Autophagy regulation and protein kinase activity of PIK3C3 controls sertoli cell polarity through its negative regulation on SCIN (scinderin).

IF 14.6 1区 生物学 Q1 CELL BIOLOGY
Autophagy Pub Date : 2023-11-01 Epub Date: 2023-07-14 DOI:10.1080/15548627.2023.2235195
Kehan Wang, Feifei Kong, Yuexin Qiu, Tao Chen, Jiayi Fu, Xin Jin, Youqiang Su, Yayun Gu, Zhibin Hu, Jing Li
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引用次数: 0

Abstract

Sertoli cells are highly polarized testicular cells that provide a nurturing environment for germ cell development and maturation during spermatogenesis. The class III phosphatidylinositol 3-kinase (PtdIns3K) plays core roles in macroautophagy in various cell types; however, its role in Sertoli cells remains unclear. Here, we generated a mouse line in which the gene encoding the catalytic subunit, Pik3c3, was specifically deleted in Sertoli cells (cKO) and found that after one round of normal spermatogenesis, the cKO mice quickly became infertile and showed disruption of Sertoli cell polarity and impaired spermiogenesis. Subsequent proteomics and phosphoproteomics analyses enriched the F-actin cytoskeleton network involved in the disorganized Sertoli-cell structure in cKO testis which we identified a significant increase of the F-actin negative regulator SCIN (scinderin) and the reduced phosphorylation of HDAC6, an α-tubulin deacetylase. Our results further demonstrated that the accumulation of SCIN in cKO Sertoli cells caused the disorder and disassembly of the F-actin cytoskeleton, which was related to the failure of SCIN degradation through the autophagy-lysosome pathway. Additionally, we found that the phosphorylation of HDAC6 at site S59 by PIK3C3 was essential for its degradation through the ubiquitin-proteasome pathway. As a result, the HDAC6 that accumulated in cKO Sertoli cells deacetylated SCIN at site K189 and led to a disorganized F-actin cytoskeleton. Taken together, our findings elucidate a new mechanism for PIK3C3 in maintaining the polarity of Sertoli cells, in which both its autophagy regulation or protein kinase activities are required for the stabilization of the actin cytoskeleton.Abbreviations: ACTB: actin, beta; AR: androgen receptor; ATG14: autophagy related 14; BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; BTB: blood-testis barrier; CASP3: caspase 3; CDC42: cell division cycle 42; CDH2: cadherin 2; CHX: cycloheximide; CTNNA1: catenin (cadherin associated protein), alpha 1; CYP11A1: cytochrome P450, family 11, subfamily A, polypeptide 1; EBSS: Earle's balanced salt solution; ES: ectoplasmic specialization; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCNA: germ cell nuclear acidic protein; GJA1: gap junction protein, alpha 1; H2AX: H2A.X variant histone; HDAC6: histone deacetylase 6; KIT: KIT proto-oncogene, receptor tyrosine kinase; LAMP1: lysosomal associated membrane protein 1; MAP3K5: mitogen-activated protein kinase kinase kinase 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OCLN: occludin; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PNA: arachis hypogaea lectin; RAC1: Rac family small GTPase 1; SCIN: scinderin; SQSTM1/p62: sequestosome 1; SSC: spermatogonia stem cell; STK11: serine/threonine kinase 11; TJP1: tight junction protein 1; TubA: tubastatin A; TUBB3: tubulin beta 3 class III; TUNEL: TdT-mediated dUTP nick-end labeling; UB: ubiquitin; UVRAG: UV radiation resistance associated gene; VIM: vimentin; WT1: WT1 transcription factor; ZBTB16: zinc finger and BTB domain containing 16.

