gene Expression in Experimental Colitis. The FASEB Journal, 20(5).
- Xu, H., Xu, H., Uno, J. K., Timmermann, B. N., Kolek, O. I., Kiela, P. R., Hines, E. R., & Ghishan, F. K. (2006). The role of tumor necrosis factor alpha in down-regulation of osteoblast Phex gene expression in experimental murine colitis.. Gastroenterology, 131(2), 497-509. doi:10.1053/j.gastro.2006.05.020More infoReduced bone mass is a common complication of inflammatory bowel disease (IBD), although the mechanisms that contribute to osteopenia are not completely understood. Tumor necrosis factor alpha (TNF-alpha) is up-regulated in patients with IBD and has detrimental effects on osteoblasts. Phex gene is expressed predominantly in osteoblasts, and its disruption results in defective bone mineralization. The aim of this study was to evaluate whether TNF-alpha regulates Phex gene expression thus contributing to the abnormal bone metabolism observed in IBD..Phex gene expression was evaluated in calvaria of 6-7-week-old mice administered with trinitrobenzene sulfonic acid (TNBS) with or without neutralizing anti-TNF-alpha antibody, dietary curcumin, or systemically with recombinant TNF-alpha. TNF-alpha-treated UMR-106 osteoblasts were also examined. Phex promoter activity was assayed in transiently transfected TNF-alpha-treated UMR-106 cells..Compared with control animals, Phex messenger RNA (mRNA) expression decreased by 40%-50% in both TNBS colitis and TNF-alpha-injected mice. Dietary curcumin and anti-TNF-alpha antibody counteracted the detrimental effect of TNBS on Phex gene expression. TNF-alpha-treated UMR-106 cells showed a concentration-dependent and transcriptionally mediated decrease in Phex mRNA and gene promoter activity, with the -133 to -74 bp region of the Phex promoter likely involved in the mechanism of TNF-alpha action. Coinciding with decreased Phex protein level, TNF-alpha drastically reduced mineralization in UMR-106 osteoblasts..Acute colitis and TNF-alpha decrease Phex mRNA and protein expression via a transcriptional mechanism. TNF-alpha-mediated reduction in Phex protein is at least in part responsible for inhibition of osteoblast mineralization, and the described mechanism may contribute to the abnormal bone metabolism associated with IBD.
- Xu, H., Yin, Y., Yang, C., Xu, H., Wang, Z., Squires, J. E., Shen, Y., Li, J., Ghishan, F. K., Fan, M. Z., & Collins, J. F. (2006). Visceral distribution of the type II sodium-dependent phosphate cotransporter (NaPi-II) isomer mRNA and the expression of NaPi-IIc mRNA along the intestinal longitudinal axis in the post-weaned pig. The FASEB Journal, 20(5).
- Xu, H., Xu, H., Ghishan, F. K., & Chen, R. (2005). Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8.. American journal of physiology. Gastrointestinal and liver physiology, 289(1), G36-41. doi:10.1152/ajpgi.00552.2004More infoApically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na(+) absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.
- Xu, H., Xu, H., Uno, J. K., Inouye, M., Ghishan, F. K., & Collins, J. F. (2005). NF1 transcriptional factor(s) is required for basal promoter activation of the human intestinal NaPi-IIb cotransporter gene.. American journal of physiology. Gastrointestinal and liver physiology, 288(2), G175-81. doi:10.1152/ajpgi.00396.2004More infoThe human intestinal type IIb Na+-P(i) cotransporter (hNaPi-IIb) gene promoter lacks a TATA box and has a high GC content in the 5'-flanking region. To understand the mechanism of hNaPi-IIb gene transcription, the current study was performed to characterize the minimal promoter region and transcriptional factor(s) necessary to activate gene expression in human intestinal cells (Caco-2). With the use of progressively shorter promoter constructs, a minimal promoter extending from bp -58 to +15 was identified and shown to direct high levels of hNaPi-IIb cotransporter expression in Caco-2 cells. Gel mobility shift assays (GMSAs) indicated that two regions could be bound by nuclear proteins from Caco-2 cells: region A at bp -26/-23 and region B at bp -44/-35. The introduction of mutations in region A abolished promoter activity, whereas mutations in region B had no effect. Deletion mutants of the same regions showed identical results. Furthermore, DNase I footprinting experiments confirmed the observation made by GMSAs. Additional studies, which used a specific nuclear factor 1 (NF1) antiserum, demonstrated that NF1 protein(s) binds to the minimal promoter at region A. These results indicated that the NF1 protein(s) is required to activate the basal transcription of hNaPi-IIb gene under normal growth conditions. This study has thus identified a new target gene in the small intestinal epithelium that is directly regulated by NF1 transcriptional factor(s).
