mTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model

Abstract

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it “tighter.” However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier “leaky.” Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII–IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier “leaky.” This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

Modulating the Blood–Testis Barrier Towards Increasing Drug Delivery

“…via oral gavage markedly enhanced adjudin uptake, reflecting a 'leakier' BTB [33], under conditions where adjudin alone at 10 mg/kg b.w. did not affect BTB function in vivo [33,34], and only acute doses of adjudin (125 or 250 mg/kg b.w.) induced irreversible BTB damage and caused infertility [64].…”

“…In vivo studies in the testis have shown that testicular cells, most notably Sertoli cells, produce several biologically active peptides through proteolytic cleavage of constituent proteins at the apical ES and basement membrane ( Figure 2) during spermatogenesis, and these peptides can effectively modulate the organization of the Sertoli cell cytoskeleton and hence BTB structure, function, and spermatogenesis through downstream signaling proteins [31]. Because these biologically active molecules modulate the BTB function, they can facilitate the transport of drugs across the BTB, as shown in studies that used a candidate non-hormonal male contraceptive drug, adjudin [32][33][34]. We discuss below these peptides and present a hypothetical model in which the activity of these peptides and associated signaling molecules can alter the BTB permeability barrier function to support the transport of developing germ cells, namely preleptotene spermatocytes, across the BTB during spermatogenesis ( Figure 2).…”

“…Indeed, this possibility was supported by overexpression studies of wild-type (WT) rpS6 and its constitutively active, phosphomimetic quadruple mutant, p-rpS6 mutant (i.e., p-rpS6-S235E/S236E/ S240E/S244E) [67] and KD studies of rpS6 using rpS6-specific short interfering RNA (siRNA) duplexes [67][68][69]. Overexpression of the p-rpS6 mutant in Sertoli cells cultured in vitro with a functional TJ permeability barrier induced BTB remodeling [70], making the barrier 'leaky' and enhancing the transport of adjudin across the BTB [34,71]. Lower doses of adjudin induced germ cell loss from the seminiferous epithelium when testes overexpressed the p-rpS6 mutant compared with WT rpS6 [71], and KD of rpS6 effectively made the TJ barrier tighter [68].…”

“…Studies using F5 peptide [66], the p-rpS6 mutant (vs WT rpS6) [34,70,71], and F5 peptide combined with p-rpS6 mutant [32,33] in the absence [33,34,71] or presence of adjudin [32][33][34]66] show that these biologically active peptides and/or their respective downstream signaling proteins can enhance drug diffusion across the BTB by altering the organization of F-actin microfilaments and MTs in Sertoli cells. These alterations in the organization of F-actin and MT cytoskeleton appear to be mediated by changes in the spatial expression of actin and MT regulatory proteins that confer integrity and plasticity to the cytoskeleton [32,33].…”

“…These alterations in the organization of F-actin and MT cytoskeleton appear to be mediated by changes in the spatial expression of actin and MT regulatory proteins that confer integrity and plasticity to the cytoskeleton [32,33]. Following F5 peptide overexpression, use of the p-rpS6 mutant, or both, the actin barbed-end capping and bundling protein, Eps8, and the branched actin nucleation protein, Arp3, were either considerably reduced in expression or mislocalized [33,34,71]. Both proteins, as well as their interacting partners, confer plasticity to the actin cytoskeleton such that F-actin bundles can be rapidly remodeled in response to cellular changes in the epithelium at different stages of the seminiferous epithelial cycle [75].…”