PTH/PTHrP Receptor Signaling Restricts Arterial Fibrosis in Diabetic LDLR −/− Mice by Inhibiting Myocardin-Related Transcription Factor Relays

Abstract

Rationale: The PTH/PTHrP receptor (PTH1R) is expressed in vascular smooth muscle (VSM), and increased VSM PTH1R signaling mitigates diet-induced arteriosclerosis in LDLR-/- mice. Objective: To study the impact of VSM PTH1R deficiency, we generated mice SM22-Cre:PTH1R(fl/fl);LDLR-/- mice (PTH1R-VKO) and Cre-negative controls (CON). Methods and Results: Immunofluorescence and western blot confirmed PTH1R expression in arterial VSM that was reduced by Cre-mediated knockout. PTH1R-VKO cohorts exhibited increased aortic collagen accumulation in vivo, and VSM cultures from PTH1R-VKO mice elaborated more collagen (2.5-fold; p = 0.01) with elevated Col3a1 and Col1a1 expression. To better understand these profibrotic responses, we performed mass spectrometry on nuclear proteins extracted from CON and PTH1R-VKO VSM. PTH1R deficiency reduced Gata6 but upregulated the MADS-box transcriptional co-regulator, myocardin-related transcription factor A (Mkl1). Co-transfection assays (Col3a1 promoter - luciferase reporter) confirmed PTH1R -mediated inhibition and Mkl1-mediated activation of Col3a1 transcription. Regulation mapped to a conserved hybrid CT(A/T)6GG MADS-box cognate in the Col3a1 promoter. Mutations of C/G's in this motif markedly reduced Col3a1 transcriptional regulation by PTH1R and Mkl1. Upregulation of Col3a1 and Col1a1 in PTH1R-VKO VSM was inhibited by siRNA targeting Mkl1, and by treatment with the Mkl1 antagonist CCG1423 or the Rock2 inhibitor KD025. Chromatin precipitation demonstrated that VSM PTH1R deficiency increased Mkl1 binding to Col3a1 and Col1a1, but not TNF, promoters. Proteomic studies of plasma extracellular vesicles (EV) and VSM from PTH1R-VKO mice identified C1r and C1s, complement proteins involved in vascular collagen metabolism, as potential biomarkers. VSM C1r protein and C1r message were increased with PTH1R deficiency, mediated by Mkl1-dependent transcription and inhibited by CCG1423 or KD025. Conclusions: PTH1R signaling restricts collagen production in the VSM lineage in part via Mkl1 regulatory circuits that control collagen gene transcription. Strategies that maintain homeostatic VSM PTH1R signaling, as reflected in EV biomarkers of VSM PTH1R/Mkl1 action, may help mitigate arteriosclerosis and vascular fibrosis.