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; P=0.01) with elevated Col3a1 and Col1a1 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 (Col3a1 promoter-luciferase reporter) confirmed PTH1R-mediated inhibition and Mkl-1-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 in this motif markedly reduced Col3a1 transcriptional regulation by PTH1R and Mkl-1. Upregulation of Col3a1 and Col1a1 in PTH1R-VKO VSM was inhibited by small interfering RNA targeting Mkl1 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 Col3a1 and Col1a1, but not TNF, 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 C1r 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.

中文翻译：

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PTH / PTHrP受体信号传导通过抑制心肌相关转录因子中继来限制糖尿病LDLR-/-小鼠的动脉纤维化。

理据PTH1R（PTH [甲状旁腺激素] / PTHrP [PTH相关蛋白]受体）在血管平滑肌（VSM）中表达，VSM PTH1R信号的增加减轻了LDLR-/-小鼠饮食引起的动脉硬化。目的为了研究VSM PTH1R缺乏的影响，我们产生了小鼠SM22-Cre：PTH1R（fl / fl）; LDLR-/-小鼠（PTH1R-VKO）和Cre阴性对照。方法和结果免疫荧光和蛋白质印迹证实了PTH1R在动脉VSM中的表达被Cre介导的敲除降低了。PTH1R-VKO队列显示体内增加的主动脉胶原蛋白积累，并且来自PTH1R-VKO小鼠的VSM培养物制备了更多胶原蛋白（2.5倍； P = 0.01），并具有Col3a1和Col1a1表达升高。为了更好地了解这些纤维变性反应，我们对从Cre阴性对照和PTH1R-VKO VSM中提取的核蛋白进行了质谱分析。PTH1R缺乏症降低了Gata6的表达，但上调了MADS（MCM1，Agamous，Deficiens和Srf DNA结合结构域）-框转录共调节子Mkl-1（巨核母细胞白血病[易位] 1）。共转染测定（Col3a1启动子-荧光素酶报告基因）证实了PTH1R介导的抑制和Mk1-1介导的Col3a1转录激活。调控映射到Col3a1启动子中的保守杂种CT（A / T）6GG MADS-box同源。C / G在此主题中的突变显着降低了PTH1R和Mkl-1的Col3a1转录调控。PTH1R-VKO VSM中Col3a1和Col1a1的上调受靶向Mkl1的小分子干扰RNA以及Mkl-1拮抗剂CCG1423或Rock（Rho相关的含有蛋白激酶的卷曲螺旋）-2抑制剂KD025的抑制。染色质沉淀表明，VSM PTH1R缺乏症增加了Mkl-1与Col3a1和Col1a1的结合，但不增强TNF的启动子。来自PTH1R-VKO小鼠的血浆细胞外囊泡和VSM的蛋白质组学研究确定了C1r（补体成分1，r）和C1s（补体成分1，s）是参与血管胶原代谢的补体蛋白，是潜在的生物标志物。VSM C1r蛋白和C1r信息随PTH1R缺乏而增加，由Mk1-1依赖性转录介导并受CCG1423或KD025抑制。结论PTH1R信号传导限制了VSM谱系中胶原蛋白的产生，部分原因是，通过控制胶原基因转录的Mkl-1调节电路。反映VSM PTH1R / Mkl-1作用的细胞外囊泡生物标志物所反映的维持稳态VSM PTH1R信号传导的策略，可能有助于减轻动脉硬化和血管纤维化。