- Xu, H., Xu, L., Xu, H., Ghishan, F. K., Dixit, M. P., Collins, J. F., & Bai, L. (2004). Effect of angiotensin-II on renal Na+/H+ exchanger-NHE3 and NHE2.. Biochimica et biophysica acta, 1664(1), 38-44. doi:10.1016/j.bbamem.2004.03.011More infoThe purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content.
- Xu, H., Xu, H., Inouye, M., Hines, E. R., Ghishan, F. K., & Collins, J. F. (2003). EGF regulation on the human intestinal NaPi-IIb gene expression involves c-Myb. Gastroenterology, 124(4), A127. doi:10.1016/s0016-5085(03)80625-x
- Xu, H., Xu, H., Inouye, M., Hines, E. R., Ghishan, F. K., & Collins, J. F. (2003). Transcriptional regulation of the human NaPi-IIb cotransporter by EGF in Caco-2 cells involves c-myb.. American journal of physiology. Cell physiology, 284(5), C1262-71. doi:10.1152/ajpcell.00456.2002More infoThe type IIb sodium-phosphate (NaP(i)-IIb) cotransporter mediates intestinal phosphate absorption. Previous work in our laboratory has shown that EGF inhibited NaP(i)-IIb cotransporter expression through transcriptional regulation. To understand this regulation, progressively shorter human NaP(i)-IIb promoter constructs were used to define the EGF response region, and gel mobility shift assays (GMSAs) were used to characterize DNA-protein interactions. Promoter analysis determined that the EGF response region was located between -784 and -729 base pair (bp) of the promoter. GMSAs and overexpression studies revealed an interaction between this promoter region and c-myb transcription factor. Inhibition of EGF receptor activation restored promoter function. Further studies suggested that MAPK, PKC, and/or PKA pathways are involved in this regulation. In conclusion, these studies suggest that EGF decreases human NaP(i)-IIb gene expression by modifying the c-myb protein such that it inhibits transcriptional activation. We further conclude that this downregulation of promoter function is mediated by EGF-activated PKC/PKA and MAPK pathways. This is the first study that demonstrates involvement of c-myb in the regulation of intestinal nutrient absorption.
- Xu, H., Xu, L., Xu, H., Uno, J. K., Inouye, M., Ghishan, F. K., & Collins, J. F. (2003). Regulation of intestinal sodium phosphate cotransporter (NaPi-IIb) gene expression by estrogen. Gastroenterology, 124(4), A104. doi:10.1016/s0016-5085(03)80514-0
- Xu, H., Xu, L., Xu, H., Uno, J. K., Inouye, M., Ghishan, F. K., Drees, J. B., & Collins, J. F. (2003). Regulation of intestinal NaPi-IIb cotransporter gene expression by estrogen.. American journal of physiology. Gastrointestinal and liver physiology, 285(6), G1317-24. doi:10.1152/ajpgi.00172.2003More infoThe current experiments were designed to study the effect of beta-estradiol on type IIb sodium-coupled phosphate (NaPi-IIb) cotransporter gene expression. Uptake studies with intestinal brush-border membrane vesicles (BBMV) showed that estrogen treatment increased sodium-dependent phosphate absorption by approximately 45% in rat intestine. Northern blot analysis indicated that NaPi-IIb mRNA expression was increased by approximately 50% after estrogen treatment. Western blot analysis also detected an increase in BBMV NaPi-IIb protein expression in estrogen-treated rats. In human intestinal Caco-2 cells, NaPi-IIb mRNA abundance was increased approximately 60% after estrogen treatment, and this increase could be abolished by inhibition of gene transcription. Transfection studies with human NaPi-IIb promoter reporter constructs showed that the promoter was responsive to estrogen treatment. These studies demonstrate for the first time that estrogen stimulates intestinal sodium-dependent phosphate absorption in female rats. This stimulation is associated with increased NaPi-IIb mRNA and protein expression. Thus the effect of estrogen on intestinal Pi absorption may be partially due to activation of NaPi-IIb gene transcription.
