Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K + efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K + efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K + and subsequent induction of stress-activated protein kinase pathways. apolipoprotein L1 | kidney | African-American | genetics | protein kinase

ADP-ribosylation is a reversible posttranslational modification mediated by poly-ADP-ribose polymerase (PARP). The results of recent studies demonstrate that ADP-ribosylation contributes to transcription regulation. Here, we report that transcription factor NFAT binds to and is ADP-ribosylated by PARP-1 in an activation-dependent manner. Mechanistically, ADP-ribosylation increases NFAT DNA binding. Functionally, NFAT-mediated interleukin-2 (IL-2) expression was reduced in T cells upon genetic ablation or pharmacological inhibition of PARP-1. Parp-1 ؊/؊ T cells also exhibit reduced expression of other NFAT-dependent cytokines, such as IL-4. Together, these results demonstrate that ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine gene transcription. These results also imply that, similar to the effect of calcineurin inhibition, PARP-1 inhibition may be beneficial in modulating immune functions.ADP-ribosylation is a reversible posttranslational modification that transfers ADP-ribose from NAD ϩ to Glu, Asp, and/or Arg amino acids of target proteins (18). Similar to ubiquitination, ADP-ribosylation modifies target proteins to various masses due to the assorted chain lengths of the ADPribose. ADP-ribosylation is inhibited by the NAD ϩ analog 3-aminobenzamide and, more specifically, by PJ-34 (45). Poly-ADP-ribose polymerase-1 (PARP-1) is a nuclear enzyme that accounts for the bulk of ADP-ribosylation in vivo (43). Indeed, only ϳ10% of PARP activity remains in Parp-1 Ϫ/Ϫ cells upon DNA damage. In addition to its role in DNA damage repair, the results of recent studies demonstrate that PARP-1 contributes to gene transcription regulation (26,40).Transcription factor NFAT is the master regulator of interleukin-2 (IL-2) gene transcription (24,42). In resting cells, NFAT resides in the cytosol. The nuclear accumulation of NFAT is regulated by calcineurin-mediated dephosphorylation (9, 15, 23). The immunosuppressant drugs cyclosporine A (CsA) and tacrolimus (FK506) inhibit calcineurin and abrogate NFAT activation. Indeed, understanding the mechanism of NFAT activation has contributed to the great advances in transplantation surgery (27). Given that immunosuppressant therapy using CsA or FK506 causes neuro-and nephrotoxicity (1, 19), further understanding of the molecular basis of NFAT activation will provide alternate therapeutic targets for the treatment of transplant patients.Once in the nucleus, NFAT interacts with coregulators to achieve optimal NFAT activation (11,21,28). These NFAT coregulators include Fos-Jun, C/EBPs, and Fox3p, which form a composite transcription complex to regulate NFAT-mediated gene transcription. In addition, transcription coactivator CREBbinding protein/p300 and class II histone deacetylases are recruited to modulate NFAT-mediated transcription (3,12,16,48). Here, we report that PARP-1 binds to and ADP-ribosylates NFAT. The ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine ge...

Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2 ؊/؊ Nfatc4 ؊/؊ compound disruption exhibit defects in fat accumulation and are lean. Nfatc2 ؊/؊ Nfatc4 ؊/؊ mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2 ؊/؊ Nfatc4 ؊/؊ mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.