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, 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.