NFAT3-FasL axis synchronously regulates apoptosis and necroptosis in murine cochlear outer hair cells after noise trauma.

Existing studies have indicated that noise induces apoptosis and necroptosis in cochlear outer hair cells (OHCs). However, the role of the extrinsic cell death pathway, initiated by death ligands in the cochlea, remains unknown. In this study, we hypothesized that noise could induce the NFAT3/FasL axis-mediated extrinsic death pathway in the cochlea. We found that NFAT3/FasL signaling was silent in normal OHCs. Noise exposure induced apoptosis and necroptosis in OHCs with specifically high FasL expression. Multiplex immunofluorescence staining revealed that NFAT3 nuclear translocation and FasL upregulation were colocalized in the apoptotic and necroptotic OHCs following noise trauma. Administration of FK506 or 11R-vivit (an specific NFAT inhibitor) blocked NFAT3 nuclear translocation, inhibited FasL expression, mitigated apoptosis and necroptosis, and protected against noise-induced hearing loss (NIHL). Finally, FasL knockdown by delivering siRNA intratympanically attenuated apoptosis and necroptosis in OHCs and alleviated NIHL, confirming the role of FasL in OHC death. Collectively, our study demonstrates that the NFAT3/FasL axis mediates noise-induced extrinsic death pathway in OHCs, leading to their apoptosis and necroptosis.

NFAT inhibitor 11R-VIVIT ameliorates mouse renal fibrosis after ischemia-reperfusion-induced acute kidney injury.

Acute kidney injury (AKI) with maladaptive tubular repair leads to renal fibrosis and progresses to chronic kidney disease (CKD). At present, there is no curative drug to interrupt AKI-to-CKD progression. The nuclear factor of the activated T cell (NFAT) family was initially identified as a transcription factor expressed in most immune cells and involved in the transcription of cytokine genes and other genes critical for the immune response. NFAT2 is also expressed in renal tubular epithelial cells (RTECs) and podocytes and plays an important regulatory role in the kidney. In this study, we investigated the renoprotective effect of 11R-VIVIT, a peptide inhibitor of NFAT, on renal fibrosis in the AKI-to-CKD transition and the underlying mechanisms. We first examined human renal biopsy tissues and found that the expression of NFAT2 was significantly increased in RTECs in patients with severe renal fibrosis. We then established a mouse model of AKI-to-CKD transition using bilateral ischemia-reperfusion injury (Bi-IRI). The mice were treated with 11R-VIVIT (5 mg/kg, i.p.) on Days 1, 3, 10, 17 and 24 after Bi-IRI. We showed that the expression of NFAT2 was markedly increased in RTECs in the AKI-to-CKD transition. 11R-VIVIT administration significantly inhibited the nuclear translocation of NFAT2 in RTECs, decreased the levels of serum creatinine and blood urea nitrogen, and attenuated renal tubulointerstitial fibrosis but had no toxic side effects on the heart and liver. In addition, we showed that 11R-VIVIT administration alleviated RTEC apoptosis after Bi-IRI. Consistently, preapplication of 11R-VIVIT (100 nM) and transfection with NFAT2-targeted siRNA markedly suppressed TGFß-induced HK-2 cell apoptosis in vitro. In conclusion, 11R-VIVIT administration inhibits IRI-induced NFAT2 activation and prevents AKI-to-CKD progression. Inhibiting NFAT2 may be a promising new therapeutic strategy for preventing renal fibrosis after IR-AKI.

Peptide 11R­VIVIT promotes fracture healing in osteoporotic rats.

Osteoporotic fracture healing is a complex clinical issue. The present study was conducted to investigate the repair properties of 11R­VIVIT on osteoporotic fractures and to examine the potential effects of 11R­VIVIT on osteoporotic bone marrow­derived mesenchymal stem cells (BMSCs), A rat model of osteoporotic femoral fracture was established, and the effects of the daily local injection of 11R­VIVIT or saline on fracture repairing were evaluated by micro­CT scans and H&E staining. Moreover, BMSCs from osteoporotic rats were treated with 11R­VIVIT, and the osteogenic and adipogenic differentiation of BMSCs was evaluated. The results revealed that 11R­VIVIT promoted bone formation and increased fracture healing. In addition, 11R­VIVIT promoted the differentiation of osteoporotic BMSCs into osteoblasts rather than adipocytes. Furthermore, mechanistic analysis revealed that 11R­VIVIT promoted autophagy by blocking the protein kinase B (AKT)/nuclear factor of activated T­cells (NFATc1) signaling pathway. Consistently, the activation and inhibition of autophagy using rapamycin and LY294002 confirmed the regulatory effects of 11R­VIVIT on autophagy. On the whole, the findings of the present study demonstrate that 11R­VIVIT promotes fracture healing in osteoporotic rats and enhances the osteogenic differentiation of osteoporotic BMSCs by dysregulating the AKT/NFATc1 signaling pathway.

Combination of antioxidants and NFAT (nuclear factor of activated T cells) inhibitor protects auditory hair cells from ototoxic insult.

Hair cell (HC) degeneration causes hearing loss in millions of people worldwide. Aminoglycoside exposure is one major cause of sensory HC damage. Aminoglycosides generate free radicals within the inner ear, permanently damaging sensory cells, and thus causing hearing loss. Hearing protection requires strategies to overcome the apparently irreversible loss of HCs in mammals. The nuclear factor of activated T cells (NFAT) inhibitor 11R-VIVIT reportedly protects HCs from gentamicin toxicity. Here we investigated whether the combination of 11R-VIVIT with the antioxidant L-carnitine or N-acetylcysteine could protect mouse cochlear HCs from gentamicin damage. Compared to single-component treatment, combined treatment with 11R-VIVIT plus L-carnitine yielded significant protection from gentamicin, and 11R-VIVIT plus N-acetylcysteine provided almost complete protection of HCs from gentamicin. Caspase activity in organ of Corti was significantly reduced by combined treatment with 11R-VIVIT + N-acetylcysteine + gentamicin, compared to 11R-VIVIT + gentamicin or gentamicin alone. Analysis of relative gene expression by qPCR revealed down-regulation of the pro-apoptotic genes Fasl and Casp9, and up-regulation of the antioxidant genes Hmox1 and Nrf2 after treatment with 11R-VIVIT + N-acetylcysteine + gentamicin, compared to single-compound treatment or gentamicin alone in cultures. Selective NFAT inhibition by 11R-VIVIT may be a good strategy for preventing gentamicin-induced HC damage. L-carnitine and N-acetylcysteine, with their ROS-reducing properties, contribute to the synergistic effectiveness with 11R-VIVIT by decreasing ROS-induced NFAT translocation. Our data suggest that a combined approach of NFAT inhibition together with an antioxidant, like N-acetylcysteine, could be useful for hearing loss treatment and/or prevention. Cover Image for this issue: https://doi.org/10.1111/jnc.14759.

Peptide 11R-VIVIT stimulates osteoblastogenesis through regulating the expression of nuclear factor of activated T cells cytoplasmic 1.

Osteoporosis is characterized by the imbalance of two relatively independent processes-osteoblastogenesis and osteoclastogenesis. Calcineurin (Cn)/nuclear factor of activated T cells (NFAT)(Cn/NFAT) signaling pathway is involved in these two processes in bone metabolism, but its potential as a target to treat osteoporosis needs to be defined. The aim of this study is to investigate the inhibition of polypeptide 11R-VIVIT onCn/NFAT signaling pathway. Rat calvaria (RC) cells were prepared from experimental model of osteoporosis in rat.11R-VIVIT wasused to treat cultured RC cells from wide type (WT) rat or from osteoporosis (OP) rat, we then measured the expressions of NFATc1, osteopontin (OPN), osteocalcin (OC), cytokines, NFκB subunit p65 by real time PCR, western blot or immunofluorescence. Then ALP expression and staining, and alizarin red S (ARS) staining were employed to study the osteoblastodifferentiation. 11R-VIVIT regulates the expression of NFATc1, and some other molecules in Cn/NFAT signaling pathway, such as OPN and OC, at transcriptional level and protein level. Further, it can regulate the expression of inflammatory cytokine like IL-1beta, IL-10 and TNF-alpha and NFκB activity. Further, 11R-VIVIT can accelerate osteoblastodifferentiation of RC cells demonstrated by ALP and ARS staining.11R-VIVIT can stimulate the bone formation by decreasing NFATc1 expression and regulating the expression of inflammation related molecules.

NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice.

BACKGROUND AND PURPOSE: Podocyte injury plays a key role in the development of diabetic nephropathy (DN). We have recently shown that 11R-VIVIT, an inhibitor of cell-permeable nuclear factor of activated T-cells (NFAT), attenuates podocyte apoptosis induced by high glucose in vitro. However, it is not known whether 11R-VIVIT has a protective effect on DN, especially podocyte injury, under in vivo diabetic conditions. Hence, we examined the renoprotective effects of 11R-VIVIT in diabetic db/db mice and the possible mechanisms underlying its protective effects on podocyte injury in vivo and in vitro. EXPERIMENTAL APPROACH: Type 2 diabetic db/db mice received i.p. injections of 11R-VIVIT (1 mg·kg(-1)) three times a week and were killed after 8 weeks. Immortalized mouse podocytes were cultured under different experimental conditions. KEY RESULTS: 11R-VIVIT treatment markedly attenuated the albuminuria in diabetic db/db mice and also alleviated mesangial matrix expansion and podocyte injury. However, body weight, food and water intake, and glucose levels were unaffected. It also attenuated the increased NFAT2 activation and enhanced urokinase-type plasminogen activator receptor (uPA receptor) expression in glomerulor podocytes. In cultured podocytes, the increased nuclear accumulation of NFAT2 and uPA receptor expression induced by high glucose treatment was prevented by 11R-VIVIT or NFAT2-knockdown; this was accompanied by improvements in the filtration barrier function of the podocyte monolayer. CONCLUSIONS AND IMPLICATIONS: The NFAT inhibitor 11R-VIVIT might be a useful therapeutic strategy for protecting podocytes and treating DN. The calcinerin/NFAT2/uPA receptor signalling pathway should be exploited as a therapeutic target for protecting podocytes from injury in DN.