Type I collagen synergistically enhances PDGF-induced smooth muscle cell proliferation through pp60src-dependent crosstalk between the α2β1 integrin and PDGFβ receptor

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Abstract

Smooth muscle cells (SMCs) are exposed to both platelet-derived growth factor (PDGF) and type I collagen (CNI) at the time of arterial injury. In these studies we explore the individual and combined effects of these agonists on human saphenous vein SMC proliferation. PDGF-BB produced a 5.5-fold increase in SMC DNA synthesis whereas CNI stimulated DNA synthesis to a much lesser extent (1.6-fold increase). Alternatively, we observed an 8.3-fold increase in DNA synthesis when SMCs were co-incubated with CNI and PDGF-BB. Furthermore, stimulation of SMCs with PDGF-BB produced a significant increase in ERK-2 activity whereas CNI alone had no effect. Co-incubation of SMCs with PDGF-BB and CNI resulted in ERK-2 activity that was markedly greater than that produced by PDGF-BB alone. In a similar fashion, PDGF-BB induced phosphorylation of the PDGF receptor β (PDGFRβ) and CNI did not, whereas concurrent agonist stimulation produced a synergistic increase in receptor activity. Blocking antibodies to the α2 and β1 subunits eliminated this synergistic interaction, implicating the α2β1 integrin as the mediator of this effect. Immunoprecipitation of the α2β1 integrin in unstimulated SMCs followed by immunoblotting for the PDGFRβ as well as Src family members, pp60src, Fyn, Lyn, and Yes demonstrated coassociation of α2β1 and the PDGFRβ as well as pp60src. Incubation of cells with CNI and/or PDGF-BB did not change the degree of association. Finally, inhibition of Src activity with SU6656 eliminated the synergistic effect of CNI on PDGF-induced PDGFRβ phosphorylation suggesting an important role for pp60src in the observed receptor crosstalk. Together, these data demonstrate that CNI synergistically enhances PDGF-induced SMC proliferation through Src-dependent crosstalk between the α2β1 integrin and the PDGFRβ.

Section snippets

Materials and methods

General materials. Human recombinant PDGF-BB was purchased from Upstate Biotechnologies (Lake Placid, NY). Collagen type I from calf skin, collagen type IV (CNIV), laminin, smooth muscle specific actin immunostaining kit, ethylene glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), adenosine 5′-triphosphate (ATP, disodium salt), sodium dodecyl sulfate (SDS), Nonidet-P40, and Triton X-100 were from Sigma

CNI synergistically enhances PDGF-BB-induced smooth muscle cell proliferation

We investigated the potential for laminin (1–50 μg/ml), CNI (1–50 μg/ml) or CNIV (1–50 μg/ml) to induce SMC proliferation. Of the ECM proteins studied, only CNI significantly enhanced tritiated thymidine incorporation. This effect was concentration dependent, first evident at 10 μg/ml (fold increase over control = 1.4 ± 0.1) and increased at 20 μg/ml (fold increase over control = 1.7 ± 0.4). The effect was most pronounced at 50 μg/ml (fold increase over control = 2.1 ± 0.5). We observed no increase in SMC

Discussion

We have characterized a novel synergistic interaction between CNI and PDGF-BB in promoting human vascular SMC proliferation. Our data demonstrate that the signaling pathways activated by PDGF-BB that lead to proliferation of vascular SMC are enhanced by CNI via an interaction that involves the α2β1 integrin. In sum, the α2β1 integrin in an interaction mediated by pp60src produces synergistic enhancement of PDGFRβ tyrosine phosphorylation as well as activation of ERK2 and consequently enhanced

Acknowledgments

This project was supported by the National Institute of Health Grants HL55465 (Kent), HL68673 (Kent), and T32 CA68971 (Hollenbeck) and the Association for Academic Surgery Research Fellowship Award (Hollenbeck).

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