Rescue of PFOS-induced human Sertoli cell injury by overexpressing a p-FAK-Y407E phosphomimetic mutant

Abstract

PFOS induces Sertoli cell injury using testicular cells isolated from rodent testes, but it remains unknown if PFOS has similar effects in humans. Herein, we maintained human Sertoli cells in a mitotically active state in vitro, thus enabling transfection experiments that altered gene expression to explore the molecular mechanism(s) underlying toxicant-induced cell injury. Human Sertoli cells obtained from men at ages 15, 23, 36 and 40 were cultured in vitro. These differentiated Sertoli cells remained mitotically active when cultured in the presence of 10% FBS (fetal bovine serum), with a replication time of ~1–3 weeks. At ~80% confluency, they were used for studies including toxicant exposure, immunoblotting, immunofluorescence analysis, tight junction (TJ)-permeability assessment, and overexpression of BTB (blood-testis barrier) regulatory genes such as FAK and its phosphomimetic mutants. PFOS was found to induce Sertoli cell injury through disruptive effects on actin microfilaments and microtubule (MT) organization across the cell cytosol. As a consequence, these cytoskeletal networks failed to support cell adhesion at the BTB. Overexpression of a FAK phosphomimetic and constitutively active mutant p-FAK-Y407E in these cells was capable of rescuing the PFOS-induced injury through corrective cellular organization of cytoskeletal elements. Summary: PFOS induces human Sertoli cell injury which can be rescued by overexpressing p-FAK-Y407E mutant.

Mechanistic Insights into PFOS-Mediated Sertoli Cell Injury

“…Although the findings above are of interest regarding the protective effects of overexpressing p-FAK-Y407E in rat Sertoli cells against PFOS-mediated Sertoli cell injury, it remains to be established if these findings are relevant to humans since the expression of FAK by human Sertoli cells was not established until recently [42]. In 2011 it was shown that primary human Sertoli cells can be maintained in vitro when cultured in F12/DMEM in the presence of 5-10% fetal calf serum (FCS) [43].…”

“…When human Sertoli cells cultured in vitro were treated with PFOS, similar to rat Sertoli cells, p-FAK-Tyr407 expression was also considerably down-regulated [42]. We prepared a phosphomimetic and constitutively active mutant of human FAK, namely p-hFAK-Y407E, and cloned into the mammalian expression vector pcDNA 3.1(+) [42]. Similar to rat Sertoli cells, PFOS induced human Sertoli cell injury as manifested by disruptive organization of actin- and MT-based cytoskeletons across human Sertoli cells ( Figure 1B ) .…”

“…Similar to rat Sertoli cells, PFOS induced human Sertoli cell injury as manifested by disruptive organization of actin- and MT-based cytoskeletons across human Sertoli cells ( Figure 1B ) . This was shown to be the result of disruptive changes in the spatial expression of branched actin-nucleation protein Arp3 and actin barbed-end capping and bundling protein Eps8, and MT stability promoting protein EB1 [42]. The net result of these changes thus perturbed proper organization of cytoskeletons versus control human Sertoli cells [42].…”

“…This was shown to be the result of disruptive changes in the spatial expression of branched actin-nucleation protein Arp3 and actin barbed-end capping and bundling protein Eps8, and MT stability promoting protein EB1 [42]. The net result of these changes thus perturbed proper organization of cytoskeletons versus control human Sertoli cells [42]. F-actin and MTs no longer stretched across the Sertoli cell cytosol as noted in control cells, but were considerably truncated and mis-aligned.…”

Modulating the Blood–Testis Barrier Towards Increasing Drug Delivery

“…The concerted actions of the N-WASP and actin-related protein (Arp3) coupled with protein Eps8 thus provide F-actin with the plasticity necessary to effectively change the organization of actin filaments across Sertoli cells in the testis, and leads to an increase in BTB permeability together with spermatid exfoliation (Figure 3) [35,36]. Indeed, in vitro studies have shown that a phosphomimetic (and constitutively active) mutant of p-FAK-Y407, designated p-FAK-Y407E (in which Tyr407 has been converted to Glu) promoted BTB integrity by making it tighter, both in rats and in human Sertoli cells in vitro [47,48].…”