Perlecan proteolysis induces an alpha2beta1 integrin- and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of focal adhesion kinase activation.
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 alpha2beta1 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 alpha2beta1 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.
Laplante
Patrick
P
Centre de Recherche du Centre Hospitalier de l'Université de Montreal, University of Montreal, 1560 Sherbrooke East, Montreal, Quebec H2L 4M1, Canada.
Raymond
Marc-André
MA
Labelle
Andrée
A
Abe
Jun-Ichi
J
Iozzo
Renato V
RV
Hébert
Marie-Josée
MJ
D000818:Animals; D017209:Apoptosis; D005347:Fibroblasts / Q000378:metabolism; D005355:Fibrosis / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000737:chemistry*; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D019061:src-Family Kinases / Q000737:chemistry*
D019812:Heparan Sulfate Proteoglycans; D038982:Integrin alpha2beta1; C071927:perlecan; D019869:Phosphatidylinositol 3-Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases