Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes

Abstract

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-RasG12V) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-RasG12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-RasG12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-RasG12V, which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes

“…Although RNA-seq analysis of EBV infected and uninfected NOKs showed minimal expression of either the NFATc1 or NFATc2 transcripts ( S1 Table ), there is detectable expression of the NFATc3 and NFAT5 transcripts. Nevertheless, NFATc1 has been reported to increase differentiation of keratinocytes, and immunosuppressant calcineurin inhibitor drugs such as cyclosporin have been proposed to induce squamous cell carcinomas in patients at least partially via their inhibitory effects on NFATc1 [ 52 , 53 ]. Since both the type 1 Akata virus and type 2 AG876 viruses used in our experiments have the NFAT-responsive Zp-V3 form of the BZLF1 IE promoter, the two viruses may respond similarly to the effects of NFAT family members in epithelial cells.…”

“…Type 1 versus Type 2 EBV infection in epithelial cells least partially via their inhibitory effects on NFATc1 [52,53]. Since both the type 1 Akata virus and type 2 AG876 viruses used in our experiments have the NFAT-responsive Zp-V3 form of the BZLF1 IE promoter, the two viruses may respond similarly to the effects of NFAT family members in epithelial cells.…”

NFATc4 knockout promotes neuroprotection and retinal ganglion cell regeneration after optic nerve injury

“…The initial increase in Ca 2+ concentration activating CaN is an obligatory step for the activation of NFAT-dependent transcription. Moreover, recent reports suggest an important contribution of NFAT to autophagy in retinal pigmental epithelial cells [91] as well as in other cell types [92]. In view of this, it is tempting to speculate that Ca 2+ -dependent activation of NFATc4 and NFATc4 downstream signaling should be placed among important events restricting axonal regeneration after mechanical injury.…”