Scott A Boitano

Interests

Research

Airway Epithelium Physiology, Pathology and Toxicology; Drug Discovery for Protease Activated Receptor-2, roles for Asthma and Pain.

Teaching

Undergraduate -- current classes include: Respiratory Physiology (PSIO450), Independent Research; past classes include: Advanced Topics in Cellular Physiology (PSIO404); Cellular Physiology (PSIO403); Cellular Biology (Univ. Wyoming); Cellular Biology Laboratory (Univ. Wyoming).Graduate Classes -- no current classes; past classes include Cellular Physiology (Univ. Wyoming) and Cellular Signaling Mechanisms (Univ. Wyoming).Medical School Classes -- no current classes; past classes include: Mechanisms in Cellular Physiology (WWAMI Program with the Univ. Washington).Textbooks -- Ganong's Review of Medical Physiology editions 23 (2010), 24 (2012), 25 (2015), co-Author with Kim E. Barrett, Susan M. Barman, Heddwen L. Brooks.

Courses

Scholarly Contributions

Journals/Publications

• Kume, M., Ahmad, A., Shiers, S., Burton, M. D., DeFea, K. A., Vagner, J., Dussor, G., Boitano, S., & Price, T. J. (2022). C781, a β-Arrestin Biased Antagonist at Protease-Activated Receptor-2 (PAR2), Displays in vivo Efficacy Against Protease-Induced Pain in Mice. The journal of pain.
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  Given the limited options and often harmful side effects of current analgesics and the suffering caused by the opioid crisis, new classes of pain therapeutics are needed. Protease-activated receptors (PARs), particularly PAR2, are implicated in a variety of pathologies, including pain. Since the discovery of the role of PAR2 in pain, development of potent and specific antagonists has been slow. In this study, we describe the in vivo characterization of a novel small molecule/peptidomimetic hybrid compound, C781, as a β-arrestin-biased PAR2 antagonist. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. Pharmacokinetic studies were done to assess pharmacokinetic/pharmacodynamic relationship in vivo. We used both prevention and reversal paradigms with protease treatment to determine whether C781 could attenuate protease-evoked pain. C781 effectively prevented and reversed mechanical and spontaneous nociceptive behaviors in response to small molecule PAR2 agonists, mast cell activators, and neutrophil elastase. The ED of C781 (intraperitoneal dosing) for inhibition of PAR2 agonist (20.9 ng 2-AT)-evoked nociception was 6.3 mg/kg. C781 was not efficacious in the carrageenan inflammation model. Pharmacokinetic studies indicated limited long-term systemic bioavailability for C781 suggesting that optimizing pharmacokinetic properties could improve in vivo efficacy. Our work demonstrates in vivo efficacy of a biased PAR2 antagonist that selectively inhibits β-arrestin/MAPK signaling downstream of PAR2. Given the importance of this signaling pathway in PAR2-evoked nociception, C781 exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development. PERSPECTIVE: Our work provides evidence that PAR2 antagonists that only block certain aspects of signaling by the receptor can be effective for blocking protease-evoked pain in mice. This is important because it creates a rationale for developing safer PAR2-targeting approaches for pain treatment.
• Pederson, W. P., Ellerman, L. M., Sandoval, E. C., Boitano, S., Frye, J. B., Doyle, K. P., Brooks, H. L., Polverino, F., & Ledford, J. G. (2022). Development of a Novel Mouse Model of Menopause-associated Asthma. American journal of respiratory cell and molecular biology, 67(5), 605-609.
• Rivas, C. M., Schiff, H. V., Moutal, A., Khanna, R., Kiela, P. R., Dussor, G., Price, T. J., Vagner, J., DeFea, K. A., & Boitano, S. (2022). Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression. Biochemical and biophysical research communications, 591, 13-19.
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  Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2 grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.
• Schiff, H. V., Rivas, C. M., Pederson, W. P., Sandoval, E., Gillman, S., Prisco, J., Kume, M., Dussor, G., Vagner, J., Ledford, J. G., Price, T. J., DeFea, K. A., & Boitano, S. (2022). β-Arrestin-biased proteinase-activated receptor-2 antagonist C781 limits allergen-induced airway hyperresponsiveness and inflammation. British journal of pharmacology.
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  Asthma is a heterogenous disease strongly associated with inflammation that has many different causes and triggers. Current asthma treatments target symptoms such as bronchoconstriction and airway inflammation. Despite recent advances in biological therapies, there remains a need for new classes of therapeutic agents with novel, upstream targets. The proteinase-activated receptor-2 (PAR2) has long been implicated in allergic airway inflammation and asthma and it remains an intriguing target for novel therapies. Here, we describe the actions of C781, a newly developed low MW PAR2 biased antagonist, in vitro and in vivo in the context of acute allergen exposure.
• Dy, A. B., Langlais, P. R., Barker, N. K., Addison, K. J., Tanyaratsrisakul, S., Boitano, S., Christenson, S. A., Kraft, M., Meyers, D., Bleecker, E. R., Li, X., & Ledford, J. G. (2021). Myeloid-associated differentiation marker is a novel SP-A-associated transmembrane protein whose expression on airway epithelial cells correlates with asthma severity. Scientific reports, 11(1), 23392.
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  Surfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma.
• Mwirigi, J., Kume, M., Hassler, S. N., Ahmad, A., Ray, P. R., Jiang, C., Chamessian, A., Mseeh, N., Ludwig, B. P., Rivera, B. D., Nieman, M. T., Van de Ven, T., Ji, R. R., Dussor, G., Boitano, S., Vagner, J., & Price, T. J. (2021). A Role for Protease Activated Receptor Type 3 (PAR3) in Nociception Demonstrated Through Development of a Novel Peptide Agonist. The journal of pain, 22(6), 692-706.
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  The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3 mice. We characterized other nociceptive phenotypes in PAR3 mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3 mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. PERSPECTIVE: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.
• Rivas, C. M., Schiff, H. V., Moutal, A., Khanna, R., Kiela, P. R., Price, T. J., Vagner, J., DeFea, K. A., & Boitano, S. (2021). Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.. Biophysical and Biochemical Research Communications, 591, 13-19. doi:10.1016/j.bbrc.2021.12.090
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  Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to initiate the physiological response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-/-) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiological responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR-/- line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2-/- grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.
• Rivas, C. M., Yee, M. C., Addison, K. J., Lovett, M., Pal, K., Ledford, J. G., Dussor, G., Price, T. J., Vagner, J., DeFea, K. A., & Boitano, S. (2021). Novel proteinase-activated receptor-2 (PAR2) antagonist C391 inhibits Alternaria-induced human airway epithelial signaling in vitro and asthma indicators in acute exposure murine models. British journal of pharmacology.
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  Despite availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in pre-clinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 induces intracellular Ca , mitogen activated protein kinase (MAPK) and β-arrestin signaling in the airway, leading to both inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 antagonism by C391 would attenuate allergen-induced acutely expressed asthma indicators in murine models.
