ABSTRACT

Macroautophagy/autophagy dysregulation has been noted in diabetic nephropathy; however, the regulatory mechanisms controlling this process remain unclear. In this study, we showed that SMAD3 (SMAD family member 3), the key effector of TGFB (transforming growth factor beta)-SMAD signaling, induces lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis. The pharmacological inhibition or genetic deletion of SMAD3 restored lysosome biogenesis activity by alleviating the suppression of TFEB, thereby protecting lysosomes from depletion and improving autophagic flux in renal tubular epithelial cells in diabetic nephropathy. Mechanistically, we found that SMAD3 directly binds to the 3ʹ-UTR of TFEB and inhibits its transcription. Silencing TFEB suppressed lysosome biogenesis and resulted in a loss of the protective effects of SMAD3 inactivation on lysosome depletion under diabetic conditions. In conclusion, SMAD3 promotes lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis; this may be an important mechanism underlying autophagy dysregulation in the progression of diabetic nephropathy.

Abbreviations: AGEs: advanced glycation end products; ATP6V1H: ATPase H+ transporting V1 subunit H; CTSB: cathepsin B; ChIP: chromatin immunoprecipitation; Co-BSA: control bovine serum albumin; DN: diabetic nephropathy; ELISA: enzyme-linked immunosorbent assay; FN1: fibronectin 1; HAVCR1/TIM1/KIM-1: hepatitis A virus cellular receptor 1; LAMP1: lysosomal associated membrane protein 1; LMP: lysosome membrane permeabilization; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NC: negative control; SIS3: specific inhibitor of SMAD3; SMAD3: SMAD family member 3; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TECs: tubular epithelial cells; TFEB: transcription factor EB; TGFB1: transforming growth factor beta 1; TGFBR1: transforming growth factor beta receptor 1; UTR: untranslated region; VPS11: VPS11 core subunit of CORVET and HOPS complexes.

摘要

在糖尿病肾病中已经注意到巨自噬/自噬失调；然而，控制这一过程的监管机制仍不清楚。在这项研究中，我们发现 SMAD3（SMAD 家族成员 3）是 TGFB（转化生长因子 β）-SMAD 信号传导的关键效应子，通过抑制 TFEB 依赖性溶酶体生物发生来诱导溶酶体耗竭。SMAD3的药理抑制或基因缺失通过减轻对TFEB的抑制来恢复溶酶体的生物合成活性，从而保护溶酶体免于消耗并改善糖尿病肾病肾小管上皮细胞的自噬通量。从机制上讲，我们发现SMAD3直接与TFEB的 3'-UTR 结合并抑制其转录。沉默TFEB抑制溶酶体的生物合成，并导致 SMAD3 失活对糖尿病条件下溶酶体耗竭的保护作用丧失。总之，SMAD3 通过抑制 TFEB 依赖的溶酶体生物发生促进溶酶体消耗；这可能是糖尿病肾病进展中自噬失调的重要机制。

缩写: AGEs：晚期糖基化终产物；ATP6V1H：ATPase H+ 转运 V1 亚基 H；CTSB：组织蛋白酶 B；ChIP：染色质免疫沉淀；Co-BSA：对照牛血清白蛋白；DN：糖尿病肾病；ELISA：酶联免疫吸附试验；FN1：纤连蛋白1；HAVCR1/TIM1/KIM-1：甲型肝炎病毒细胞受体1；LAMP1：溶酶体相关膜蛋白 1；LMP：溶酶体膜透化；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；NC：阴性对照；SIS3：SMAD3的特异性抑制剂；SMAD3：SMAD家族成员3；siRNA：小干扰RNA；SQSTM1/p62：隔离体 1；TECs：肾小管上皮细胞；TFEB：转录因子EB；TGFB1：转化生长因子β1；TGFBR1：转化生长因子β受体1；UTR：未翻译区；VPS11：