TGF-β reduces megalin cell surface stability by promoting shedding and subsequent transcriptional downregulation of the receptor in alveolar epithelial cells.

Rationale:

Protein-rich alveolar edema is a hallmark of acute respiratory distress syndrome (ARDS) and elevated alveolar protein concentrations are associated with worse outcomes. We have recently shown that the endocytic receptor megalin plays a central role in alveolar protein clearance and that TGF-β, which plays an important role in the pathogenesis of ARDS, rapidly (within 30 min.), reduces protein transport through induction of megalin endocytosis and degradation. However, long term effects of TGF-β (more than 24 hours) on megalin cell surface stability have not been established. Considering that megalin expression can be regulated in a Notch-like manner, the aim of the current project was to elucidate the role of TGF-β in this process.

Results:

TGF-β treatment of alveolar epithelial cells resulted in transcriptional downregulation of megalin after 24 to 48h, which was mediated by shedding of the receptor and a subsequent increase of the megalin c-terminal fragment (MCTF) abundance, a negative regulator of megalin gene expression, with a maximum release between 8 and 12h after TGF-β exposure. Protein kinase C (PKC) has been described to activate matrix metalloproteases (MMPs) that are responsible for megalin shedding. Importantly, MCTF abundance was markedly reduced in the presence of a PKC inhibitor (gö6976) and specific knockdown of the kinase prevented the TGF-β-induced megalin endocytosis and rescued megalin function. Similar results were obtained when γ-secretase activity, which is responsible for regulated intramembrane proteolysis (RIP) of megalin in response to shedding, was inhibited. In order to identify the MMPs involved in the shedding of megalin, MMP-2, -9 and -14 expression, release and activation upon TGF-β treatment were assessed. An increase in protein expression of all three MMPs was observed in whole cell lysates, however only MMP-2 release and activity were upregulated in cell culture supernatants. Furthermore, TGF-β increased co-localization of MMP-14 and PKC at the plasma membrane. Finally, genetic inhibition of each of these enzymes rescued megalin cell surface stability and function in the presence of TGF-β.

Conclusions:

TGF-β downregulates megalin transcriptional expression by increasing shedding and RIP of the receptor in a process dependent on PKC, γ-secretase and MMPs activity. As lower protein concentrations in the ELF are associated with better outcomes in ARDS, interfering with this newly identified signaling pattern may hold a therapeutic promise.