• Hassler, S. N., Kume, M., Mwirigi, J. M., Ahmad, A., Shiers, S., Wangzhou, A., Ray, P. R., Belugin, S. N., Naik, D. K., Burton, M. D., Vagner, J., Boitano, S., Akopian, A. N., Dussor, G., & Price, T. J. (2020). The cellular basis of protease-activated receptor 2-evoked mechanical and affective pain. JCI insight, 5(11).
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  Protease-activated receptor 2 (PAR2) has long been implicated in inflammatory and visceral pain, but the cellular basis of PAR2-evoked pain has not been delineated. Although PAR2-evoked pain has been attributed to sensory neuron expression, RNA-sequencing experiments show ambiguous F2rl1 mRNA detection. Moreover, many pharmacological tools for PAR2 are nonspecific, acting also on the Mas-related GPCR family (Mrg) that are highly enriched in sensory neurons. We sought to clarify the cellular basis of PAR2-evoked pain. We developed a PAR2-conditional knockout mouse and specifically deleted PAR2 in all sensory neurons using the PirtCre mouse line. Our behavioral findings show that PAR2 agonist-evoked mechanical hyperalgesia and facial grimacing, but not thermal hyperalgesia, are dependent on PAR2 expression in sensory neurons that project to the hind paw in male and female mice. F2rl1 mRNA is expressed in a discrete population (~4%) of mostly small-diameter sensory neurons that coexpress the Nppb and IL31ra genes. This cell population has been implicated in itch, but our work shows that PAR2 activation in these cells causes clear pain-related behaviors from the skin. Our findings show that a discrete population of DRG sensory neurons mediate PAR2-evoked pain.
• Nguyen, C. H., Zeng, C., Boitano, S. A., Field, J. A., & Sierra Alvarez, M. R. (2020). Assessment of Gallium- and Indium-Based Nanoparticles Toward Human Bronchial Epithelial Cells Using an Impedance-Based Real-Time Cell Analyzer. International Journal of Toxicology, 39, 218-231.
• Cotter, M. L., Cotter, M. L., Boitano, S. A., Boitano, S. A., Lampe, P. D., Lampe, P. D., Solan, J. L., Solan, J. L., Vagner, J., Vagner, J., Ek Vitorin, J. F., Ek Vitorin, J. F., Burt, J. M., & Burt, J. M. (2019). The lipidated connexin mimetic peptide SRPTEKT-Hdc is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho-isoform. Am J Physiol Cell Physiol, 317(4), C825-C842.
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  Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 microM, 1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl (Hdc) lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT-Hdc) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca(2+)-wave propagation (IC50 64.8 pM) and HCh-mediated dye uptake (IC50 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT-Hdc inhibition of Ca(2+)-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca(2+)-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT-Hdc potently inhibited GJCh-mediated Ca(2+)-wave propagation (IC50 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca(2+)-wave propagation, dye coupling, and dye uptake were largely unaffected (IC50 12.3 muM) by SRPTEKT-Hdc in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT-Hdc is a potent inhibitor of physiological Ca(2+)-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.
• Dy, A. C., Arif, M. Z., Addison, K. J., Que, L. G., Boitano, S. A., Kraft, M., & Ledford, J. (2019). Genetic Variation in Surfactant Protein-A2 Delays Resolution of Eosinophilia in Asthma. J Immunol, 203(5), 1122-1130.
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  Surfactant protein-A (SP-A) is an important mediator of pulmonary immunity. A specific genetic variation in SP-A2, corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 of the lectin domain, was shown to alter the ability of SP-A to inhibit eosinophil degranulation. Because a large subgroup of asthmatics have associated eosinophilia, often accompanied by inflammation associated with delayed clearance, our goal was to define how SP-A mediates eosinophil resolution in allergic airways and whether genetic variation affects this activity. Wild-type, SP-A knockout (SP-A KO) and humanized (SP-A2 223Q/Q, SP-A2 223K/K) C57BL/6 mice were challenged in an allergic OVA model, and parameters of inflammation were examined. Peripheral blood eosinophils were isolated to assess the effect of SP-A genetic variation on apoptosis and chemotaxis. Five days postchallenge, SP-A KO and humanized SP-A2 223K/K mice had persistent eosinophilia in bronchoalveolar lavage fluid compared with wild-type and SP-A2 223Q/Q mice, suggesting an impairment in eosinophil resolution. In vitro, human SP-A containing either the 223Q or the 223K allele was chemoattractant for eosinophils whereas only 223Q resulted in decreased eosinophil viability. Our results suggest that SP-A aids in the resolution of allergic airway inflammation by promoting eosinophil clearance from lung tissue through chemotaxis, independent of SP-A2 Q223K, and by inducing apoptosis of eosinophils, which is altered by the polymorphism.
• Hassler, S., Ahmad, F. B., Burgos-Vega, C. C., Boitano, S. A., Vagner, J., Price, T. J., & Dussor, G. (2019). Protease activated receptor 2 (PAR2) activation casues migraine-like pain behaviors in mice. Cephalalgia, 39(1), 111-122. doi:10.1177/0333102418779548
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  BACKGROUND: Pain is the most debilitating symptom of migraine. The cause of migraine pain likely requires activation of meningeal nociceptors. Mast cell degranulation, with subsequent meningeal nociceptor activation, has been implicated in migraine pathophysiology. Degranulating mast cells release serine proteases that can cleave and activate protease activated receptors. The purpose of these studies was to investigate whether protease activated receptor 2 is a potential generator of nociceptive input from the meninges by using selective pharmacological agents and knockout mice. METHODS: Ratiometric Ca(++) imaging was performed on primary trigeminal and dural cell cultures after application of 2at-LIGRL-NH2, a specific protease activated receptor 2 agonist. Cutaneous hypersensitivity and facial grimace was measured in wild-type and protease activated receptor 2(-/-) mice after dural application of 2at-LIGRL-NH2 or compound 48-80, a mast cell degranulator. Behavioral experiments were also conducted in mice after dural application of 2at-LIGRL-NH2 (2AT) in the presence of either C391, a selective protease activated receptor 2 antagonist, or sumatriptan. RESULTS: 2at-LIGRL-NH2 evoked Ca(2+) signaling in mouse trigeminal neurons, dural fibroblasts and in meningeal afferents. Dural application of 2at-LIGRL-NH2 or 48-80 caused dose-dependent grimace behavior and mechanical allodynia that were attenuated by either local or systemic application of C391 as well as in protease activated receptor 2(-/-) mice. Nociceptive behavior after dural injection of 2at-LIGRL-NH2 was also attenuated by sumatriptan. CONCLUSIONS: Functional protease activated receptor 2 receptors are expressed on both dural afferents and fibroblasts and activation of dural protease activated receptor 2 produces migraine-like behavioral responses. Protease activated receptor 2 may link resident immune cells to meningeal nociceptor activation, driving migraine-like pain and implicating protease activated receptor 2 as a therapeutic target for migraine in humans.
