rpS6 regulates blood-testis barrier dynamics via its effects on MMP-9 mediated by Akt signaling

Abstract

Mammalian target of rapamycin complex 1 (mTORC1) is an emerging regulator of blood-tissue barriers that utilizes ribosomal protein S6 (rpS6) as the downstream signaling molecule. To explore the role of rpS6 in blood-testis barrier (BTB) function, a constitutively active quadruple rpS6 phosphomimetic mutant was constructed in mammalian expression vector and overexpressed in Sertoli cells cultured in vitro that mimicked the BTB in vivo. Using this quadruple phosphomimetic mutant, phosphorylated (p)-rpS6 was shown to disrupt IGF-1/insulin signaling, thereby abolishing Akt phosphorylation, which led to an induction of MMP-9. This increase in MMP-9 secretion perturbed the Sertoli cell tight junction permeability barrier by proteolysis-mediated downregulation of tight junction proteins at the BTB. These findings were confirmed by the use of a specific MMP-9 inhibitor that blocked the disruption of the tight junction permeability barrier by the rpS6 mutant. Additionally, RNA interference (RNAi)-mediated Akt silencing was able to mimic the results of rpS6 mutant overexpression in Sertoli cells, further confirming this p-rpS6-Akt-MMP-9 signaling pathway. In conclusion, these data support a new concept of mTORC1-mediated BTB regulation, that is possibly also applicable to other blood-tissue barriers.

rpS6 Regulates Blood-Testis Barrier Dynamics Through Arp3-Mediated Actin Microfilament Organization in Rat Sertoli Cells. An In Vitro Study

“…This conclusion was reached based on findings here in which a knockdown of Akt1/2 by RNAi mimics the phenotype detected by overexpressing phosphomimetic rpS6 mutant in Sertoli cells, such as a disruption of actin microfilaments in Sertoli cells, including truncation and misorganization, as well as an up-regulation of p-FAK-Tyr 407 . Furthermore, studies using specific inhibitors also support the notion that Akt is a regulator that modulates rpS6-mediated Sertoli cell BTB function (21). Additionally, Akt is also a downstream regulator of mTORC2 signaling complex (39,62).…”

“…Dual-labeled immunofluorescence analysis was performed using frozen sections of testes (7-m thickness) and corresponding primary antibodies (Supplemental Table 1) and Alexa Fluor-conjugated secondary antibodies (red fluorescence, Alexa Fluor 555 and green fluorescence, Alexa Fluor 488; Invitrogen) (16,21). To visualize F-actin, sections or cells were incubated with Cy3 or fluorescein isothiocyanate-conjugated phalloidin (Sigma-Aldrich) at 1:50 dilution together with corresponding secondary antibodies.…”

“…Here, a constitutively active quadruple phosphomimetic mutant of p-rpS6 was prepared to assess its effects on F-actin organization at the Sertoli cell BTB after transfection vs the use of wild-type (WT) rpS6 and control (empty mammalian expression vector). We also examined whether the recently identified upstream regulator of Akt (transforming oncogene of v-akt, also known as protein kinase B) of mTORC1 (21) is also involved in these events. In short, we sought to test the hypothesis that p-rpS6 which promotes Sertoli cell TJbarrier disruption is exerting its effects on the organization of actin microfilaments, mediated by the intricate action of branched actin polymerization-inducing protein Actin-related protein 2/3 (Arp2/3)/Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) complex.…”

Transport of germ cells across the seminiferous epithelium during spermatogenesis—the involvement of both actin- and microtubule-based cytoskeletons

“…[18][19][20] Some are recently shown to be actively involved in F-actin organization at the basal ES at the Sertoli cell-cell interface, such as Akt1/2, 21,22 which was earlier shown to be a putative component of the apical and basal ES 23 in the rat testis. In short, the MT-based tracks are analogous to 2-way highways that modulate in-coming and outgoing traffic to maintain cellular homeostasis.…”

G protein‐dependent signaling triggers a β‐arrestin‐scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation

“…Of relevance to this study, in the male gonad, natural FSH targets Sertoli cells, which are are polarized cells where this phenomenon is likely to occur. Interestingly, rpS6 has been shown to play an important role in the regulation of the blood-testis formation, which is formed by the tight junctions between Sertoli cells (85)(86)(87). Of note, 59TOP mRNA encodes proteins involved in the translational machinery such as eIF4B or eIF3A, as well as ribosomal components, and as such represent about 25% of all the RNA abundance in a cell (88).…”