Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2.

NFAT5 is the only known mammalian tonicity-responsive transcription factor with an essential role in cellular adaptation to hypertonic stress. It is also implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity, but the underlying mechanisms remain elusive. Here, we demonstrate that NFAT5 enters the nucleus via the nuclear pore complex. We found that NFAT5 utilizes a unique nuclear localization signal (NFAT5-NLS) for nuclear import. siRNA screening revealed that only karyopherin ß1 (KPNB1), but not karyopherin α, is responsible for the nuclear import of NFAT5 via direct interaction with the NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is driven by exportin-T (XPOT), where the process requires RuvB-like AAA-type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified an unconventional tonicity-dependent nucleocytoplasmic trafficking pathway for NFAT5 that represents a critical step in orchestrating rapid cellular adaptation to change in extracellular tonicity. These findings offer an opportunity for the development of novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

RUVBL1/2 Complex Regulates Pro-Inflammatory Responses in Macrophages via Regulating Histone H3K4 Trimethylation.

Macrophages play an important role in the host defense mechanism. In response to infection, macrophages activate a genetic program of pro-inflammatory response to kill any invading pathogen, and initiate an adaptive immune response. We have identified RUVBL2 - an ATP-binding protein belonging to the AAA+ (ATPase associated with diverse cellular activities) superfamily of ATPases - as a novel regulator in pro-inflammatory response of macrophages. Gene knockdown of Ruvbl2, or pharmacological inhibition of RUVBL1/2 activity, compromises type-2 nitric oxide synthase (Nos2) gene expression, nitric oxide production and anti-bacterial activity of mouse macrophages in response to lipopolysaccharides (LPS). RUVBL1/2 inhibitor similarly inhibits pro-inflammatory response in human monocytes, suggesting functional conservation of RUVBL1/2 in humans. Transcriptome analysis further revealed that major LPS-induced pro-inflammatory pathways in macrophages are regulated in a RUVBL1/2-dependent manner. Furthermore, RUVBL1/2 inhibition significantly reduced the level of histone H3K4me3 at the promoter region of Nos2 and Il6, two prototypical pro-inflammatory genes, and diminished the recruitment of NF-kappaB to the corresponding enhancers. Our study reveals RUVBL1/2 as an integral component of macrophage pro-inflammatory responses through epigenetic regulations, and the therapeutic potentials of RUVBL1/2 inhibitors in the treatment of diseases caused by aberrant activation of pro-inflammatory pathways.