• Witten, M. L., Chau, B., Saez, E., Boitano, S. A., & Lantz, R. C. (2019). Early life inhalation exposure to mine tailings dust affects lung development. Toxicology and Applied Pharmacology.
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  Exposure to mine tailings dust from active and abandoned mining operations may be a very significant health hazard, especially to sensitive populations living in arid and semi-arid climates like the desert southwest of the US. It is anticipated that early life exposures during sensitive times of development can lead to adult disease. However, very few studies have investigated the effects of inhalation exposure to real world dusts during lung development. Using a mouse model, we have examined the effect(s) of inhalation of real world mine tailing dusts under three separate conditions: (1) Exposure only during in utero development (exposure of the pregnant moms) (2) exposure only after birth and (3) exposures that occurred continuously during in utero development, through gestation and birth until the mice reached adulthood (28days old). We found that the most significant changes in lung structure and function were observed in male mice when exposure occurred continuously throughout development. These changes included increased airway hyper-reactivity, increased expression of epithelial to mesenchymal (EMT) transition protein markers and increased expression of cytokines related to eosinophils. The data also indicate that in utero exposures through maternal inhalation can prime the lung of male mice for more severe responses to subsequent postnatal exposures. This may be due to epigenetic alterations in gene regulation, immune response, molecular signaling, and growth factors involved in lung development that may make the neonatal lung more susceptible to continued dust exposure.
• Yee, M. C., Yee, M. C., Nichols, H. L., Nichols, H. L., Polley, D., Polley, D., Mahmoud, S., Mahmoud, S., Pal, K., Pal, K., Lee, K., Lee, K., Wilson, E. H., Wilson, E. H., Daines, M. O., Daines, M. O., Hollenberg, M. D., Hollenberg, M. D., Boitano, S. A., , Boitano, S. A., et al. (2018). Protease-activated Receptor-2 Signaling through beta-Arrestin-2 Mediates Alternaria Alkaline Serine Protease-induced Airway Inflammation. Am J Physiol Lung Cell Mol Physiol. doi:10.1152/ajplung.00196.2018
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  Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similar to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor, protease-activated receptor-2 (PAR2), to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/beta-arrestin signaling axis, to identify the protease activity responsible for PAR2 signaling and determine if protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/beta-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2(-/-) and beta-arrestin-2(-/-) mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type mice, but not PAR2(-/-) or beta-arrestin-2(-/-) mice. Protease was isolated from Alternaria preparations and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria Alkaline Serine Protease (AASP), was sufficient to fully activate PAR2 signaling and induce beta-arrestin-2-dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/beta-arrestin signaling axis. Thus, beta-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.
• Moy, J. K., Khoutorsky, A. A., Asiedu, M. N., Black, B. J., Kuhn, J. L., Barragan-Iglesias, P., Megat, S., Burton, M. D., Burgos-Vega, C. C., Melemedjian, O. K., Boitano, S., Vagner, J., Gkogkas, C. G., Pancrazio, J. J., Mogil, J. S., Dussor, G. G., Sonenberg, N., & Price, T. J. (2017). The MNK - eIF4E signaling axis controls injury-induced nociceptive plasticity and the transition to chronic pain. Journal of Neuroscience, 37(31), 7481-7499. doi:10.1523/JNEUROSCI.0220-17.2017
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  Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4E(S209A) ). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca(2+) imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4E(S209A) mice. These effects were recapitulated in Mnk1/2(-/-) mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4E(S209A) and Mnk1/2(-/-) mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in the excitability of peripheral nociceptive neurons, but the precise mechanisms controlling these changes are not elucidated. Emerging evidence demonstrates that mRNA translation regulation pathways are key factors in changes in nociceptor excitability. Our work demonstrates that a single phosphorylation site on the 5' cap-binding protein eIF4E is a critical mechanism for changes in nociceptor excitability that drive the development of chronic pain. We reveal a new mechanistic target for the development of a chronic pain state and propose that targeting the upstream kinase, MAPK interacting kinase 1/2, could be used as a therapeutic approach for chronic pain.
• Polley, D., Mihara, K., Ramachandran, R., Vliagoftis, H., Renaux, B., Saifeddine, M., Daines, M. O., Boitano, S., & Hollenberg, H. (2017). Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2. Clinical and Experimental Allergy, 47(7), 946-960. doi:10.1111/cea.12921
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  BACKGROUND: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES: We aimed to identify, isolate and characterize the trypsin-like proteinases in a German cockroach allergen extract used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50); (2) its amino acid sequence and (3) its ability to activate calcium signaling and/or ERK1/2 phosphorylation via PAR2. METHODS: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signaling. FINDINGS: Each of the three serine proteinase-activity-based probe-labelled enzymes isolated were biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57 to 71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signaling via PAR2. CONCLUSIONS AND RELEVANCE: We have identified three distinct allergen proteinases from the German cockroach that may play different roles for allergen-sensitization in vivo via PAR2 and may represent attractive therapeutic targets for asthma.
• Sherwood, C. L., & Boitano, S. (2016). Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine. Respiratory research, 17(1), 57.
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  The potential for adverse respiratory effects following exposure to electronic (e-) cigarette liquid (e-liquid) flavorings remains largely unexplored. Given the multitude of flavor permutations on the market, identification of those flavor constituents that negatively impact the respiratory tract is a daunting task. In this study we examined the impact of common e-liquid flavoring chemicals on the airway epithelium, the cellular monolayer that provides the first line of defense against inhaled particulates, pathogens, and toxicants.
• Boitano, S., Hoffman, J., Flynn, A. N., Asiedu, M. N., Tillu, D. V., Zhang, Z., Sherwood, C. L., Rivas, C. M., DeFea, K. A., Vagner, J., & Price, T. J. (2015). The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo. British journal of pharmacology.
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  Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described.
• Burgess, J. L., Kurzius-Spencer, M., Poplin, G. S., Littau, S. R., Kopplin, M. J., Stürup, S., Boitano, S., & Clark Lantz, R. (2015). Environmental arsenic exposure, selenium and sputum alpha-1 antitrypsin. Journal of exposure science & environmental epidemiology, 24(2), 150-5.
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  Exposure to arsenic in drinking water is associated with increased respiratory disease. Alpha-1 antitrypsin (AAT) protects the lung against tissue destruction. The objective of this study was to determine whether arsenic exposure is associated with changes in airway AAT concentration and whether this relationship is modified by selenium. A total of 55 subjects were evaluated in Ajo and Tucson, Arizona. Tap water and first morning void urine were analyzed for arsenic species, induced sputum for AAT and toenails for selenium and arsenic. Household tap-water arsenic, toenail arsenic and urinary inorganic arsenic and metabolites were significantly higher in Ajo (20.6±3.5 μg/l, 0.54±0.77 μg/g and 27.7±21.2 μg/l, respectively) than in Tucson (3.9±2.5 μg/l, 0.16±0.20 μg/g and 13.0±13.8 μg/l, respectively). In multivariable models, urinary monomethylarsonic acid (MMA) was negatively, and toenail selenium positively associated with sputum AAT (P=0.004 and P=0.002, respectively). In analyses stratified by town, these relationships remained significant only in Ajo, with the higher arsenic exposure. Reduction in AAT may be a means by which arsenic induces respiratory disease, and selenium may protect against this adverse effect.