- Xu, H., Xu, H., Ghishan, F. K., Collins, J. F., & Bai, L. (2002). Age-dependent regulation of rat intestinal type IIb sodium-phosphate cotransporter by 1,25-(OH)(2) vitamin D(3).. American journal of physiology. Cell physiology, 282(3), C487-93. doi:10.1152/ajpcell.00412.2001More infoThe current studies were designed to characterize type IIb sodium-inorganic phosphate (P(i)) cotransporter (NaP(i)-IIb) expression and to assess the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb gene expression during rat ontogeny. Sodium-dependent P(i) absorption by intestinal brush-border membrane vesicles (BBMVs) decreased with age, and NaP(i)-IIb gene expression also decreased proportionally with age. 1,25-(OH)(2) vitamin D(3) treatment increased intestinal BBMV P(i) absorption by approximately 2.5-fold in suckling rats and by approximately 2.1-fold in adult rats. 1,25-(OH)(2) vitamin D(3) treatment also increased NaP(i)-IIb mRNA abundance by approximately 2-fold in 14-day-old rats but had no effect on mRNA expression in adults. Furthermore, in rat intestinal epithelial (RIE) cells, 1,25-(OH)(2) vitamin D(3) increased NaP(i)-IIb mRNA abundance, an effect that was abolished by actinomycin D. Additionally, human NaP(i)-IIb gene promoter activity in transiently transfected RIE cells showed approximately 1.6-fold increase after 1,25-(OH)(2) vitamin D(3) treatment. In conclusion, we demonstrate that the age-related decrease in intestinal sodium-dependent P(i) absorption correlates with decreased NaP(i)-IIb mRNA expression. Our data also suggest that the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb expression is at least partially mediated by gene transcription in suckling rats.
- Xu, H., Xu, H., Missey, T., Inouye, M., Ghishan, F. K., & Collins, J. F. (2002). Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes.. Biochimica et biophysica acta, 1567(1-2), 97-105. doi:10.1016/s0005-2736(02)00604-1More infoThe recently cloned NaPi-IIb cotransporter is an apical membrane protein that is involved in the absorption of phosphate in the intestine. To expedite functional and structural studies, the human intestinal NaPi-IIb cotransporter was stably expressed in hamster fibroblast (PS120) cells. The hNaPi-IIb cDNA stably transfected cells exhibited a 1.8-fold higher sodium-dependent phosphate uptake than vector DNA transfected cells, and had a K(m) for Pi of approximately 106 microM and a K(m) for Na(+) of approximately 34 mM. The hNaPi-IIb cotransporter was also expressed in Xenopus oocytes and it exhibited a K(m) for Pi of approximately 113 microM and a K(m) for Na(+) of approximately 65 mM. The hNaPi-IIb cotransporter expressed in both PS120 cells and oocytes was inhibited by high external pH. Furthermore, the phosphate uptake mediated by the hNaPi-IIb cotransporter was inhibited by 5 mM phosphonoformic acid (PFA), 1 mM arsenate and 100 nM phorbol myristate acetate (PMA). These results demonstrate that the human intestinal NaPi-IIb cotransporter is functional when expressed in hamster fibroblasts, and that this model system may be useful in the future to identify NaPi-IIb cotransporter-specific inhibitors.
- Xu, H., Collins, J. F., & Bal, L. (2001). Regulation of intestinal sodium phosphate cotransporter (NaPi-IIb) gene expression by vitamin D3 is age dependent. Gastroenterology, 120(5), A41-A42. doi:10.1016/s0016-5085(08)80203-x
- Xu, H., Lynch, R. M., Xu, H., Lynch, R. M., Kiela, P. R., Collins, J. F., & Bai, L. (2001). EGF regulation of rat intestinal sodium hydrogen exchanger isoform 2 (NHE 2). Gastroenterology, 120(5), A304. doi:10.1016/s0016-5085(08)81510-7
- Xu, H., Lynch, R. M., Xu, H., Lynch, R. M., Kiela, P. R., Ghishan, F. K., Collins, J. F., & Bai, L. (2001). Epidermal growth factor regulation of rat NHE2 gene expression.. American journal of physiology. Cell physiology, 281(2), C504-13. doi:10.1152/ajpcell.2001.281.2.c504More infoEpidermal growth factor (EGF) is involved in acute regulation of Na(+)/H(+) exchangers (NHEs), but the effect of chronic EGF administration on NHE gene expression is unknown. The present studies showed that EGF treatment increased NHE2-mediated intestinal brush-border membrane vesicle Na(+) absorption and NHE2 mRNA abundance by nearly twofold in 19-day-old rats. However, no changes were observed in renal NHE2 mRNA or intestinal and renal NHE3 mRNA abundance. To understand the mechanism of this regulation, we developed the rat intestinal epithelial (RIE) cell as an in vitro model to study the effect of EGF on NHE2 gene expression. EGF increased functional NHE2 activity and mRNA abundance in cultured RIE cells, and this stimulation could be blocked by actinomycin D (a transcriptional inhibitor). Additionally, NHE2 promoter reporter gene assays in transiently transfected RIE cells showed an almost twofold increase in promoter activity after EGF treatment. We conclude that rat NHE2 activity can be stimulated by chronic EGF treatment and that this response is at least partially mediated by gene transcription.
