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

Abstract

The PTH1R (PTH [parathyroid hormone]/PTHrP [PTH-related protein] receptor) is expressed in vascular smooth muscle (VSM) and increased VSM PTH1R signaling mitigates diet-induced arteriosclerosis in LDLR^-/- mice. To study the impact of VSM PTH1R deficiency, we generated mice SM22-Cre:PTH1R(fl/fl);LDLR^-/- mice (PTH1R-VKO) and Cre-negative controls. 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; <i>P</i>=0.01) with elevated <i>Col3a1</i> and <i>Col1a1</i> expression. To better understand these profibrotic responses, we performed mass spectrometry on nuclear proteins extracted from Cre-negative controls and PTH1R-VKO VSM. PTH1R deficiency reduced Gata6 but upregulated the MADS (MCM1, Agamous, Deficiens, and Srf DNA-binding domain)-box transcriptional co-regulator, Mkl-1 (megakaryoblastic leukemia [translocation] 1). Co-transfection assays (<i>Col3a1</i> promoter-luciferase reporter) confirmed PTH1R-mediated inhibition and Mkl-1-mediated activation of <i>Col3a1</i> transcription. Regulation mapped to a conserved hybrid CT(A/T)₆GG MADS-box cognate in the <i>Col3a1</i> promoter. Mutations of C/G in this motif markedly reduced <i>Col3a1</i> transcriptional regulation by PTH1R and Mkl-1. Upregulation of <i>Col3a1</i> and <i>Col1a1</i> in PTH1R-VKO VSM was inhibited by small interfering RNA targeting <i>Mkl1</i> and by treatment with the Mkl-1 antagonist CCG1423 or the Rock (Rho-associated coiled-coil containing protein kinase)-2 inhibitor KD025. Chromatin precipitation demonstrated that VSM PTH1R deficiency increased Mkl-1 binding to <i>Col3a1 and Col1a1</i>, but not <i>TNF</i>, promoters. Proteomic studies of plasma extracellular vesicles and VSM from PTH1R-VKO mice identified C1r (complement component 1, r) and C1s (complement component 1, s), complement proteins involved in vascular collagen metabolism, as potential biomarkers. VSM C1r protein and <i>C1r</i> message were increased with PTH1R deficiency, mediated by Mkl-1-dependent transcription and inhibited by CCG1423 or KD025. PTH1R signaling restricts collagen production in the VSM lineage, in part, via Mkl-1 regulatory circuits that control collagen gene transcription. Strategies that maintain homeostatic VSM PTH1R signaling, as reflected in extracellular vesicle biomarkers of VSM PTH1R/Mkl-1 action, may help mitigate arteriosclerosis and vascular fibrosis.