• Kurzius-Spencer, M., Foster, K., Littau, S., Richey, K. J., Clark, B. M., Sherrill, D., Boitano, S., Caruso, D. M., & Burgess, J. L. (2015). Tracheobronchial protease inhibitors, body surface area burns, and mortality in smoke inhalation. Journal of burn care & research : official publication of the American Burn Association, 30(5), 824-31.
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  The objective of this study was to assess tracheobronchial protease inhibitor concentrations longitudinally and determine whether initial concentrations predict subsequent lung injury and mortality in intubated burn victims. Tracheobronchial suction fluid was collected every 2 hours for 36 hours. Alpha-1-antitrypsin (AAT), secretory leukocyte peptidase inhibitor (SLPI), alpha-2-macroglobulin (A2M), and cell and differential counts were assayed. Partial pressure of oxygen in arterial blood/fraction of inspired oxygen (PaO2/FIO2) and peak airway pressure (PAP) were recorded for 72 hours. Standard statistics were used to evaluate cross-sectional relationships; random coefficient (mixed) models were used to evaluate temporal trends in marker concentrations and relation to clinical outcomes. Among 29 patients, 24 (83%) developed hypoxemia (PaO2/FIO2 35% TBSA burn (P = .010 and .033, respectively), when compared with patients with less severe burns. However, patients with increased A2M in combination with >35% TBSA burn had a 6-fold (95% CI: 1.8-20) increased relative risk of death. Tracheobronchial AAT and A2M levels were significantly lower in patients with more severe burns and increased over time. Initial SLPI levels predicted subsequent PAP. Increased early A2M in combination with extensive burn predicted early mortality.
• Kurzius-Spencer, M., Foster, K., Littau, S., Richey, K. J., Clark, B. M., Sherrill, D., Goodman, R. B., Boitano, S., & Burgess, J. L. (2015). Tracheobronchial markers of lung injury in smoke inhalation victims. Journal of burn care & research : official publication of the American Burn Association, 29(2), 311-8.
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  Although smoke inhalation injury victims frequently develop severe hypoxemia and are at increased risk of acute respiratory distress syndrome (ARDS), no early prognostic tests are currently available. The objectives were to determine early longitudinal changes in tracheobronchial fluid inflammatory markers and assess the value of initial concentrations as predictors of subsequent lung injury. Partial pressure of arterial oxygen (Pao2) and the fraction of inspired oxygen (Fio2) were recorded approximately every 6 hours from intubated smoke inhalation victims admitted to a regional burn center. Tracheobronchial suction fluid was collected every 2 hours and assayed for interleukins (IL-1beta, -8, and -10), tumor necrosis factor-alpha, transforming growth factor-beta1, soluble Fas ligand (sFasL), and complement factor 5a. Temporal trends in marker concentrations during 36 hours and the relations between initial concentrations and lowest Pao2/Fio2 or ARDS within 72 hours were assessed using random coefficients modeling and cross-sectional analysis. In 21 subjects with tracheobronchial samples collected within 6.5 hours of intubation, 14 (66.7%) developed acute hypoxemia (Pao2/Fio2 < or =200) within 72 hours of exposure and nine (42.9%) developed ARDS, as defined by the American-European consensus conference on ARDS. IL-8 increased sharply in the first 6.5 hours postexposure (P < .001), and IL-1beta in the first 6.1 hours (P < .001). No significant temporal trends in IL-10, tumor necrosis factor-alpha, transforming growth factor-beta1, sFasL, or complement factor 5a were found. Only initial IL-8 was associated with increased Pao2/Fio2 (P = .013) and with a minimum Pao2/Fio2 >200 (P = .042) during 72 hours. In smoke inhalation victims, tracheobronchial IL-1beta and IL-8 increase rapidly and high initial IL-8 may predict improved oxygenation.
• Maura, C. L., Boitano, S. A., Vagner, J., & Burt, J. M. (2017). Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca2+-wave propagation. American Journal of Physiology: Cell Physiology, 315(2), C141-C154. doi:10.1152/ajpcell.00156.2017
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  Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap 27: SRPTEKTIFII, and Gap36: KRDPCHQVDCFLSRPTEK) have been used as reversible, Cx-specific inhibitors of gap junction intercellular communication. Typically, these peptides require relatively high concentrations (i.e., 100–200 μM with IC50s ~20–30 μM) to achieve inhibition. We hypothesized that lipidation of Cx mimetic peptides would improve their ability to concentrate in the plasma membrane and thus more effectively interfere with intercellular signaling. A hexadecyl (Hdc) lipid tail was added to the conserved SRPTEKT peptide sequence of Gap27 (SRPTEKT-Hdc) and its efficacy in inhibiting mechanically-induced intercellular Ca2+-wave propagation in confluent cultures of both Madin-Darby canine kidney epithelial cells expressing Cx43 (MDCK43) and primary-cultured rabbit tracheal epithelial cells (RTECs) assessed. SRPTEKT-Hdc reversibly inhibited in a time- and concentration- dependent manner intercellular Ca2+-wave propagation in both cell types with an IC50 in the picomolar range (i.e., 31.8–138.9 pM), five orders of magnitude lower than the non-lipidated Gap27 peptide. SRPTEKT-Hdc was less effective at inhibiting dye coupling. Scrambled and reverse sequence lipidated peptides had no detectable inhibitory effect on Ca2+-wave propagation. Immunoblot analysis revealed no change in Cx43 expression, but a small reduction in plaque-associated Cx43 following SRPTEKT-Hdc incubation. Our results suggest SRPTEKT-Hdc potently inhibits, in a channel conformation-specific manner, intercellular IP3 signaling at concentrations that minimally affect dye (electrical) coupling. In summary, lipidation of mimetic peptides represents a paradigm for development of highly potent, efficacious and selective inhibitors of gap junction-mediated intercellular signaling.
• Tillu, D. V., Hassler, S. N., Burgos-Vega, C. C., Quinn, T. L., Sorge, R. E., Dussor, G., Boitano, S., Vagner, J., & Price, T. J. (2015). Protease activated receptor 2 (PAR2) activation is sufficient to induce the transition to a chronic pain state. Pain.
