PP2A phosphatase inhibition is anti-fibrotic through Ser77 phosphorylation-mediated ARNT/ARNT homodimer formation.

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.

Pharmacological induction of hypoxia-inducible transcription factor ARNT attenuates chronic kidney failure.

Progression of chronic kidney disease associated with progressive fibrosis and impaired tubular epithelial regeneration is still an unmet biomedical challenge because, once chronic lesions have manifested, no effective therapies are available as of yet for clinical use. Prompted by various studies across multiple organs demonstrating that preconditioning regimens to induce endogenous regenerative mechanisms protect various organs from later incurring acute injuries, we here aimed to gain insights into the molecular mechanisms underlying successful protection and to explore whether such pathways could be utilized to inhibit progression of chronic organ injury. We identified a protective mechanism controlled by the transcription factor ARNT that effectively inhibits progression of chronic kidney injury by transcriptional induction of ALK3, the principal mediator of antifibrotic and proregenerative bone morphogenetic protein-signaling (BMP-signaling) responses. We further report that ARNT expression itself is controlled by the FKBP12/YY1 transcriptional repressor complex and that disruption of such FKBP12/YY1 complexes by picomolar FK506 at subimmunosuppressive doses increases ARNT expression, subsequently leading to homodimeric ARNT-induced ALK3 transcription. Direct targeting of FKBP12/YY1 with in vivo morpholino approaches or small molecule inhibitors, including GPI-1046, was equally effective for inducing ARNT expression, with subsequent activation of ALK3-dependent canonical BMP-signaling responses and attenuated chronic organ failure in models of chronic kidney disease, and also cardiac and liver injuries. In summary, we report an organ-protective mechanism that can be pharmacologically modulated by immunophilin ligands FK506 and GPI-1046 or therapeutically targeted by in vivo morpholino approaches.