TLR4 signalling protects Sertoli cells from cell stress via reprogramming inflammasome and autophagy pathways in MAPK1/ERK2 dependent way

Sertoli cells constitutively maintain the spermatogonial stem cell niche and blood-testis barrier via impenetrable tight junctions under hormonal, growth factor and cytokine control. Immature Sertoli cell proliferation and survival benefit from FSH, NF-κB, and IL-1α-dependent signalling effective via the MAPK family of pathways and related to p38 MAPK dependent Ca2+ and K+ fluxes. Little is known about how the NF-κB and MAPK axis interact with pro-inflammatory NOD1/TLR4 and different voltage-gated channels. Silencing MAPK1, we found that MAPK1/ERK2 has a synergistic role with TLR4 in restricting the pro-inflammatory NF-κB/Caspase-1/IL-1β axis while having an antagonistic effect on TLR4 in promoting adult 15 P-1 Sertoli cell line proliferation and migration. In MAPK1/ERK2 absence, there was an unexpected TLR4-NF-κB behaviour, showing a strong NF-κB transactivation, assessed using a pNIFTY-SEAP reporter plasmid, accompanied by significantly activated cell migration and proliferation, as demonstrated by the wound healing assay. Voltage-gated potassium flux, most likely via Kv1.3, was significantly increased after TLR4 and NOD1 challenge in ATP DAMP signal presence, associated with inflammasome activation. TLR4 challenge also activated macroautophagy while inhibiting mitophagy. Thus, TLR4 activation restricted the NF-κB and Caspase-1/IL-1β pro-inflammatory axis, cell migration and proliferation, voltage-gated potassium efflux, and shifted mitophagy to macroautophagy (towards an inflammasome pathway-related cell death), promoting cell survival. MAPK1/ERK2 had a differential effect, as in its absence, TLR4 activation had the opposite effect on NF-κB transactivation and cell migration and proliferation. Thus, TLR4/MAPK1/NF-κB/Kv1.3 interaction provides optimal Sertoli cell maintenance conditions and allows cell death only in extreme circumstances of cumulative DAMP/PAMP signalling stimulation.