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  Protease Activated Receptor Type 2 (PAR2) is known to play an important role in inflammatory, visceral and cancer-evoked pain based on studies using PAR2 knockout (PAR2) mice. Here we have tested the hypothesis that specific activation of PAR2 is sufficient to induce a chronic pain state via extracellular signal-regulated kinase (ERK) signaling to protein synthesis machinery. We have further tested whether the maintenance of this chronic pain state involves a brain-derived neurotrophic factor (BDNF) / tropomyosin related kinase B (trkB) / atypical protein kinase C (aPKC) signaling axis. We observed that intraplantar injection of the novel, highly specific PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at), evokes a long-lasting acute mechanical hypersensitivity (ED50 ∼ 12 pmoles), facial grimacing and causes robust hyperalgesic priming as revealed by a subsequent mechanical hypersensitivity and facial grimacing to prostaglandin E2 (PGE2) injection. The pro-mechanical hypersensitivity effect of 2-at is completely absent in PAR2 mice as is hyperalgesic priming. Intraplantar injection of the upstream ERK inhibitor, U0126 and the eukaryotic initiation factor (eIF) 4F complex inhibitor, 4EGI-1, prevented the development of acute mechanical hypersensitivity and hyperalgesic priming following 2-at injection. Systemic injection of the trkB antagonist ANA-12 likewise inhibited PAR2-mediated mechanical hypersensitivity, grimacing and hyperalgesic priming. Inhibition of aPKC (intrathecal delivery of ZIP) or trkB (systemic administration of ANA-12) after the resolution of 2-at-induced mechanical hypersensitivity reversed the maintenance of hyperalgesic priming. Hence, PAR2 activation is sufficient to induce neuronal plasticity leading to a chronic pain state, the maintenance of which is dependent on a BDNF/trkB/aPKC signaling axis.
• Boitano, S., Hoffman, J., Tillu, D. V., Asiedu, M. N., Zhang, Z., Sherwood, C. L., Wang, Y., Dong, X., Price, T. J., & Vagner, J. (2014). Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering. PloS one, 9(6), e99140.
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  Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.
• Sherwood, C. L., Daines, M. O., Price, T. J., Vagner, J., & Boitano, S. (2014). A highly potent agonist to protease-activated receptor-2 reveals apical activation of the airway epithelium resulting in Ca2+-regulated ion conductance. American journal of physiology. Cell physiology, 307(8), C718-26.
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  The airway epithelium provides a barrier that separates inhaled air and its various particulates from the underlying tissues. It provides key physiological functions in both sensing the environment and initiating appropriate innate immune defenses to protect the lung. Protease-activated receptor-2 (PAR2) is expressed both apically and basolaterally throughout the airway epithelium. One consequence of basolateral PAR2 activation is the rapid, Ca(2+)-dependent ion flux that favors secretion in the normally absorptive airway epithelium. However, roles for apically expressed PAR2 activation have not been demonstrated, in part due to the lack of specific, high-potency PAR2 ligands. In the present study, we used the newly developed PAR2 ligand 2at-LIGRLO(PEG3-Pam)-NH2 in combination with well-differentiated, primary cultured airway epithelial cells from wild-type and PAR2 (-/-) mice to examine the physiological role of PAR2 in the conducting airway after apical activation. Using digital imaging microscopy of intracellular Ca(2+) concentration changes, we verified ligand potency on PAR2 in primary cultured airway cells. Examination of airway epithelial tissue in an Ussing chamber showed that apical activation of PAR2 by 2at-LIGRLO(PEG3-Pam)-NH2 resulted in a transient decrease in transepithelial resistance that was due to increased apical ion efflux. We determined pharmacologically that this increase in ion conductance was through Ca(2+)-activated Cl(-) and large-conductance K(+) channels that were blocked with a Ca(2+)-activated Cl(-) channel inhibitor and clotrimazole, respectively. Stimulation of Cl(-) efflux via PAR2 activation at the airway epithelial surface can increase airway surface liquid that would aid in clearing the airway of noxious inhaled agents.
• Boitano, S., Sherwood, C. L., Lantz, R. C., & Boitano, S. A. (2013). Chronic arsenic exposure in nanomolar concentrations compromises wound response and intercellular signaling in airway epithelial cells. Toxicological sciences : an official journal of the Society of Toxicology, 132(1).
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  Paracrine ATP signaling in the lung epithelium participates in a variety of innate immune functions, including mucociliary clearance, bactericide production, and as an initiating signal in wound repair. We evaluated the effects of chronic low-dose arsenic relevant to U.S. drinking water standards (i.e., 10 ppb [130nM]) on airway epithelial cells. Immortalized human bronchial epithelial cells (16HBE14o-) were exposed to 0, 130, or 330nM arsenic (as Na-arsenite) for 4-5 weeks and examined for wound repair efficiency and ATP-mediated Ca(2+) signaling. We found that chronic arsenic exposure at these low doses slows wound repair and reduces ATP-mediated Ca(2+) signaling. We further show that arsenic compromises ATP-mediated Ca(2+) signaling by altering both Ca(2+) release from intracellular stores (via metabotropic P2Y receptors) and Ca(2+) influx mechanisms (via ionotropic P2X receptors). To better model the effects of arsenic on ATP-mediated Ca(2+) signaling under conditions of natural exposure, we cultured tracheal epithelial cells obtained from mice exposed to control or 50 ppb Na-arsenite supplemented drinking water for 4 weeks. Tracheal epithelial cells from arsenic-exposed mice displayed reduced ATP-mediated Ca(2+) signaling dynamics similar to our in vitro chronic exposure. Our findings demonstrate that chronic arsenic exposure at levels that are commonly found in drinking water (i.e., 10-50 ppb) alters cellular mechanisms critical to airway innate immunity.
• Flynn, A. N., Hoffman, J., Tillu, D. V., Sherwood, C. L., Zhang, Z., Patek, R., Asiedu, M. N., Vagner, J., Price, T. J., & Boitano, S. (2013). Development of highly potent protease-activated receptor 2 agonists via synthetic lipid tethering. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 27(4), 1498-510.
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  Protease-activated receptor-2 (PAR₂) is a G-protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR₂ is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR₂ is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR₂ agonist (2-aminothiazol-4-yl-LIGRL-NH₂) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high-throughput physiological assay, these STL agonists displayed EC₅₀ values as low as 1.47 nM, representing a ∼200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR₂: EC₅₀ for Ca(2+) response as low as 3.95 nM; EC₅₀ for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC₅₀ of 14.4 pmol. STLs failed to elicit responses in PAR2(-/-) cells at agonist concentrations of >300-fold their EC₅₀ values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR₂ and represent opportunities for drug development at other protease activated receptors and across GPCRs.
• Sherwood, C. L., Liguori, A. E., Olsen, C. E., Lantz, R. C., Burgess, J. L., & Boitano, S. (2013). Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures. PloS one, 8(12), e82970.
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  Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {
• Hoffman, J., Flynn, A. N., Tillu, D. V., Zhang, Z., Patek, R., Price, T. J., Vagner, J., & Boitano, S. (2012). Lanthanide labeling of a potent protease activated receptor-2 agonist for time-resolved fluorescence analysis. Bioconjugate chemistry, 23(10), 2098-104.
