Activated alpha(2)-macroglobulin promotes mitogenesis in rat vascular smooth muscle cells by a mechanism that is independent of growth-factor-carrier activity

Vascular smooth muscle cell (VSMC) proliferation is important in atherosclerosis. We previously demonstrated that methylamine-activated alpha-macrogolobulin (alpha(2)M) and transforming growth factor beta 1 (TGF-beta 1) cause a synergistic proliferative response in quiescent rat aortic vSMCs [Stouffer, G. A., LaMarre, J., Gonias, S. L. & Owens, G. K. (1993) J. Biol. Chern. 268, 18340-18344]. The first goal of this study was to determine whether the synergy is due to the ability of alpha(2)M-methylamine (alpha(2)M-MeNH(2)) to bind TGF-beta 1 and target the growth factor to vSMCs that express the alpha(2)M receptor. Receptor-recognized alpha(2)M derivatives without TGF-beta 1-binding activity, including ternary alpha(2)M-trypsin, an 18-kDa proteolytic fragment of the alpha(2)M subunit, and the corresponding recombinant receptor-binding fragment (rRBF) increased VSMC [H-3]thymidine incorporation and cell number in a manner similar to alpha(2)M-MeNH(2). In combination with TGF-beta 1, each alpha(2)M derivative caused a synergistic vSMC proliferative response. vSMCs responded comparably when treated with alpha(2)M-MeNH(2), and TGF-beta 1 simultaneously or in sequence. Furthermore, alpha(2)M-MeNH(2)-TGF-beta 1 complexes increased [H-3]thymidine incorporation no more than alpha(2)M-MeNH(2) alone. These results indicate that TGF-beta 1 binding to alpha(2)M is not responsible for the synergistic mitogenic activity. Additional studies were undertaken to determine whether activated alpha(2)M independently induces a signal-transduction response in vSMCs. alpha(2)M-MeNH(2) and rRBF caused a rapid, transient increase in VSMC inositol 1,4,5-trisphosphate. This response was pertussis-toxin insensitive. Receptor-associated protein (RAP; 170 nmol/L) inhibited 91-95% of the specific binding of I-125-alpha(2)M-MeNH(2) and I-125-rRBF to vSMCs; however, RAP did not affect the inositol 2,4,5-trisphosphate response or the mitogenic response. These studies suggest that vSMCs express a receptor, other than low-density-lipoprotein-receptor-related protein, that transduces a signal in response to activated (alpha(2)M. This receptor may mediate the mitogenic activity of alpha(2)M in VSMC culture.