- Xu, H., Xu, H., Ghishan, F. K., Collins, J. F., & Bai, L. (2001). Molecular and functional analysis of a novel neuronal vesicular glutamate transporter.. The Journal of biological chemistry, 276(39), 36764-9. doi:10.1074/jbc.m104578200More infoGlutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.
- Xu, H., Xu, H., Ghishan, F. K., Collins, J. F., & Bai, L. (2001). Molecular cloning and functional characterization of a murine type III intestinal sodium dependent phosphate cotransporter (mPit-2) promoter. Gastroenterology, 120(5), A526. doi:10.1016/s0016-5085(08)82613-3
- Xu, H., Xu, H., Ghishan, F. K., Collins, J. F., & Bai, L. (2001). Transcriptional regulation of rat Na(+)/H(+) exchanger isoform-2 (NHE-2) gene by Sp1 transcription factor.. American journal of physiology. Cell physiology, 280(5), C1168-75. doi:10.1152/ajpcell.2001.280.5.c1168More infoThe rat Na(+)/H(+) exchanger isoform-2 (NHE-2) gene promoter lacks a TATA box and is very GC rich. A minimal promoter extending from bp -36 to +116 directs high-level expression of NHE-2 in mouse inner medullary collecting duct (mIMCD-3) cells. Four Sp1 consensus elements were found in this region. The introduction of mutations within these Sp1 consensus elements and DNA footprinting revealed that only two of them were utilized and are critical for basal transcriptional activation in mIMCD-3 cells. The use of Sp1, Sp3, and Sp4 antisera in electrophoretic mobility shift assays demonstrated that Sp1, Sp3, and Sp4 bound to this minimal promoter. We further analyzed the transcriptional regulation of NHE-2 by members of the Sp1 multigene family. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activated transcription over 100-fold, suggesting that Sp1 is critical for transcriptional regulation. However, neither Sp3 nor Sp4 was able to activate transcription in these cells. Furthermore, in mIMCD-3 cells, Sp1-mediated transcriptional activation was repressed by expression of Sp3 and Sp4. These data suggest that Sp1 is critical for the basal promoter function of rat NHE-2 and that Sp3 and Sp4 may repress transcriptional activation by competing with Sp1 for binding to core cis-elements.
- Xu, H., Xu, H., Kiela, P. R., Ghishan, F. K., Collins, J. F., & Bai, L. (2001). Regulation of the human sodium-phosphate cotransporter NaP(i)-IIb gene promoter by epidermal growth factor.. American journal of physiology. Cell physiology, 280(3), C628-36. doi:10.1152/ajpcell.2001.280.3.c628More infoThe intestinal sodium-phosphate cotransporter (NaP(i)-IIb) plays a major role in intestinal P(i) absorption. Epidermal growth factor (EGF) is involved in the regulation of P(i) homeostasis. However, the role of EGF in intestinal NaP(i)-IIb regulation is not clear. The current studies showed that EGF decreased NaP(i)-IIb mRNA abundance by 40-50% in both rat intestine and Caco-2 cells. To understand the mechanism of this regulation, we cloned the human NaP(i)-IIb gene and promoter region and studied the effect of EGF on NaP(i)-IIb gene transcription. The human NaP(i)-IIb gene has 12 exons and 11 introns. Two transcription initiation sites were identified by primer extension. Additionally, 2.8 kb of the 5'-flanking region of the gene was characterized as a functional promoter in human intestinal (Caco-2) and human lung (A549) cells. Additional studies showed that EGF inhibited promoter activity by 40-50% in Caco-2 cells and that actinomycin D treatment abolished this inhibition. EGF had no effect on promoter activity in lung (A549) cells. We conclude that the human NaP(i)-IIb gene promoter is functional in Caco-2 and A549 cells and that the gene is responsive to EGF by a transcriptionally mediated mechanism in intestinal cells.
