Perlecan Proteolysis Induces an α2β1 Integrin- and Src Family Kinase-dependent Anti-apoptotic Pathway in Fibroblasts in the Absence of Focal Adhesion Kinase Activation

Abstract

Dysregulation of apoptosis in endothelial cells (EC) and fibroblasts contributes to fibrosis. We have shown previously that apoptosis of EC triggers the proteolysis of extracellular matrix components and the release of a C-terminal fragment of perlecan, which in turn inhibits apoptosis of fibroblasts. Here we have defined the receptors and pathways implicated in this anti-apoptotic response in fibroblasts. Neutralizing ␣2␤1 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or a recombinant perlecan C-terminal fragment (LG3) prevented resistance to apoptosis and is associated with decreased levels of Akt phosphorylation. Co-incubation of fibroblasts for 24 h with SSC or LG3 in the presence of PP2 (AG1879), a biochemical inhibitor of Src family kinases (SFKs) and focal adhesion kinase, showed a significantly decreased anti-apoptotic response. However, focal adhesion kinase gene silencing with RNA interference did not inhibit the anti-apoptotic response in fibroblasts. Src phosphorylation was increased in fibroblasts exposed to SSC, and transfection of fibroblasts with constitutively active Src mutants induced an anti-apoptotic response that was not further increased by SSC. Also, Src ؊/؊ Fyn ؊/؊ fibroblasts failed to mount an anti-apoptotic response in presence of SSC for 24 h but developed a complete anti-apoptotic response when exposed to SSC for 7 days. These results suggest that extracellular matrix fragments produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an ␣2␤1 integrin/SFK (Src and Fyn)/phosphatidylinositol 3-kinase (PI3K)-dependent pathway. In the long term, additional SFK members are recruited for sustaining the anti-apoptotic response, which could play crucial roles in abnormal fibrogenic healing.

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

“…Co-incubation with PP2 and LY2940002, inhibitors of SFK and PI3K, respectively, blocked CTGFdependent aSMA upregulation in human and mouse fibroblasts ( Figure 7b and Supplementary Figure 5). In agreement with our previous work, 15,16 heightened Akt phosphorylation was found in fibroblasts exposed to SSC (Figure 7c). CTGF also increased Akt phosphorylation in fibroblasts, which was inhibited in the presence of PP2 (Figure 7c).…”

“…We showed previously that caspase-3 activation in ECs triggers the release of the C-terminal laminin G motif of perlecan (LG3). 8,16 Yet, fibroblasts exposed to LG3 in vitro failed to differentiate into myofibroblasts (Supplementary Figure 3). This suggested that caspase-3 activation supports the release of various mediators active on target cells.…”

“…SSC, SSC-ZVAD and SSC-DMSO were generated from C3H ECs, as described for HUVECs. 8,15,16 In brief, an equal number of ECs were exposed to the vehicle (DMSO) or ZVAD-FMK (100 mM) for 2 h, washed, kept in SS for 4 h and harvested. For all experiments, equal volumes of media conditioned by an equal number of cells (apoptotic or not) were compared.…”

“…15,16 In the present work, we aimed to establish the fibrogenic role of this paracrine loop in vivo and to identify the paracrine mediators mediating this fibrogenic activity. We demonstrate that caspase-3 activation in apoptotic ECs triggers CTGF secretion, which in turn induces myofibroblast differentiation and fibrogenesis through TGF-b1-independent pathways.…”

The bone morphogenetic protein 1/Tolloid-like metalloproteinases

“…The BMP1/TLD-like proteinase cleavage site for LG3 is identical to the site at which perlecan is cleaved in vivo to produce LG3 fragments found in the urine of patients with end-stage renal disease (Oda et al, 1996) and in the amniotic fluid of pregnant women with ruptured fetal membranes (Vuadens et al, 2003), suggesting that cleavage by these proteinases is physiologically relevant. Recently, the LG3 domain was also shown to promote apoptosis-resistance in fibroblasts via interactions with α2β1 integrins (Laplante et al, 2006). As accumulation of apoptosis resistant fibroblasts may be a hallmark of fibrosis (Arora and McCulloch, 1999;Desmouliere et al, 1995), proteolytic processing of perlecan by BMP1/TLD-like proteinases may have functional implications in both angiogenesis and fibrosis.…”