The acute respiratory distress syndrome (ARDS) is a severe clinical condition characterized by impaired gas exchange due to inflammation and disruption of the alveolar-capillary barrier, which leads to accumulation of protein-rich edema into the alveolar space exacerbating the damage and reducing survival. The ability to remove excess proteins from the distal airways has been associated with positive prognosis; however, there are no effective pharmacological approaches able to facilitate alveolar protein clearance. For this reason, further research is necessary in order to shed light on the molecular mechanisms responsible of regulating the pathogenesis and resolution of ARDS.Here we provide evidence that transforming growth factor-beta (TGF-beta), a key regulator of the pathogenesis of ARDS, significantly impairs alveolar protein clearance by downregulation of the endocytic receptor megalin in alveolar epithelial cells. Megalin function was found to be critical for maintenance of homeostasis in many organs where negative effects of TGF-beta on megalin function have been described. However, the exact mechanisms underlying TGF-beta-dependent megalin downregulation remain unclear. Our data suggest that TGF-beta induces rapid megalin endocytosis and subsequently promotes its degradation in an ubiquitin-proteasome-dependent manner. Furthermore, prolonged exposition to this cytokine promotes megalin ectodomain shedding and intramembrane proteolysis of the remaining fragment, resulting in the release of a soluble variant of megalin c-terminal tail that translocates into the nucleus and regulates gene expression, including repression of its own mRNA transcription. We also demonstrate that TGF-beta-induced megalin shedding and regulated intramembrane proteolysis requires protein kinase C and gamma-secretase activities; as well as regulation of the expression, activity and localization of matrix-metalloproteases (MMPs)-2, -9 and -14. Remarkably, we propose for the first time MMP-2 and MMP-14 as novel sheddases of megalin.Short- and long-term effects of TGF-beta on megalin downregulation significantly contribute to the impairment of alveolar protein clearance, which impairs the healing of the alveolar-capillary barrier and restoration of proper lung function. Understanding how to interfere with the molecular mechanisms underlying TGF-beta-induced megalin downregulation may, thus, hold a therapeutic promise.
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