An early component of atherogenesis is abnormal vascular smooth muscle cell (VSMC) proliferation. The presence of Chlamydia pneumoniae in many atherosclerotic lesions raises the possibility that this organism plays a causal role in atherogenesis. In this study, C pneumoniae elementary bodies (EBs) rapidly activated p44/p42 mitogen-activated protein kinases (MAPKs) and stimulated proliferation of VSMCs in vitro. Exposure of VSMCs derived from human saphenous vein to C pneumoniae EBs (3x10(7) inclusion forming units/mL) enhanced bromodeoxyuridine (BrdU) incorporation 12+/-3-fold. UV- and heat-inactivated C pneumoniae EBs also stimulated VSMC proliferation, indicating a role of direct stimulation by chlamydial antigens. However, the mitogenic activity of C pneumoniae was heat-labile, thus excluding a role of lipopolysaccharide. Chlamydial hsp60 (25 microg/mL) replicated the effect of C pneumoniae, stimulating BrdU incorporation 7+/-3-fold. Exposure to C pneumoniae or chlamydial hsp60 rapidly activated p44/p42 MAPK, within 5 to 10 minutes of exposure. In addition, PD98059 and U0126, which are two distinct inhibitors of upstream MAPK kinase 1/2 (MEK1/2), abolished the mitogenic effect of C pneumoniae and chlamydial hsp60. Toll-like receptors (TLRs) act as sensors for microbial antigens and can signal via the p44/p42 MAPK pathway. Human VSMCs were shown to express TLR4 mRNA and protein, and a TLR4 antagonist abolished chlamydial hsp60-induced VSMC proliferation and attenuated C pneumoniae-induced MAPK activation and VSMC proliferation. Together these results indicate that C pneumoniae and chlamydial hsp60 are potent inducers of human VSMC proliferation and that these effects are mediated, at least in part, by rapid TLR4-mediated activation of p44/p42 MAPK.