Pathogenic role of PDE4 in hepatic stellate cell activation and TGF signaling

不同原因的肝损伤导致创面愈合过程，涉及肝星状细胞的激活 (HSCs)。然而，持续的肝细胞损伤和炎症导致不受控制的激活和 肝星状细胞的增殖和肝纤维化的发展导致肝硬变甚至肝细胞癌。 尽管取得了进展，但我们对这一进程所涉及的机制的理解仍然存在差距。 HSC由静止表型向激活表型转化。最近，我们发现 磷酸二酯酶4(PDE4)亚家族在胆汁淤积症发病中的作用 肝脏损伤和纤维化。值得注意的是，PDE4不存在于静止的HSC中，在 体外激活。此外，PDE4特异性抑制剂罗利普兰有效地减弱了αSMA，胶原蛋白 HSCs的表达及伴随的形态变化。PDE4是阵营中最大的子家族- 降解PDE，严格调节细胞内cAMP水平。CAMP，通过其效应分子 蛋白激酶A(PKA)和环磷酸腺苷(CAMP)直接激活的交换蛋白(EPAC)，已被证明下调- 调控细胞因子诱导的非肝细胞纤维化基因。因此，我们假设PDE4的诱导 表达和活性通过降低cAMP-PKA/EPAC活性在HSC激活中起因果作用 促进纤维化信号转导。我们推测，在HSC激活过程中，启动子相关的表观遗传学 改变和翻译后修饰在PDE4表达和表达的调节中发挥重要作用 活动。我们还推测，抑制PDE4可以恢复PKA/EPAC的活性，并减弱转化生长因子β-SMAD 通过以下途径发出信号：(I)相关MAPK的失活；以及(Ii)去抑制PPARγ，导致减少 α、SMA和COL1A1的表达。重要的是，抑制PDE4活性可能是一种重要的治疗方法 治疗肝纤维化的方法。这项提议的具体目的是：1)确定PDE4在 肝干细胞中纤维化信号的调控；2)确定启动子相关的表观遗传修饰 有助于在HSC激活过程中诱导PDE4亚型；以及3)确定翻译后 在HSC激活过程中与PDE4亚型功能相关的修饰(PTM)。重要的是，这一结果 科布雷资助的项目将为体内翻译研究提供原则证明和机制基础 (R01)研究PDE4针对预防和治疗肝纤维化的策略。

Liver injury of different etiologies leads to a wound healing process involving activation of hepatic stellate cells (HSCs). However, an ongoing hepatocyte injury and inflammation results in an uncontrolled activation and proliferation of HSCs and development of hepatic fibrosis leading to cirrhosis and even hepatocellular cancer. Despite the advances made, gaps remain in our understanding of the mechanisms involved in the process of HSC transformation from quiescent to activated phenotype. Recently, we discovered that the phosphodiesterase 4 (PDE4) subfamily of enzymes play a pathogenic role in the development of cholestatic liver injury and fibrosis. Notably, PDE4s are not present in quiescent HSCs and are rapidly induced upon activation in vitro. Further, the PDE4 specific inhibitor, rolipram, effectively attenuates αSMA, collagen expression and accompanying morphological changes in HSCs. PDE4 is the largest sub-family among cAMP- hydrolyzing PDEs, which tightly regulate the levels of cellular cAMP. cAMP, through its effector molecules protein kinase A (PKA) and Exchange Protein directly Activated by cAMP (EPAC), has been shown to down- regulate cytokine induced fibrogenic genes in non-hepatic cells. Hence, we hypothesize that induction of PDE4 expression and activity plays a causal role in HSC activation by decreasing cAMP-PKA/EPAC activities and promoting fibrogenic signaling. We postulate that during HSC activation, promoter associated epigenetic changes and post-translational modifications play a significant role in the regulation of PDE4 expression and activity. We also postulate that PDE4 inhibition will restore PKA/EPAC activities and attenuate TGFβ-Smad signaling through: (i) inactivation of relevant MAPKs; and (ii) de-repressing PPARγ leading to decreased expression of αSMA and Col1A1. Importantly, inhibition of PDE4 activity may be a significant therapeutic approach for liver fibrosis. The specific aims of this proposal are to: 1) Determine the role of PDE4 in the regulation of fibrogenic signaling in HSCs; 2) Determine promoter-associated epigenetic modifications contributing to the induction of PDE4 isoforms during HSC activation; and 3) Determine the post-translational modifications (PTMs) relevant for PDE4 isoform function during HSC activation. Importantly, the results of this COBRE-funded project will provide proof-of-principle and mechanistic rationale for in vivo translational studies (R01) to examine PDE4 targeted strategies for prevention and treatment of hepatic fibrosis.