Investigation of the proteins SPARC and HMGB1 in chronic airways disease

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The matricellular protein, secreted protein acidic and rich in cysteine (SPARC), mediates the interaction between cells and their surrounding extracellular matrix (ECM) but does not contribute structurally to the matrix. It regulates basic cellular functions such as cell adhesion and proliferation, as well as the processing and deposition of ECM proteins. SPARC is overexpressed in many fibrotic tissues including the lung. SPARC also serves as a down-stream mediator of transforming growth factor-beta (TGF-β), a key driver of airway remodeling in chronic airways disease, and demonstrates context-dependent immunoregulatory functions. Although airway inflammation and remodeling are prominent features of asthma and chronic obstructive pulmonary disease (COPD), the role of SPARC in these conditions has not been studied. In this thesis, we investigated the expression of SPARC in airway structural cells including airway epithelial cells (AECs) and airway smooth muscle (ASM) cells, and also determined if its expression is altered in cells derived from subjects with asthma or COPD. We demonstrated that TGF-β increases SPARC expression and release in AECs and ASM cells, although to a lesser extent in the former. We observed that type 1 and type 2 cytokines tend to suppress basal and TGF-β-mediated SPARC expression in AECs, and showed that TGF-β-induced SPARC expression in ASM cells is regulated by the unfolded protein response (UPR). Notably, we observed distinct abnormalities in SPARC expression in asthma and COPD. Our preliminary studies suggest SPARC is overexpressed in AECs from subjects with asthma. In contrast, there was a trend for reduced SPARC expression in ASM cells from COPD subjects, compared to those from non-COPD subjects. Functional studies indicate SPARC does not impart immunoregulatory functions or regulate changes in airway epithelial cell phenotype, although this requires further validation. Our studies herein also explored the potential homeostatic role of extracellular high mobility group box 1 (HMGB1) in AECs. HMGB1 is a danger-associated molecular pattern (DAMP) that normally resides in the intracellular compartment, and is released into the extracellular space upon cellular injury, stress or death to orchestrate inflammatory responses. Although it is implicated as a mediator of the airway inflammatory response, its physiological role in lung homeostasis has received little attention. Interestingly, we detected HMGB1 in the culture supernatant of AECs under basal conditions, and found that it presents exclusively as a constituent of protein complexes. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic approaches, we generated an unbiased profile of HMGB1-binding proteins in the extracellular space of unstimulated AECs. Protein network analysis of identified binding proteins indicates a role for extracellular HMGB1 in epithelial cell homeostasis and airway mucosal immunity. In summary, findings in this thesis suggest aberrant regulation of SPARC expression in airway structural cells may be a contributing factor to the pathogenesis of chronic airways disease. Our studies also provide a new understanding of the extracellular functions of HMGB1 in AECs and opens new research directions for its use as a therapeutic target.
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