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  Protease activated receptor-2 (PAR(2)) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR(2) can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR(2) to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR(2) peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH(2) (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR(2) agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH(2), 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH(2), 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH(2), 6-an-LIGRL) and PAR(2). A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH(2), 3-ia-LIGRL) that does not activate PAR(2) signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR(2) binding. In summary, we have developed a europium-containing PAR(2) agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR(2) ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR(2) ligands.
• Otero-González, L., Sierra-Alvarez, R., Boitano, S., & Field, J. A. (2012). Application and validation of an impedance-based real time cell analyzer to measure the toxicity of nanoparticles impacting human bronchial epithelial cells. Environmental science & technology, 46(18), 10271-8.
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  Nanomaterials are increasingly used in a variety of industrial processes and consumer products. There are growing concerns about the potential impacts for public health and environment of engineered nanoparticles. The aim of this work was to evaluate a novel impedance-based real time cell analyzer (RTCA) as a high-throughput method for screening the cytotoxicity of nanoparticles and to validate the RTCA results using a conventional cytotoxicity test (MTT). A collection of 11 inorganic nanomaterials (Ag(0), Al(2)O(3), CeO(2), Fe(0), Fe(2)O(3), HfO(2), Mn(2)O(3), SiO(2), TiO(2), ZnO, and ZrO(2)) were tested for potential cytotoxicity to a human bronchial epithelial cell, 16HBE14o-. The data collected by the RTCA system was compared to results obtained using a more traditional methyl tetrazolium (MTT) cytotoxicity assay at selected time points following application of nanomaterials. The most toxic nanoparticles were ZnO, Mn(2)O(3) and Ag(0), with 50% response at concentrations lower than 75 mg/L. There was a good correlation in cytotoxicity measurements between the two methods; however, the RTCA method maintained a distinct advantage in continually following cytotoxicity over time. The results demonstrate the potential and validity of the impedance-based RTCA technique to rapidly screen for nanoparticle toxicity.
• Boitano, S., Flynn, A. N., Schulz, S. M., Hoffman, J., Price, T. J., & Vagner, J. (2011). Potent agonists of the protease activated receptor 2 (PAR2). Journal of medicinal chemistry, 54(5), 1308-13.
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  Novel peptidomimetic pharmacophores to PAR(2) were designed based on the known activating peptide SLIGRL-NH(2). A set of 15 analogues was evaluated with a model cell line (16HBE14o-) that highly expresses PAR(2). Cells exposed to the PAR(2) activating peptide with N-terminal 2-furoyl modification (2-furoyl-LIGRLO-NH(2)) initiated increases in intracellular calcium concentration ([Ca(2+)](i) EC(50) = 0.84 μM) and in vitro physiological responses as measured by the xCELLigence real time cell analyzer (RTCA EC(50) = 138 nM). We discovered two selective PAR(2) agonists with comparable potency: compound 1 (2-aminothiazol-4-yl; Ca(2+) EC(50) = 1.77 μM, RTCA EC(50) = 142 nM) and compound 2 (6-aminonicotinyl; Ca(2+) EC(50) = 2.60 μM, RTCA EC(50) = 311 nM). Unlike the previously described agonist, these novel agonists are devoid of the metabolically unstable 2-furoyl modification and thus provide potential advantages for PAR(2) peptide design for in vitro and in vivo studies. The novel compounds described herein also serve as a starting point for structure-activity relationship (SAR) design and are, for the first time, evaluated via a unique high throughput in vitro physiological assay. Together these will lead to discovery of more potent agonists and antagonists of PAR(2).
• Boitano, S., Flynn, A. N., Sherwood, C. L., Schulz, S. M., Hoffman, J., Gruzinova, I., & Daines, M. O. (2011). Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR2. American journal of physiology. Lung cellular and molecular physiology, 300(4), L605-14.
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  Allergens are diverse proteins from mammals, birds, arthropods, plants, and fungi. Allergens associated with asthma (asthmagens) share a common protease activity that may directly impact respiratory epithelial biology and lead to symptoms of asthma. Alternaria alternata is a strong asthmagen in semiarid regions. We examined the impact of proteases from A. alternata on lung inflammation in vivo and on cleaving protease-activated receptor-2 (PAR(2)) in vitro. A. alternata filtrate applied to the airway in nonsensitized Balb/c mice induced a protease-dependent lung inflammation. Moreover, A. alternata filtrate applied to human bronchial epithelial cells (16HBE14o-) induced changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), consistent with PAR(2) activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR(2) specific ligands SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2), but not the PAR(1)-specific ligand TFLLR-NH(2). Desensitization of PAR(2) in 16HBE14o- cells with 2-furoyl-LIGRLO-NH(2) or trypsin prevented A. alternata-induced [Ca(2+)](i) changes while desensitization of PAR(1), PAR(3), and PAR(4) with thrombin had no effect on A. alternata-induced Ca(2+) responses. Furthermore, the Ca(2+) response to A. alternata filtrates was dependent on PAR(2) expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR(2) to induce rapid increases in human airway epithelial [Ca(2+)](i) in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.
• Boitano, S., Sherwood, C. L., Lantz, R. C., Burgess, J. L., & Boitano, S. A. (2011). Arsenic alters ATP-dependent Ca²+ signaling in human airway epithelial cell wound response. Toxicological sciences : an official journal of the Society of Toxicology, 121(1).
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  Arsenic is a natural metalloid toxicant that is associated with occupational inhalation injury and contaminates drinking water worldwide. Both inhalation of arsenic and consumption of arsenic-tainted water are correlated with malignant and nonmalignant lung diseases. Despite strong links between arsenic and respiratory illness, underlying cell responses to arsenic remain unclear. We hypothesized that arsenic may elicit some of its detrimental effects on the airway through limitation of innate immune function and, specifically, through alteration of paracrine ATP (purinergic) Ca²+ signaling in the airway epithelium. We examined the effects of acute (24 h) exposure with environmentally relevant levels of arsenic (i.e., < 4 μM as Na-arsenite) on wound-induced Ca²+ signaling pathways in human bronchial epithelial cell line (16HBE14o-). We found that arsenic reduces purinergic Ca²+ signaling in a dose-dependent manner and results in a reshaping of the Ca²+ signaling response to localized wounds. We next examined arsenic effects on two purinergic receptor types: the metabotropic P2Y and ionotropic P2X receptors. Arsenic inhibited both P2Y- and P2X-mediated Ca²+ signaling responses to ATP. Both inhaled and ingested arsenic can rapidly reach the airway epithelium where purinergic signaling is essential in innate immune functions (e.g., ciliary beat, salt and water transport, bactericide production, and wound repair). Arsenic-induced compromise of such airway defense mechanisms may be an underlying contributor to chronic lung disease.
• Field, J. A., Luna-Velasco, A., Boitano, S. A., Shadman, F., Ratner, B. D., Barnes, C., & Sierra-Alvarez, R. (2011). Cytotoxicity and physicochemical properties of hafnium oxide nanoparticles. Chemosphere, 84(10), 1401-7.
