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

RATIONALE

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.

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.

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; =0.01) with elevated and 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 ( promoter-luciferase reporter) confirmed PTH1R-mediated inhibition and Mkl-1-mediated activation of transcription. Regulation mapped to a conserved hybrid CT(A/T)GG MADS-box cognate in the promoter. Mutations of C/G in this motif markedly reduced transcriptional regulation by PTH1R and Mkl-1. Upregulation of and in PTH1R-VKO VSM was inhibited by small interfering RNA targeting 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 , but not , 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 message were increased with PTH1R deficiency, mediated by Mkl-1-dependent transcription and inhibited by CCG1423 or KD025.

CONCLUSIONS

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.