- Xu, H., Xu, L., Xu, H., Ghishan, F. K., Collins, J. F., & Bai, L. (2001). Molecular cloning of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter.. Biochimica et biophysica acta, 1522(1), 42-5. doi:10.1016/s0167-4781(01)00297-4More infoWe report the novel cloning and preliminary characterization of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter. Five promoter/luciferase reporter gene constructs, -1816/+61, -1620/+61, -1223/+61, -600/+61 and -225/+61, showed significant luciferase activity (6-14-fold over background) when transfected into human colon carcinoma (Caco-2) and opossum kidney (OKP) cells.
- Xu, H., Ghishan, F. K., & Collins, J. F. (2000). Human, intestinal type ii sodium phosphate transporter (HNPT) gene organization and characterization of the promoter region. Gastroenterology, 118(4), A296-A297. doi:10.1016/s0016-5085(00)83274-6
- Xu, H., Xu, H., Kiela, P. R., Ghishan, F. K., & Collins, J. F. (2000). Expression of rat, renal NHE2 and NHE3 during postnatal development.. Biochimica et biophysica acta, 1464(1), 7-17. doi:10.1016/s0005-2736(99)00241-2More infoThe current studies were designed to characterize the expression of sodium-hydrogen exchangers NHE2 and NHE3 during rat, renal ontogeny. NHE2 mRNA and immunoreactive protein were more highly expressed at 2 and 3 weeks of age, with declining levels into adulthood. In situ hybridization of NHE2 mRNA localized the message to the renal inner cortex and outer medullary regions and suggested higher mRNA levels in suckling animals as compared to adults. Immunohistochemical analysis of rat kidney with the NHE2 antiserum showed specific staining of the distal convoluted tubules. In contrast, NHE3 mRNA expression was lowest in 2-week animals and higher in older rats, while NHE3 immunoreactive protein showed constant expression levels during development. Additionally uptake experiments utilizing HOE694 showed no change in NHE2 or NHE3 functional protein expression in 2-week-old rats versus adults. We conclude that the developmental increase in NHE2 mRNA and immunoreactive protein expression cannot be detected by functional assays, which suggests that NHE2 does not play a role in sodium absorption by the renal tubules (as has been previously suggested). Additionally, molecular changes seen in NHE3 mRNA expression do not affect functional protein activity, suggesting increased mRNA translational efficiency or protein stability in suckling rats.
- Xu, H., Xu, H., Kiela, P. R., Guner, Y. S., Ghishan, F. K., & Collins, J. F. (2000). Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine.. American journal of physiology. Cell physiology, 278(4), C629-37. doi:10.1152/ajpcell.2000.278.4.c629More infoOf the two known apical isoforms of the Na(+)/H(+) exchanger (NHE) family, only the NHE3 gene is regulated by glucocorticoids. The aim of these studies was to investigate the mechanisms underlying the effects of methylprednisolone (MP) on expression of NHE3 in the proximal and distal small intestine of suckling and adult rats. Immunoblots showed that the glucocorticoid responsiveness in the proximal small intestine was greatest in suckling animals (NHE3/beta-actin: 0.43 +/- 0.09 control vs. 1.57 +/- 0.15 MP; P < 0. 001), and responsiveness decreased with age with no effect in adults (0.56 +/- 0.14 vs. 0.64 +/- 0.17). Distal small intestine was responsive only in adult rats (0.49 +/- 0.13 vs. 1.65 +/- 0.09; P < 0.001). This pattern was confirmed at the mRNA level and by (22)Na(+) uptake. Western blot and [(3)H]dexamethasone mesylate binding showed that the responsiveness of NHE3 to glucocorticoids is directly related to the expression of glucocorticoid receptor (GR) in the small intestine. These studies suggest that loss and gain of glucocorticoid responsiveness in the proximal and distal small intestine, respectively, are related to age- and segment-dependent expression of GR.
- Xu, H., Xu, H., Ghishan, F. K., Collins, J. F., & Bai, L. (1999). Molecular cloning, functional characterization, tissue distribution, and chromosomal localization of a human, small intestinal sodium-phosphate (Na+-Pi) transporter (SLC34A2).. Genomics, 62(2), 281-4. doi:10.1006/geno.1999.6009More infoPhosphate plays a crucial role in cellular metabolism, and its homeostatic regulation in intestinal and renal epithelia is critical. Apically expressed sodium-phosphate (Na(+)-P(i)) transporters play a critical role in this regulation. We have isolated a cDNA (HGMW-approved symbol SLC34A2) encoding a novel human small intestinal Na(+)-P(i) transporter. The cDNA is shown to be 4135 bp in length with an open reading frame that predicts a 689-amino-acid polypeptide. The putative protein has 76% homology to mouse intestinal type II Na(+)-P(i) transporter (Na/Pi-IIb) and lower homologies with renal type II Na(+)-P(i) transporters. Northern blots showed a singular transcript of 5.0 kb in human lung, small intestine, and kidney. Computer analysis suggests a protein with 11 transmembrane domains and several potential posttranslational modification sites. Functional characterization in Xenopus laevis oocytes showed that this cDNA encodes a functional Na(+)-P(i) transporter. Furthermore, the gene encoding this cDNA was mapped to human chromosome 4p15.1-p15.3 by the FISH method.