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  Nano-sized hafnium oxide (HfO(2)) particles are being considered for applications within the semiconductor industry. However, little is known about their cytotoxicity. The objective of this work was to assess several HfO(2) nanoparticles (NPs) samples for their acute cytotoxicity. Dynamic light scattering analysis of the samples indicated that the average particle size of the HfO(2) in aqueous dispersions was in the submicron range with a fraction of particles having nano-dimensions. The media used in the toxicity assays decreased or increased the average particle size of HfO(2) NPs due to dispersion or agglomeration. Static time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed numerous surface contaminants on the NPs. Only one HfO(2) sample caused moderate cytotoxicity to human cell lines. The inhibitory sample caused a 50% response in the Live/Dead assay with HaCaT skin cells at 2200 mg L(-1); and a 50% response in the mitochondrial toxicity test at 300 mg L(-1). A microbial inhibition assay based on methanogenic activity also revealed that another HFO(2) sample caused moderate inhibition. The difference in toxicity between samples could not be attributed to size. Instead the difference in toxicity was likely due to differences in the contaminants of the HfO(2). The ToF-SIMS analysis indicated unique signatures of Br and P in the sample toxic to human cell lines suggesting a distinct synthesis was used for that sample which may have been accompanied by inhibitory impurities. The results taken as a whole indicate that HfO(2) itself is relatively non-toxic.
• Flynn, A. N., Tillu, D. V., Asiedu, M. N., Hoffman, J., Vagner, J., Price, T. J., & Boitano, S. (2011). The protease-activated receptor-2-specific agonists 2-aminothiazol-4-yl-LIGRL-NH2 and 6-aminonicotinyl-LIGRL-NH2 stimulate multiple signaling pathways to induce physiological responses in vitro and in vivo. The Journal of biological chemistry, 286(21), 19076-88.
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  Protease-activated receptor-2 (PAR(2)) is one of four protease-activated G-protein-coupled receptors. PAR(2) is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR(2) reveals a tethered ligand that activates PAR(2) and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca(2+) signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR(2) (e.g. SLIGRL-NH(2) and SLIGKV-NH(2)) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH(2) (2-f-LIGRLO-NH(2)) underscores the use of peptidomimetic PAR(2) ligands as a mechanism to enhance pharmacological action at PAR(2), 2-f-LIGRLO-NH(2) has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH(2) and two recently described pentapeptidomimetic PAR(2)-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH(2) (2-at-LIGRL-NH(2)) and 6-aminonicotinyl-LIGRL-NH(2) (6-an-LIGRL-NH(2)). All peptidomimetic agonists stimulated PAR(2)-dependent in vitro physiological responses, MAPK signaling, and Ca(2+) signaling with an overall rank order of potency of 2-f-LIGRLO-NH(2) ≈ 2-at-LIGRL-NH(2) > 6-an-LIGRL-NH(2) ≫ SLIGRL-NH(2). Because PAR(2) plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca(2+) signaling in nociceptors in vitro, and both 2-at-LIGRL-NH(2) and 2-f-LIGRLO-NH(2) stimulated PAR(2)-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR(2) in a variety of systems and pathologies.
• Ramanathan, S., Mazzalupo, S., Boitano, S., & Montfort, W. R. (2011). Thrombospondin-1 and angiotensin II inhibit soluble guanylyl cyclase through an increase in intracellular calcium concentration. Biochemistry, 50(36), 7787-99.
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  Nitric oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca(2+)](i)), and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells and that inhibition requires an increase in [Ca(2+)](i). Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca(2+)](i), up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca(2+)](i), also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca(2+) remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca(2+)](i) in response to NO, inducing vasodilation, but also is inhibited by high [Ca(2+)](i), providing a fine balance between signals for vasodilation and vasoconstriction.
• Sherwood, C. L., Lantz, R. C., Burgess, J. L., & Boitano, S. A. (2011). Arsenic alters ATP-dependent Ca 2+ signaling in human airway epithelial cell wound response. Toxicological Sciences, 121(1), 191-206.
• Straub, A. C., Johnstone, S. R., Heberlein, K. R., Rizzo, M. J., Best, A. K., Boitano, S., & Isakson, B. E. (2010). Site-specific connexin phosphorylation is associated with reduced heterocellular communication between smooth muscle and endothelium. Journal of vascular research, 47(4), 277-86.
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  Myoendothelial junctions (MEJs) represent a specialized signaling domain between vascular smooth muscle cells (VSMC) and endothelial cells (EC). The functional consequences of phosphorylation state of the connexins (Cx) at the MEJ have not been explored.
• Kurzius-Spencer, M., Foster, K., Littau, S. R., Richey, K. J., Clark, B. M., Sherrill, D., Boitano, S. A., Caruso, D. M., & Burgess, J. L. (2009). Tracheobronchial protease inhibitors, body surface area burns, and mortality in smoke inhalation. Journal of Burn Care and Research, 30(5), 824-831.
• Boitano, S., Omsland, A., Miranda, K. M., Friedman, R. L., & Boitano, S. A. (2008). Bordetella bronchiseptica responses to physiological reactive nitrogen and oxygen stresses. FEMS microbiology letters, 284(1).
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  Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.
• Burgess, J. L., Kurzius-Spencer, M., Foster, K., Littau, S. R., Richey, K. J., Clark, B. M., Sherrill, D. L., Goodman, R. B., & Boitano, S. A. (2008). Tracheobronchial markers of lung injury in smoke inhalation victims. Journal of burn care & research : official publication of the American Burn Association, 29(2).
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  Although smoke inhalation injury victims frequently develop severe hypoxemia and are at increased risk of acute respiratory distress syndrome (ARDS), no early prognostic tests are currently available. The objectives were to determine early longitudinal changes in tracheobronchial fluid inflammatory markers and assess the value of initial concentrations as predictors of subsequent lung injury. Partial pressure of arterial oxygen (Pao2) and the fraction of inspired oxygen (Fio2) were recorded approximately every 6 hours from intubated smoke inhalation victims admitted to a regional burn center. Tracheobronchial suction fluid was collected every 2 hours and assayed for interleukins (IL-1beta, -8, and -10), tumor necrosis factor-alpha, transforming growth factor-beta1, soluble Fas ligand (sFasL), and complement factor 5a. Temporal trends in marker concentrations during 36 hours and the relations between initial concentrations and lowest Pao2/Fio2 or ARDS within 72 hours were assessed using random coefficients modeling and cross-sectional analysis. In 21 subjects with tracheobronchial samples collected within 6.5 hours of intubation, 14 (66.7%) developed acute hypoxemia (Pao2/Fio2 < or =200) within 72 hours of exposure and nine (42.9%) developed ARDS, as defined by the American-European consensus conference on ARDS. IL-8 increased sharply in the first 6.5 hours postexposure (P < .001), and IL-1beta in the first 6.1 hours (P < .001). No significant temporal trends in IL-10, tumor necrosis factor-alpha, transforming growth factor-beta1, sFasL, or complement factor 5a were found. Only initial IL-8 was associated with increased Pao2/Fio2 (P = .013) and with a minimum Pao2/Fio2 >200 (P = .042) during 72 hours. In smoke inhalation victims, tracheobronchial IL-1beta and IL-8 increase rapidly and high initial IL-8 may predict improved oxygenation.
