beta-catenin C-terminal Domain/Sphingosine-1-Phosphate Receptor 1 Axis is a Potential Therapeutic Target in Vascular Remodeling

Vascular smooth muscle cells (SMCs), in part via activation of the Wnt/β-catenin signaling pathway, contribute importantly to vascular remodeling. While the C-terminal domain of β-catenin links the pathway to gene expression, how this domain and related molecular mechanisms are involved in vascular remodeling is unknown. Inhibitors targeting specifically the C-terminal domain of β-catenin have been developed, but their use in obstructive vascular disease is not yet justified because of this gap of knowledge. Our group has previously shown that β-catenin C-terminal domain in SMCs is essential for arterial wall assembly during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Therefore, the objective of this study was to define the importance of β-catenin C-terminal domain in SMCs during vascular remodeling and the underlying molecular mechanisms. To test the hypothesis that β-catenin C-terminal domain in SMCs is required for vascular remodeling, mice bearing a mutant β-catenin allele (ΔC) that prevents the transcription of the C-terminal domain were crossed with the SMC-specific SMA-CreERT2 line to generate mice bearing one β-catenin flox allele and one knockin mutant (Ctnnb1 ) or wt (Ctnnb1 ) allele. In this system, tamoxifen administration removes the flox allele, resulting in mice bearing the mutant allele (SMβC mice) or the wt allele (SMβC mice) as the only sources of β-catenin in SMCs. To induce vascular remodeling, we performed carotid artery ligation one week after tamoxifen injection in male and female adult mice. Carotid arteries from both male and female SMβC mice showed reduced neointima formation after injury induced by ligation compared to SMβC mice. Arteries from SMβC mice also showed reduced SMC proliferation and increased SMC apoptosis and differentiation markers. We also isolated mouse aortic SMCs (MASMCs) from Ctnnb1 mice, and transduced these cells with GFP- or Cre-expressing adenovirus to obtain control (βC ) or cells expressing β-catenin (βC ) and found that βC MASMCs show reduced growth compared to βC . To gain further insight on the mechanisms underlying the effects of β-catenin C-terminus, we performed whole-transcriptome analysis by RNA-seq of βC and βC MASMCs and found a downregulation of the sphingosine-1-phosphate receptor 1 (S1pr1) gene in βC MASMCs. This receptor is known to be important in vascular remodeling, but its regulation is not fully elucidated. Interestingly, we found decreased S1PR1 expression in SMCs of injured arteries from SMβC mice. Consistent with this observation, βC MASMCs showed reduced S1PR1 mRNA and protein levels and decreased S1pr1 promoter activity, which were all rescued by the transfection of a constitutively active form of β-catenin. By using a S1PR1 conditional gain-of-function mouse line, we found that overexpression of S1PR1 in SMCs rescues the injury-induced neointima formation in SMβC mice. Consistently, overexpression of S1PR1 in SMCs restored βC MASMC growth towards βC levels. Taken together, our results define an essential SMC β-catenin C-terminal domain-S1PR1 axis that drives neointima formation after arterial injury, and identify multiple points for potential therapeutic intervention to control vascular remodeling in disease.