- Xu, H., Xu, H., Kiela, P. R., Guner, Y. S., Ghishan, F. K., & Collins, J. F. (1999). Differential regulation of renal sodium-phosphate transporter by glucocorticoids during rat ontogeny.. The American journal of physiology, 277(5), C884-90. doi:10.1152/ajpcell.1999.277.5.c884More infoThe effects of chronic administration of methylprednisolone (MP) were studied on the ontogeny of the renal type II Na-P(i) transporter (NaPi-2). Immunoblot analysis showed that MP did not alter the expression of NaPi-2 protein levels in suckling and weanling rats; however, there was an approximately 50% decrease in adolescent and adult rats. There was no change in Na-dependent P(i) uptake in brush-border membrane vesicles in suckling rats, but there was an almost twofold decrease in adolescent rats induced by MP treatment. MP administration did not alter mRNA levels in suckling or adolescent rats. Dual injections with the glucocorticoid receptor blocker RU-486 (mifepristone) and MP did not reverse the downregulation of NaPi-2 immunoreactive protein levels in adolescent rats. To control for RU-486 antagonism efficiency, Na/H exchanger isoform 3 (NHE3) protein levels were also assayed after injection with RU-486 and MP. As expected, NHE3 protein levels increased after MP injection; however, the increase was blocked in adolescent rats by RU-486. We conclude that there is an age-dependent responsiveness to glucocorticoids and that the marked decrease in NaPi-2 immunoreactive protein levels and activity in adolescent rats is due to posttranscriptional mechanisms.
- Xu, H., Xu, H., Muller, Y. L., Kiela, P. R., Ghishan, F. K., Collins, J. F., & Bai, L. (1999). Characterization of cis-elements required for osmotic response of rat Na(+)/H(+) exchanger-2 (NHE-2) gene.. The American journal of physiology, 277(4), R1112-9. doi:10.1152/ajpregu.1999.277.4.r1112More infoThe Na(+)/H(+) exchanger (NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na(+) across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD-3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 5'-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic-responsive element, bp -808 to -791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp -1201 to -1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2 gene. Electrophoretic mobility shift assays suggest that different DNA-protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of trans-acting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.
- Xu, H., Xu, H., Muller, Y. L., Ghishan, F. K., Collins, J. F., & Bai, L. (1998). Molecular cloning and characterization of the rat NHE-2 gene promoter.. Biochimica et biophysica acta, 1442(2-3), 314-9. doi:10.1016/s0167-4781(98)00191-2More infoTo understand the molecular mechanisms underlying NHE-2 regulation in the mammalian kidney and intestine, we cloned and sequenced 5.6 kb of the 5'-flanking region of the rat NHE-2 gene. DNA sequence analysis revealed multiple putative cis-acting regulatory elements including SP1, CK, NFY-CBF, Tant, GCN4, and one progesterone and several retinoic acid response elements. The upstream sequence lacked TATA and CAAT boxes, but contained a high G/C rich region within the first 300 bp. A single transcriptional initiation site was identified by primer extension in rat kidney and small intestine, approximately 103 bp upstream of the previously identified 5'-end of the rat NHE-2 cDNA. Various regions of the promoter (from [-]5567 to [+]105 bp) were tested for their ability to drive expression of the luciferase reporter gene in transiently transfected murine Inner Medullary Collecting Duct (mIMCD-3) cells. Results demonstrated that [-]289, [-]1271 and [-]2630 bp constructs showed promoter activity that was significantly higher than the negative control construct (20-fold). These results also demonstrated that basal cis-acting elements are contained within [-]289 bp of the transcriptional start site. However, the functional activity of the [-]5567 bp construct was not significantly different from the negative control, suggesting that a negative regulatory element may be present between [-]2630 and [-]5567 bp of the promoter region.
