Cytokine Signaling in Renal Fibrosis

描述（由申请人提供）：通过该基金的资助，我们在TGF-β信号传导方面取得了几项关键发现，这些发现将我们的研究重点从晚期肾脏疾病中记录良好的肾小球和间质纤维化结局转向足细胞中新型促凋亡和生长抑制性TGF-β信号传导。在这个修订的更新申请中，我们提出TGF-β在肾小球损伤的早期阶段有助于足细胞耗竭。我们的新假说指出，Gadd 45 b的负调控Smad 2沉默促凋亡p38 MARK在适度的生理TGF-0活性的背景下。相反，当TGF-β活性超过临界阈值时，Smad 2对Gadd 45 b的负调节被抑制，导致Gadd 45 b的去抑制。如果Gadd 45 b的细胞内浓度超过临界信号阈值，则其选择性激活促凋亡p38 MARK通路。为了验证这一假设，我们提出了三个具体的目标：1）确定Smad 2是否作为促凋亡Gadd 45 b和p38/JNK MARK和足细胞凋亡的负调节因子，并检查Smad 2是否因此保护足细胞耗竭和肾小球硬化。2)确定Gadd 45 b是否介导促凋亡p38/JNK MARK信号转导和足细胞凋亡的激活，并检查Gadd 45 b缺乏是否阻止Cd 2ap-/-小鼠足细胞耗竭和FSGS。3)使用强力霉素诱导的条件永生化足细胞中Gadd 45 b蛋白丰度的三次计数，确定足细胞中Gadd 45 b蛋白丰度的阈值是否足以指定促凋亡p38/JNK通路的有效激活以介导凋亡。 从长远来看，这项工作将阐明分子信号机制，确定特定的，上下文依赖性的细胞反应，如凋亡和生长停滞，这可能是由不同浓度的TGF-β在不同类型的细胞受到慢性进行性肾脏疾病损伤触发。

DESCRIPTION (provided by applicant): With funding from this grant, we have achieved several key discoveries in TGF-beta signaling that redirected our research focus from well-documented, glomerular and interstitial fibrosis outcomes in advanced renal disease to novel proapoptotic and growth inhibitory TGF-beta signaling in podocytes. In this revised renewal application, we propose that TGF-beta contributes to podocyte depletion during early stages of glomerular injury. Our new hypothesis states that negative regulation of Gadd45b by Smad2 silences proapoptotic p38 MARK in the context of moderate physiological TGF-0 activity. In contrast, when TGF-(3 activity exceeds a critical threshold, negative regulation of Gadd45b by Smad2 becomes suppressed resulting in derepression of Gadd45b. Gadd45b selectively activates the proapoptotic p38 MARK pathway if its intracellular concentrations exceed a critical signaling threshold. To test this hypothesis, we propose three SPECIFIC AIMS: 1) Determine whether Smad2 functions as negative regulator of proapoptotic Gadd45b and p38/JNK MARK and podocyte apoptosis, and examine whether Smad2 thereby protects against podocyte depletion and glomerulosclerosis. 2) Determine whether Gadd45b mediates activation of proapoptotic p38/JNK MARK signaling and podocyte apoptosis, and examine whether Gadd45b-deficiency prevents podocyte depletion and FSGS in Cd2ap-/- mice. 3) Determine whether an abundance threshold of Gadd45b protein in podocytes is sufficient to specify effective activation of proapoptotic p38/JNK pathways to mediate apoptosis, using doxycycline-inducible tritation of abundance of Gadd45b protein in conditionally-immortalized podocytes. In the long-term, this work will elucidate molecular signaling mechanisms determining specific, contextdependent cellular responses, such as apoptosis and growth arrest, that may be triggered by different concentrations of TGF-beta in different cell types subject to injury in chronic progressive renal disease.