• Caldwell, P. T., Thorne, P. A., Johnson, P. D., Boitano, S., Runyan, R. B., & Selmin, O. (2008). Trichloroethylene disrupts cardiac gene expression and calcium homeostasis in rat myocytes. Toxicological sciences : an official journal of the Society of Toxicology, 104(1), 135-43.
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  We have been investigating the molecular mechanisms by which trichloroethylene (TCE) might induce cardiac malformations in the embryonic heart. Previous results indicated that TCE disrupted expression of genes encoding proteins involved in regulation of intracellular Ca2+, [Ca2+](i), in cardiac cells, including ryanodine receptor isoform 2 (Ryr2), and sarcoendoplasmatic reticulum Ca2+ ATPase, Serca2a. These observations are important in light of the notion that altered cardiac contractility can produce morphological defects. The hypothesis tested in this study is that the TCE-induced changes in gene expression of Ca2+-associated proteins resulted in altered Ca2+ flux regulation. We used real-time PCR and digital imaging microscopy to characterize effects of various doses of TCE on gene expression and Ca2+ response to vasopressin (VP) in rat cardiac H9c2 myocytes. We observed a reduction in Serca2a and Ryr2 expression at 12 and 48 h after exposure to TCE. In addition, we found significant differences in Ca2+ response to VP in cells treated with TCE doses as low as 10 parts per billion. Taken all together, our data strongly indicate that exposure to TCE disrupts the ability of myocytes to regulate cellular Ca2+ fluxes. Perturbation of calcium signaling alters cardiac cell physiology and signal transduction and may hint to morphogenetic consequences in the context of heart development. These results point to a novel area of TCE biology and, if confirmed in vivo, may help to explain the apparent cardio-specific toxicity of TCE exposure in the rodent embryo.
• Olsen, C. E., Liguori, A. E., Zong, Y., Lantz, R. C., Burgess, J. L., & Boitano, S. (2008). Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells. American journal of physiology. Lung cellular and molecular physiology, 295(2), L293-302.
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  As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.
• Olsen, C. E., Liguori, A. E., Zong, Y., Lantz, R. C., Burgess, J. L., & Boitano, S. A. (2008). Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells. American Journal of Physiology - Lung Cellular and Molecular Physiology, 295(2), L293-L302.
• Lantz, R. C., Lynch, B. J., Boitano, S., Poplin, G. S., Littau, S., Tsaprailis, G., & Burgess, J. L. (2007). Pulmonary biomarkers based on alterations in protein expression after exposure to arsenic. Environmental health perspectives, 115(4), 586-91.
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  Environmental exposure to arsenic results in multiple adverse effects in the lung. Our objective was to identify potential pulmonary protein biomarkers in the lung-lining fluid of mice chronically exposed to low-dose As and to validate these protein changes in human populations exposed to As.
• Clark, J. A., Doelle, S. M., Halpern, M. D., Saunders, T. A., Holubec, H., Dvorak, K., Boitano, S. A., & Dvorak, B. (2006). Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment. American journal of physiology. Gastrointestinal and liver physiology, 291(5), G938-49.
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  Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function.
• Isakson, B. E., Olsen, C. E., & Boitano, S. (2006). Laminin-332 alters connexin profile, dye coupling and intercellular Ca2+ waves in ciliated tracheal epithelial cells. Respiratory research, 7, 105.
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  Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin alpha3beta3gamma2 (LM-332) proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC).
• Boitano, S., Olsen, C. O., Isakson, B. E., Seedorf, G. J., Lubman, R. L., & Boitano, S. A. (2005). Extracellular matrix-driven alveolar epithelial cell differentiation in vitro. Experimental lung research, 31(5).
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  During homeostasis and in response to injury, alveolar type II (AT2) cells serve as progenitor cells to proliferate, migrate, differentiate, and re-establish both alveolar type I (AT1) and AT2 cells into a functional alveolar epithelium. To understand specific changes in cell differentiation, we monitored morphological characteristics and cell-specific protein markers over time for isolated rat AT2 cells cultured on combinations of collagen, fibronectin and/or laminin-5 (Ln5). For all matrices tested, cultured AT2 cells displayed reduced expression of AT2 cell-specific markers from days 1 to 4 and increased expression of AT1-specific markers by day 3, with continued expression until at least day 5. Over days 5 to 7 in culture, cells took on an AT1-like phenotype (on collagen/fibronectin alone; collagen alone; or Ln5 alone), an AT2-like phenotype (on collagen/fibronectin/Ln5; or collagen/Ln5), or both AT1-like and AT2-like phenotypes (on collagen/fibronectin matrix with a subsaturating amount of Ln5). Cells transferred between matrices at day 4 of culture retained the ability to alter day 7 phenotype. We conclude that in vitro, (1) AT2 cells exhibited phenotype plasticity that included an intermediate cell type with both AT1 and AT2 cell characteristics independent of day 7 phenotype; (2) both collagen and Ln5 were needed to promote the development of an AT2-like phenotype at day 7; and (3) components of the extracellular matrix (ECM) contribute to phenotypic switching of alveolar cells in culture. The described tissue culture models provide accessible models for studying changes in alveolar epithelial cell physiology from AT2 cell progenitors to the establishment of alveolar epithelial monolayers that represent AT1-like, AT2-like, or a mix of AT1- and AT2-like cells.
• Boitano, S., Isakson, B. E., Seedorf, G. J., Lubman, R. L., Evans, W. H., & Boitano, S. A. (2003). Cell-cell communication in heterocellular cultures of alveolar epithelial cells. American journal of respiratory cell and molecular biology, 29(5).
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  The mammalian alveolar epithelium is composed of alveolar type I (AT1) and alveolar type II (AT2) cells that together coordinate tissue function. We used a heterocellular culture model of AT1 and AT2 cells to determine pathways for intercellular signaling between these two phenotypes. Gap junction protein (connexin) profiles of AT1 and AT2 cells in heterocellular cultures were similar to those seen in rat lung alveolar sections. Dye coupling studies revealed functional gap junctions between and among each cell phenotype. Localized mechanical stimulation resulted in propagated changes of intracellular Ca2+ to AT1 or AT2 cells independent of the stimulated cell phenotype. Ca2+ communication that originated after AT1 cell stimulation was inhibited by gap junction blockers, but not by an inhibitor of extracellular nucleotide signaling (apyrase). Conversely, Ca2+ communication after stimulation of AT2 cells was not significantly reduced by gap junction inhibitors. However, apyrase significantly reduced Ca2+ communication from AT2 to AT1 cells, but not from AT2 to AT2 cells. In conclusion, AT1 and AT2 cells have unique connexin profiles that allow for functional coupling and distinct intercellular pathways for coordination of Ca2+ signaling.