PTH/PTHrP信号通路和TGFβII型受体的新功能在糖尿病肾脏疾病中的作用

Recent studies showed a previously unrecognized function for transforming growth factor (TGF)-β type II receptor (TβRII). TβRII directly phosphorylates the parathyroid hormone receptor (PTH1R) cytoplasmic domain, forms an endocytic complex with PTH1R in response to PTH (1-34), and induces endocytosis of the PTH1R-TβRII complex. Parathyroid hormone-related protein (PTHrP) shares significant N-terminal amino acid sequence homology with PTH, particularly within the first 13 residues. In addition to PTH, PTH1R also recognizes PTHrP. PTHrP acts as a paracrine or autocrine factor, and play a physiological role in development, differentiation, cell proliferation and survival. Recently, PTHrP was identified as a factor which can control the differentiation and survival of β cells. PTHrP is produced in the normal pancreatic islet by β cells. PTHrP increased the content and mRNA level of insulin in a mouse β cell line, MIN6, and primary-cultured mouse islets, which suggested the future therapeutic potential of PTHrP for the treatment of diabetes. PTHrP and its receptor, PTH1R, are widely expressed in the kidney, where PTHrP exerts a modulatory action on renal function. PTHrP is known to be upregulated in several experimental nephropathies such as diabetic kidney disease (DKD), but its role remains unclear. It has been reported that PTHrP acts as an important mediator of high glucose-induced human mesangial cell hypertrophy by modulating TGFβ1. On the other hand, PTH treatment of LLCP-K1 porcine renal tubular cells (0-40 min) increased ERK1/2 activation, while prolonging PTH treatment (90-120 min) caused an inhibition of ERK1/2. Furthermore, elimination of PTH1R phosphorylation caused sustained PTH activation of ERK1/2. It suggests that PTH downregulation of the ERK1/2 pathway is largely dependent on PTH1R phosphorylation. The present study is to observe whether short PTH or PTHrP treatment (0-2 h) can induce endocytosis of the PTH1R-TβRII complex in rat mesangial cells, attenuate the sensitivity of mesangial cells to TGFβ1 and thereby inhibit the upregulation of extracellular matrix protein (ECM) induced by TGFβ1 and high glucose. Next step is to observe whether long PTH or PTHrP treatment (24-72 h) can induce cell proliferation or hypertrophy in rat mesangial cells. Whether PTH or PTHrP involves in high glucose-induced TGFβ1 and ECM upregulation will also be investigated. Streptozocin (STZ)-induced diabetic rats are used to elucidate the different roles of short or long treatment of PTH, PTHrP or non-peptide PTH1R antagonist (JB4250) in mesangial cell hypertrophy, TGFβ1 and ECM upregulation. Recombinant adenoviruses carrying short hairpin RNA targeting PTH and PTHrP will also be used to elucidate the complicated mechanisms of PTH and PTHrP in glomerular sclerosis. Taken together, our study may illuminate the interactions between PTH/PTHrP and TGFβ1 pathway, and provide the new strategy for the therapy of diabetic kidney disease.

最近发现了TGFβII型受体的新功能，在甲状旁腺激素多肽片段PTH(1-34)诱导下可结合并磷酸化PTH I型受体，共同内吞至胞浆。甲状旁腺激素相关蛋白(PTHrP)的氨基末端和PTH的同源性很高，而且通过共同的受体PTH I型受体发挥作用，提示PTHrP也可能与TGFβ通路存在相互作用。PTHrP在糖尿病的治疗方面颇有前景，但在糖尿病肾脏疾病中的作用机制尚不清楚。另一方面，PTH(1-34)短期处理猪肾小管细胞可激活ERK1/2，长期孵育则抑制ERK1/2通路，说明PTH(1-34)的短期和长期效应存在差异。因此，本课题提出PTH/PTHrP早期拮抗TGFβ信号通路，晚期则激活TGFβ信号通路的假说，并从糖尿病肾脏疾病细胞模型和动物模型两方面观察和验证PTH/PTHrP的双重作用，拓宽对肾小球硬化信号途径的认识，为寻找防治糖尿病肾脏疾病的新策略奠定基础。

PTHrP在糖尿病的治疗方面颇有前景，但在糖尿病肾病中的作用机制尚不清楚。本课题提出PTHrP早期拮抗TGFβ信号通路，晚期则激活TGFβ信号通路的假说，并从糖尿病肾病细胞模型和动物模型两方面进行观察和验证。基于PTHrP和PTH有较高的同源性，且可通过共同的受体PTH I型受体发挥作用，本课题主要采用PTHrP和PTH的N末端短肽进行研究。实验结果发现PTH/PTHrP在细胞水平具有双重作用，PTH/PTHrP处理系膜细胞3小时内可诱导PTH1R-TβRII复合体内吞，减少细胞膜表面的TβRII，从而抑制TGFβ诱导的Smad通路激活及细胞外基质蛋白上调。但是内吞后的受体并未降解，而是重新回到细胞膜表面，因此PTH/PTHrP长期处理后不能下调细胞膜表面的TβRII，也不能抑制TGFβ诱导的Smad通路激活及细胞外基质蛋白上调。另一方面，实验结果发现PTHrP长期处理系膜细胞时可诱导TGFβ及其受体的表达上调，说明PTH/PTHrP长期处理可激活TGFβ信号通路。此外，PTH/PTHrP诱导的纤连蛋白表达上调早于TGFβ及其受体的表达上调，说明PTH/PTHrP有不依赖于TGFβ信号通路的作用机制。进一步研究发现，PTH/PTHrP可诱导EGFR反式激活，通过PI3K/Akt 通路和ERK1/2通路诱导细胞外基质蛋白的上调。PTH以内分泌的方式发挥作用，而PTHrP主要以旁分泌或自分泌方式发挥作用。考虑到PTH的作用广泛，我们在STZ诱导的糖尿病大鼠模型中主要验证了PTHrP是否也发挥双重作用，实验结果发现PTHrP处理2周可抑制肾小球细胞外基质蛋白的上调，但是PTHrP长期处理（3月）促进了肾小球细胞外基质蛋白的上调，可能参与糖尿病肾病肾小球硬化。进一步构建PTHrP的siRNA腺病毒表达质粒，利用293细胞包装、生成腺病毒；用腺病毒感染大鼠肾组织，再建立糖尿病动物模型，发现PTHrP基因沉默可以延缓糖尿病肾病肾小球硬化。本课题拓宽了对肾小球硬化信号途径的认识，有助于寻找防治糖尿病肾病的新策略。