- Xu, H., Xu, H., Vanderhoof, J. A., Michail, S., Kaufman, S. S., Ghishan, F. K., & Collins, J. F. (1998). Abnormal expression of brush-border membrane transporters in the duodenal mucosa of two patients with microvillus inclusion disease.. Journal of pediatric gastroenterology and nutrition, 27(5), 536-42. doi:10.1097/00005176-199811000-00008More infoMicrovillus inclusion disease is a congenital disorder characterized by secretory diarrhea. Patients demonstrate villus atrophy, loss of microvilli, and internalized inclusions of microvilli within the cytoplasm of small intestinal enterocytes. The exact molecular defect in these patients is not known. Two infants are described in this report with microvillus inclusion disease. Case 1 was a 3-month-old boy who developed secretory diarrhea shortly after birth. Case 2 was a 9-month-old boy who had abrupt onset diarrhea at 2 weeks of age resulting in weight loss and dehydration. Light microscopy revealed total villus atrophy with minimal crypt hyperplasia, and electron microscopic examination revealed variably shortened microvilli and cytoplasmic microvillus inclusions in both patients..Poly (A)+ RNA was purified from duodenal biopsies and RT-PCR reactions were performed. Normal human intestinal RNA was used as a positive control. Primers specific for human NHE-1, NHE-2, NHE-3 (2 sets), sodium-glucose transporter (SGLT1), and beta-actin were used..Results showed that NHE-1 and beta-actin cDNAs amplified to similar levels in both patient and control samples. However, the expression of NHE-2 and SGLT1 was much higher in the control sample than in the patient samples. Additionally, NHE-3 mRNA was not detected in the patient samples using two sets of NHE-3 specific primers..The patients with microvillus inclusion disease have defects in apical but not basolateral membrane transport systems, and these defects are related to the pathogenesis of the disease.
- Xu, H., Zeng, J., Xu, H., Kiela, P. R., Ghishan, F. K., & Collins, J. F. (1998). Increased NHE2 expression in rat intestinal epithelium during ontogeny is transcriptionally mediated.. The American journal of physiology, 275(4), C1143-50. doi:10.1152/ajpcell.1998.275.4.c1143More infoWe have previously described changes in intestinal brush-border membrane vesicle (BBMV) Na+/H+ exchange activity and characterized Na+/H+ exchanger (NHE3) expression during rat ontogeny. The current studies were designed to investigate developmental changes in NHE2 expression in rat intestine. In previous studies, pH-dependent uptake of Na+ in jejunal BBMV utilizing HOE-694 inhibition demonstrated that NHE2 functional protein levels were lowest in 2-wk-old rats, higher in 3-wk-old and adult rats, and highest in 6-wk-old rats [Collins et al. Am. J. Physiol. 273 (Cell Physiol. 42): C1937-C1946, 1997]. In the current investigation, Northern blot analyses showed that NHE2 mRNA levels in the jejunum were similar in 6-wk-old, adult, and 3-wk-old rats and three- to fivefold lower in 2-wk-old rats. In situ hybridization of 2- and 6-wk-old rat intestine with NHE2-specific probes confirmed Northern blot observations. Polyclonal antibodies were developed against an NHE2-specific peptide from amino acids 652-661. Western blots with NHE2 antiserum showed that the intensity of a specific 90-kDa band was lowest in 2-wk-old animals and four- to sixfold higher in 3- and 6-wk-old and adult animals. Immunohistochemical analysis showed specific staining of NHE2 antiserum to only the apical intestinal membrane. Furthermore, nuclear run-on analyses showed a 1.7-fold higher NHE2 transcription rate in 6-wk-old rats than in 2-wk-old rats. Overall, the current data suggest that increases in NHE2 expression upon weaning are mediated by increased gene transcription.
