Cytokine Signaling in Renal Fibrosis

描述（由申请人提供）：在该基金的资助下，我们在tgf - β信号方面取得了几项关键发现，将我们的研究重点从文献充分的晚期肾脏疾病肾小球和间质纤维化结果转向了足细胞中新的促凋亡和生长抑制tgf - β信号。在这个修订后的更新申请中，我们提出tgf - β在肾小球损伤的早期阶段有助于足细胞的消耗。我们的新假设表明，在适度的生理TGF-0活性的背景下，Smad2对Gadd45b的负调控使促凋亡的p38 MARK沉默。相反，当TGF-(3)活性超过临界阈值时，Smad2对Gadd45b的负调控被抑制，导致Gadd45b的下调。如果Gadd45b的细胞内浓度超过一个关键的信号阈值，它会选择性地激活促凋亡的p38 MARK途径。为了验证这一假设，我们提出了三个特定的目的：1)确定Smad2是否作为促凋亡的Gadd45b和p38/JNK MARK以及足细胞凋亡的负调节因子，并研究Smad2是否因此保护足细胞耗损和肾小球硬化。2)确定Gadd45b是否介导促凋亡p38/JNK MARK信号的激活和足细胞凋亡，并检测Gadd45b缺乏是否能预防Cd2ap-/-小鼠足细胞耗竭和FSGS。3)利用强力霉素诱导的条件永生化足细胞中Gadd45b蛋白丰度的滴定，确定足细胞中Gadd45b蛋白丰度阈值是否足以明确促凋亡p38/JNK通路的有效激活，从而介导细胞凋亡。

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.