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

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, <i>Pik3c3</i>, 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. <b>Abbreviations:</b> 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.

支持细胞（Sertoli cells）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与成熟提供滋养微环境。III类磷脂酰肌醇3-激酶（class III phosphatidylinositol 3-kinase, PtdIns3K）在多种细胞类型的巨自噬过程中发挥核心作用，但其在支持细胞中的功能仍不明确。本研究构建了在支持细胞中特异性敲除其催化亚基编码基因Pik3c3的条件性敲除（cKO）小鼠模型，观察到该模型小鼠在经历一轮正常精子发生后迅速出现不育表型，同时伴随支持细胞极性破坏与精子发生障碍。后续蛋白质组学与磷酸化蛋白质组学分析显示，条件性敲除小鼠睾丸中支持细胞结构紊乱所涉及的F-肌动蛋白细胞骨架网络发生显著富集，我们在此网络中发现F-肌动蛋白负调控因子凝溶胶蛋白（scinderin, SCIN）的表达水平显著升高，以及α-微管蛋白去乙酰化酶HDAC6（histone deacetylase 6）的磷酸化水平降低。进一步实验证实，条件性敲除小鼠支持细胞中SCIN的积累会引发F-肌动蛋白细胞骨架的紊乱与解聚，这一过程与自噬-溶酶体通路介导的SCIN降解失败密切相关。此外，我们发现PIK3C3对HDAC6第S59位点的磷酸化修饰，是其通过泛素-蛋白酶体通路完成降解的必要条件。由此，在条件性敲除小鼠支持细胞中积累的HDAC6可对SCIN第K189位点进行去乙酰化修饰，进而导致F-肌动蛋白细胞骨架结构紊乱。综上，本研究阐明了PIK3C3在维持支持细胞极性中的全新机制：其既需要调控自噬的功能活性，也需要蛋白激酶活性，才能稳定肌动蛋白细胞骨架。

缩写说明：
ACTB：肌动蛋白β（actin, beta）；AR：雄激素受体（androgen receptor）；ATG14：自噬相关14（autophagy related 14）；BafA1：巴弗洛霉素A₁（bafilomycin A₁）；BECN1：自噬相关蛋白Beclin 1（beclin 1, autophagy related）；BTB：血-睾屏障（blood-testis barrier）；CASP3：半胱氨酸天冬氨酸蛋白酶3（caspase 3）；CDC42：细胞分裂周期蛋白42（cell division cycle 42）；CDH2：钙粘蛋白2（cadherin 2）；CHX：放线菌酮（cycloheximide）；CTNNA1：连环蛋白α1（catenin (cadherin associated protein), alpha 1）；CYP11A1：细胞色素P450家族11亚家族A多肽1（cytochrome P450, family 11, subfamily A, polypeptide 1）；EBSS：Earle平衡盐溶液（Earle’s balanced salt solution）；ES：外质特化（ectoplasmic specialization）；FITC：异硫氰酸荧光素（fluorescein isothiocyanate）；GAPDH：甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；GCNA：生殖细胞核酸性蛋白（germ cell nuclear acidic protein）；GJA1：间隙连接蛋白α1（gap junction protein, alpha 1）；H2AX：组蛋白H2A.X变体（H2A.X variant histone）；HDAC6：组蛋白去乙酰化酶6（histone deacetylase 6）；KIT：KIT原癌基因受体酪氨酸激酶（KIT proto-oncogene, receptor tyrosine kinase）；LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）；MAP3K5：丝裂原活化蛋白激酶激酶激酶5（mitogen-activated protein kinase kinase kinase 5）；MAP1LC3B：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）；OCLN：闭合蛋白（occludin）；PIK3C3：III类磷脂酰肌醇3-激酶催化亚基3型（phosphatidylinositol 3-kinase catalytic subunit type 3）；PIK3R4：磷脂酰肌醇3-激酶调节亚基4（phosphoinositide-3-kinase regulatory subunit 4）；PNA：花生凝集素（arachis hypogaea lectin）；RAC1：Rac家族小GTP酶1（Rac family small GTPase 1）；SCIN：凝溶胶蛋白（scinderin）；SQSTM1/p62：自噬衔接蛋白p62（sequestosome 1）；SSC：精原干细胞（spermatogonia stem cell）；STK11：丝氨酸/苏氨酸激酶11（serine/threonine kinase 11）；TJP1：紧密连接蛋白1（tight junction protein 1）；TubA：他司他汀A（tubastatin A）；TUBB3：微管蛋白β3类III（tubulin beta 3 class III）；TUNEL：脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TdT-mediated dUTP nick-end labeling）；UB：泛素（ubiquitin）；UVRAG：紫外线抵抗相关基因（UV radiation resistance associated gene）；VIM：波形蛋白（vimentin）；WT1：WT1转录因子（WT1 transcription factor）；ZBTB16：锌指和BTB结构域包含蛋白16（zinc finger and BTB domain containing 16）。