- Xu, H., Zeng, J., Xu, H., Kiela, P. R., Ghishan, F. K., & Collins, J. F. (1998). Ontogeny of basolateral membrane sodium-hydrogen exchange (NHE) activity and mRNA expression of NHE-1 and NHE-4 in rat kidney and jejunum.. Biochimica et biophysica acta, 1369(2), 247-58. doi:10.1016/s0005-2736(97)00226-5More infoNa+/H+ exchange (NHE) activity varies with ontogenic state in rat intestinal basolateral membrane vesicles (BLMV). The current investigation sought to determine if these observations are due to differential expression of BLM NHE isoforms, NHE-1 and NHE-4. In rat kidney, BLMV sodium uptake levels were similar in 2, 3 and 6 week rats (13.28+/-0.68, 14.03+/-0.84, and 11.71+/-0.66 nmol Na+/mg protein/30 s, respectively), and lower in adults (5.53+/-0.24) (n=4; p
- Xu, H., Zeng, J., Xu, H., Kiela, P. R., Ghishan, F. K., & Collins, J. F. (1997). Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium.. The American journal of physiology, 273(6), C1937-46. doi:10.1152/ajpcell.1997.273.6.c1937More infoOntogenic changes occur in intestinal brush-border membrane vesicle (BBMV) Na+/H+ exchange activity. The present studies were designed to investigate ontogenic changes in Na+/H+ exchanger (NHE) isoform 3 in rat jejunum. pH-dependent Na+ uptake was assayed in four age groups of rats in the presence of 0, 50, or 800 microM HOE-694, a specific NHE inhibitor with differential sensitivities for NHE2 [inhibition constant (Ki) = 5 microM in PS120 fibroblasts] and NHE3 (Ki = 650 microM). Results showed that NHE2 and NHE3 contribute to basal BBMV uptake at all ages. Uptake levels were highest in 6-wk-old rats, lower in adult rats, and lowest in 2-wk-old (suckling) and 3-wk-old (weanling) rats, NHE3 contribution ranged from 92% at 6 wk of age to 59% at 2 and 3 wk of age. NHE3 inhibition by 800 microM HOE-694 was 38-45%. Statistical analysis showed that HOE-694 had a significant effect at both concentrations at all ages and that differences were present between all ages except 2- and 3-wk rats (at all HOE-694 concentrations). Northern blot analyses of jejunal mucosa showed lowest NHE3 mRNA levels in 2-wk animals and higher levels in all other age groups. Polyclonal antibodies were developed against an NHE3 COOH-terminal fusion protein, and antiserum was characterized with NHE3-transfected PS120 cells and by immunohistochemistry. Western blot analyses showed lowest protein levels in 2-wk animals and higher levels in the other ages. Suckling rats were subcutaneously injected with methylprednisone (MP) for 2 days and killed 1 day later. Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment. Immunoblot analyses showed a 2.5-fold increase in NHE3 immunoreactive protein levels with MP injection. Overall, these data suggest that NHE3 is regulated during ontogeny and that ontogenic changes are most apparent around the time of weaning. Furthermore, the data suggest that NHE3 is regulated at transcriptional and posttranscriptional levels during mammalian intestinal development.
Presentations
- Bernardazzi, C., Figliuolo, V., Tong, H., Ghishan, F., & Xu, H. (2020, May). THE ROLE OF BUTYRATE ON NHE8-DEFICIENT GOBLET CELLS: AN IN VIVO AND IN VITRO STUDY. DDW 2020.
- Tong, H., Bernardazzi, C., Ghishan, F., & Xu, H. (2020, May). NHE8 EXPRESSION IMPACTS RESPONSE TO CARBON TETRACHLORIDE INDUCED ACUTE LIVER INJURY. DDW 2020.
- Xu, H., Bernardazzi, C., Li, J., & Ghishan, F. (2019, May). LOSS OF NHE8 IMPAIRS GOBLET CELL DIFFERENTIATION/MATURATION IN THE COLON. DDW 2019.
- Xu, H. (2018, Sept). Short Chain Fatty Acid and Digestive Health. 2018 IFFSH 食品科学与健康国际学术论坛. Changsha, China: 湖南农业大学.
- Xu, H., Li, J., & Ghishan, F. (2017, May). LOSS OF NHE8 EXPRESSION IMPAIRS CRYPT FUNCTION IN THE COLON. DDW.
- Gurney, M. A., Laubitz, D., Xu, H., Ghishan, F. K., & Kiela, P. R. (2016, April). Intrinsic Effects of Reduced NHE3 Activity in Intestinal Epithelial Cells. Digestive Disease Week. San Diego, USA: American Gastroenterological Association.
- Xu, H., Li, J., & Ghishan, F. (2016, Apr). NHE8 is required for goblet cell function. Experimental Biology.
- Xu, H., Li, J., & Ghishan, F. (2016, May). NHE8 has a protective role from colon cancer development. Digestive Disease Week.
- Xu, H., Bernardazzi, C., & Ghishan, F. (2018, December). EFFECTS OF BUTYRATE SUPPLEMENTATION ON NHE8KO MICE. DDW 2019.
Poster Presentations
- Xu, H., Li, J., Chen, H., & Ghishan, F. (2018, June 2-5). INCREASED COLONIC LGR5 EXPRESSION IS ASSOCIATED WITH NHE8 DEFICIENCY. DDW2018.