From the Internal Medicine, Endocrine Division (A.B., D.Z., L.L., S.-L.C., M.M., B.R., P.S., D.A.T.), UT Southwestern Medical Center, Dallas, TX.
Biochemistry (M.G., A.L.), UT Southwestern Medical Center, Dallas, TX.
Circ Res. 2020 May 8;126(10):1363-1378. doi: 10.1161/CIRCRESAHA.119.316141. Epub 2020 Mar 11.
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; =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.
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.
甲状旁腺激素 1 型受体（PTH1R[甲状旁腺激素]/PTHrP[甲状旁腺激素相关蛋白]受体）在血管平滑肌（VSM）中表达，增加 VSM 的 PTH1R 信号可减轻 LDLR 小鼠的饮食诱导的动脉粥样硬化。
为了研究 VSM PTH1R 缺失的影响，我们生成了 SM22-Cre：PTH1R（fl/fl）；LDLR 小鼠（PTH1R-VKO）和 Cre 阴性对照小鼠。
免疫荧光和 Western blot 证实 PTH1R 在动脉 VSM 中有表达，Cre 介导的敲除使其减少。PTH1R-VKO 队列表现出体内主动脉胶原积累增加，并且 PTH1R-VKO 小鼠的 VSM 培养物表达更多的胶原（2.5 倍；=0.01）， 和 表达升高。为了更好地理解这些促纤维化反应，我们对 Cre 阴性对照和 PTH1R-VKO VSM 提取的核蛋白进行了质谱分析。PTH1R 缺失降低了 Gata6，但上调了 MADS（MCM1、Agamous、Deficiens 和 Srf DNA 结合域）盒转录共调节因子 Mkl-1（巨核细胞白血病[易位]1）。转染实验（ 启动子-荧光素酶报告基因）证实 PTH1R 介导的抑制和 Mkl-1 介导的 转录激活。调控映射到 启动子中保守的混合 CT（A/T）GG MADS 盒同源物。该模体中 C/G 的突变显著降低了 PTH1R 和 Mkl-1 对 转录的调控。PTH1R-VKO VSM 中的 和 上调被靶向 的小干扰 RNA 以及 Mkl-1 拮抗剂 CCG1423 或 Rock（Rho 相关卷曲螺旋蛋白激酶-2 抑制剂）KD025 处理所抑制。染色质沉淀表明，VSM PTH1R 缺失增加了 Mkl-1 与 启动子的结合，但不与 启动子结合。从 PTH1R-VKO 小鼠的血浆细胞外囊泡和 VSM 进行的蛋白质组学研究鉴定出 C1r（补体成分 1，r）和 C1s（补体成分 1，s），作为潜在的生物标志物，这些补体蛋白参与血管胶原代谢。VSM C1r 蛋白和 消息随着 PTH1R 缺失而增加，这是由 Mkl-1 依赖性转录介导的，并被 CCG1423 或 KD025 抑制。
PTH1R 信号通过 Mkl-1 调节回路限制 VSM 谱系中的胶原产生，该调节回路控制胶原基因转录。维持血管平滑肌 PTH1R 信号的稳态的策略，如血管平滑肌 PTH1R/Mkl-1 作用的细胞外囊泡生物标志物所反映的那样，可能有助于减轻动脉粥样硬化和血管纤维化。