PIK3C3的自噬调节和蛋白激酶活性通过其对SCIN(闪烁蛋白)的负调控来控制支持细胞的极性。
支持细胞是高度极化的睾丸细胞,在精子发生过程中为生殖细胞的发育和成熟提供了培养环境。III类磷脂酰肌醇3-激酶(PtdIns3K)在各种细胞类型的大自噬中发挥核心作用;然而,它在支持细胞中的作用尚不清楚。在这里,我们产生了一个小鼠系,其中编码催化亚基Pik3c3的基因在支持细胞(cKO)中被特异性缺失,并发现在一轮正常精子发生后,cKO小鼠很快变得不育,并表现出支持细胞极性的破坏和精子生成受损。随后的蛋白质组学和磷酸蛋白质组学分析丰富了参与cKO睾丸支持细胞结构紊乱的F-肌动蛋白细胞骨架网络,我们发现F-肌动蛋白负调控因子SCIN(闪烁蛋白)显著增加,HDAC6(一种α-微管蛋白脱乙酰酶)磷酸化减少。我们的结果进一步证明,SCIN在cKO支持细胞中的积累导致F-肌动蛋白细胞骨架的紊乱和分解,这与SCIN未能通过自噬-溶酶体途径降解有关。此外,我们发现PIK3C3在S59位点磷酸化HDAC6对其通过泛素-蛋白酶体途径降解至关重要。结果,在cKO支持细胞中积累的HDAC6在K189位点脱乙酰SCIN,并导致F-肌动蛋白细胞骨架紊乱。总之,我们的发现阐明了PIK3C3维持支持细胞极性的新机制,其中其自噬调节或蛋白激酶活性是肌动蛋白细胞骨架稳定所必需的。缩写:ACTB:肌动蛋白,β;AR:雄激素受体;ATG14:自噬相关14;BafA1:巴非霉素A1;BECN1:beclin 1,自噬相关;BTB:血睾丸屏障;CASP3:胱天蛋白酶3;CDC42:细胞分裂周期42;CDH2:钙粘蛋白2;CHX:环己酰亚胺;CTNNA1:连环蛋白(钙粘蛋白相关蛋白),α1;CYP11A1:细胞色素P450,家族11,亚家族A,多肽1;EBSS:厄尔平衡盐溶液;ES:胞质特化;FITC:异硫氰酸荧光素;GAPDH:甘油醛-3-磷酸脱氢酶;GCNA:生殖细胞核酸性蛋白;GJA1:间隙连接蛋白,α1;H2AX:H2A。X变体组蛋白;HDAC6:组蛋白脱乙酰酶6;KIT:KIT原癌基因,受体酪氨酸激酶;LAMP1:溶酶体相关膜蛋白1;MAP3K5:丝裂原活化蛋白激酶激酶5;MAP1LC3B:微管相关蛋白1轻链3β;OCLN:occludin;PIK3C3:磷脂酰肌醇3-激酶催化亚基3型;PIK3R4:磷酸肌醇-3-激酶调节亚单位4;PNA:花生凝集素;RAC1:Rac家族小GTPase 1;SCIN:闪烁蛋白;SQSTM1/p62:螯合体1;SSC:精原干细胞;STK11:丝氨酸/苏氨酸激酶11;TJP1:紧密连接蛋白1;TubA:tubastatin A;TUB3:微管蛋白β3 III类;TUNEL:TdT介导的dUTP缺口末端标记;UB:泛素;UVRAG:紫外线辐射抗性相关基因;VIM:波形蛋白;WT1:WT1转录因子;ZBTB16:锌指和含有16。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Autophagy
Autophagy 生物-细胞生物学
CiteScore
21.30
自引率
2.30%
发文量
277
审稿时长
1 months
期刊介绍: Autophagy is a peer-reviewed journal that publishes research on autophagic processes, including the lysosome/vacuole dependent degradation of intracellular material. It aims to be the premier journal in the field and covers various connections between autophagy and human health and disease, such as cancer, neurodegeneration, aging, diabetes, myopathies, and heart disease. Autophagy is interested in all experimental systems, from yeast to human. Suggestions for specialized topics are welcome. The journal accepts the following types of articles: Original research, Reviews, Technical papers, Brief Reports, Addenda, Letters to the Editor, Commentaries and Views, and Articles on science and art. Autophagy is abstracted/indexed in Adis International Ltd (Reactions Weekly), EBSCOhost (Biological Abstracts), Elsevier BV (EMBASE and Scopus), PubMed, Biological Abstracts, Science Citation Index Expanded, Web of Science, and MEDLINE.
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