Abstract
Aryl hydrocarbon receptor nuclear translocator (ARNT) mediates anti-fibrotic activity in kidney and liver through induction of ALK3-receptor expression and subsequently increased Smad1/5/8 signaling. While expression of ARNT can be pharmacologically induced by sub-immunosuppressive doses of FK506 or by GPI1046, its anti-fibrotic activity is only realized when ARNT-ARNT homodimers form, as opposed to formation of ARNT-AHR or ARNT-HIF1α heterodimers. Mechanisms underlying ARNTs dimerization decision to specifically form ARNT–ARNT homodimers and possible cues to specifically induce ARNT homodimerization have been previously unknown. Here, we demonstrate that phosphorylation of the Ser77 residue is critical for ARNT–ARNT homodimer formation and stabilization. We further demonstrate that inhibition of PP2A phosphatase activity by LB100 enhances ARNT–ARNT homodimers both in vivo and in vitro (mouse tubular epithelial cells and human embryonic kidney cells). In murine models of kidney fibrosis, and also of liver fibrosis, combinations of FK506 or GPI1046 (to induce ARNT expression) with LB100 (to enhance ARNT homodimerization) elicit additive anti-fibrotic activities. Our study provides additional evidence for the anti-fibrotic activity of ARNT–ARNT homodimers and reveals Ser77 phosphorylation as a novel pharmacological target to realize the therapeutic potential of increased ARNT transactivation activity.
Highlights
Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor of the family of basic helix-loophelix-Per-ARNT-Sim proteins, which elicits its transcription factor activity upon d imerization[1,2]
Relative mRNA levels were determined after normalization to GAPDH, and data are shown as the fold change compared with DMSO treated control cells
GAPDH and Lamin A/C serve as loading controls for cytoplasmic and nuclear fractions. (h, i) Representative immunoblotting for co-immunoprecipitation of ARNT-myc and ARNT-EGFP in HEK293 cells treated with control DMSO, LB100 (5 μM), Okadaic acid (OA; 50 nM) and Tautomycetin (TMC; 200 nM) for 4 h. n = 3. (j) Intensity of co-immunoprecipitation display as graph after compared with DMSO treated control cells
Summary
Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor of the family of basic helix-loophelix-Per-ARNT-Sim (bHLH-PAS) proteins, which elicits its transcription factor activity upon d imerization[1,2]. (a) ARNT protein level in MCT cells indicated different concentrations following treatment with LB100, OA and TMC relative to control DMSO treated cells. (c) Relative expression level of ARNT in MCT cells is determined by qRT-PCR after 5 μM of LB100 treatment at indicated time points. Relative mRNA levels were determined after normalization to GAPDH, and data are shown as the fold change compared with DMSO treated control cells. We further demonstrate that formation of ARNT homodimers can be enhanced by the PP2A phosphatase inhibitor LB100, and that administration of LB100 has an additive protective effect to FK506/GPI1046 in murine models of kidney and liver fibrosis
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