Wild-type LRP6 inhibits, whereas atherosclerosis-linked LRP6R611C increases PDGF-dependent vascular smooth muscle cell proliferation

Abstract

Vascular smooth muscle cell (VSMC) proliferation is an important event in atherosclerosis and other vasculopathies. PDGF signaling is a key mediator of SMC proliferation, but the mechanisms that control its activity remain unclear. We previously identified a mutation in LDL receptor-related protein 6 (LRP6), LRP6(R611C), that causes early atherosclerosis. Examination of human atherosclerotic coronary arteries showed markedly increased expression of LRP6 and colocalization with PDGF receptor β (PDGFR-β). Further investigation showed that wild-type LRP6 inhibits but LRP6(R611C) promotes VSMC proliferation in response to PDGF. We found that wild-type LRP6 forms a complex with PDGFR-β and enhances its lysosomal degradation, functions that are severely impaired in LRP6(R611C). Further, we observed that wild-type and mutant LRP6 regulate cell-cycle activity by triggering differential effects on PDGF-dependent pathways. These findings implicate LRP6 as a critical modulator of PDGF-dependent regulation of cell cycle in smooth muscle and indicate that loss of this function contributes to development of early atherosclerosis in humans.

Fig. 1.

PDGFR-β and LRP6 expression in normal and atherosclerotic coronary arteries. Immunofluorescent staining in a cross-section of a normal coronary artery shows that LRP6 and PDGFR-β are expressed at very low levels in the intima (I) and muscularis (M) layers, respectively (Upper). In the cross-section of the atherosclerotic coronary artery, LRP6 and PDGFR-β are highly expressed and colocalized in the subintima (S) and the muscularis (M) layers (Lower) (n = 5). A, adventicia.

Fig. 2.

Effect of LRP6_WT and LRP6_R611C on cyclin D1 expression and cell proliferation. (A) After 12-h stimulation with Wnt3a, mRNA expression levels of cyclin D1 were about threefold lower in cells expressing LRP6_R611C than in uninfected cells (P < 0.0001) or cells expressing LRP6_WT (P < 0.001). (B) Wnt3a stimulated the proliferation rate in aortic smooth muscle cells overexpressing LRP6_WT and LRP6_R611C. Percent BrdU incorporation in cells expressing LRP6_R611C after stimulation with Wnt3a remained unchanged and was considerably lower than in cells infected with empty vector (Control) (P < 0.001) or cells expressing LRP6_WT (P < 0.001).

Fig. 3.

Increased cell-cycle activity in human aortic smooth muscle cells expressing LRP6_R611C or LRP6 shRNA. (A) Percent change in BrdU incorporation is considerably higher in cells expressing LRP6_R611C in response to PDGF and is considerably lower in cells expressing LRP6_WT cells (P < 0.05) than in cells infected with empty vector (P < 0.001). (B) Fold change in cyclin D1 expression levels. After stimulation with PDGF, cyclin D1 expression levels were significantly increased in cells expressing LRP6_R611C (P < 0.01) but remained unchanged in cells expressing LRP6_WT. (C) Increased proliferation rate in cells infected with LRP6-specific shRNA (sh-LRP6). RNA interference significantly increased the cell proliferation rate both without (P < 0.05) and with PDGF stimulation compared with scrambled shRNA (sh-CTL) (P < 0.001).

Fig. 4.

Expression levels of PDGFR-β in human aortic smooth muscle cells expressing LRP6_WT and LRP6_R611C. (A) LRP6_WT significantly reduces PDGFR-β expression levels (P = 0.001). This effect is diminished significantly in the presence of LRP6_R611C. (B) Dose effect of LRP6_WT on endogenous PDGFR-β expression. There is an inverse relationship between the multiplicity of infection of viral particles expressing LRP6_WT-HA2 and PDGFR-β expression. (C) LRP6 knock down by RNA interference (shLRP6). Knocking down LRP6 by RNA interference raised PDGFR-β levels (P = 0.01).

Fig. 5.

LRP6_WT and LRP6_R611C form complexes with PDGFR-β. (A) Proteins from cell lysates were immunoprecipitated with either anti-HA or anti–PDGFR-β antibodies followed by Western blotting with either anti–PDGFR-β or anti-HA antibodies, respectively. LRP6_WT and LRP6_R611C but not IgG or NPC2 (used as control) coimmunoprecipitated with PDGFR-β. (B) LRP6_WT ubiquitinates PDGFR-β. Ubiquitination of PDGFR-β following MG132 treatment in cells overexpressing LRP6_R611C or LRP6_WT and in cells infected with empty vector was compared. LRP6_WT but not LRP6_R611C ubiquitinates PDGFR-β. (C) Lysosomal degradation of PDGFR-β. The lysosomal inhibitor NH₄Cl rescues reduced expression of PDGFR-β (P = 0.001). (D) LRP6_WT reduces and LRP6_R611C increases STAT1 tyrosine phosphorylation. After PDGF stimulation, tyrosine phosphorylation of STAT1 is significantly higher in cells expressing LRP6_R611C (P < 0.001) and is significantly lower in cells expressing LRP6_WT (P < 0.001) than in cells transfected with empty plasmid.