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.
