Tacrolimus-induced chronic nephrotoxicity (TICN) hinders its long-term use, but its mechanism remains unclear. Tacrolimus exerts its pharmacological effect by inhibiting calcineurin and its substrate NFAT. Whether the inhibition of other calcineurin substrates is related to TICN remains to be explored. Transcription factor EB (TFEB), a substrate of calcineurin, plays a crucial role in various homeostasis. Herein, we found that tacrolimus inhibited TFEB nuclear translocation and activity in mouse kidneys and HK-2 cells. Then, TFEB gain- and loss-of-function rescued the effect of tacrolimus in HK-2 cells. Furthermore, TFEB activation both by phosphorylation sites mutation and agonist rescued TICN in mice. To elucidate the mechanism of TFEB, we analyzed ChIP-seq data. Growth arrest and DNA damage-inducible 45α (GADD45α) was identified as a transcriptional target of TFEB via chromatin immunoprecipitation and dual luciferase reporter assays. And then we revealed that GADD45α overexpression rescued DNA damage and kidney injury caused by tacrolimus or TFEB knockdown in vitro, and vise versa. The protective effect of GADD45α against TICN and DNA damage was further demonstrated by overexpressing it in mice. In conclusion, the persistent inhibition of TFEB-GADD45α pathway by tacrolimus contributes to TICN. This study identifies a specific target for intervention of TICN.
Cerebral ischemia–reperfusion (I/R) injury remains a major therapeutic challenge, primarily due to complex mechanisms involving oxidative stress and apoptosis. Growth arrest and DNA damage-inducible protein α (Gadd45α), a stress sensor linked to cellular stress responses, has been implicated in I/R injury, yet its precise role in ischemic stroke is incompletely understood. This study aimed to elucidate the function and underlying mechanisms of Gadd45α in cerebral I/R injury using both in vivo and in vitro models. In rats subjected to middle cerebral artery occlusion (MCAO) and in neurons exposed to oxygen–glucose deprivation/reperfusion (OGD/R), Gadd45α expression was significantly upregulated. Lentivirus-mediated knockdown of Gadd45α (sh-Gadd45α) reduced infarct volumes, improved neurological function and increased miniature excitatory postsynaptic current (mEPSC) amplitude. In primary cortical neurons exposed to OGD/R, Gadd45α knockdown decreased reactive oxygen species (ROS) production, DNA damage, and apoptosis, while Gadd45α overexpression exacerbated these effects. Mechanistically, Gadd45α directly interacts with forkhead box O1 (FOXO1) and positively regulates its transcriptional activity. Gadd45α knockdown attenuated the ischemia-induced upregulation of both total FOXO1 and its phosphorylated form (p-FOXO1), thereby suppressing FOXO1 signaling and mitigating cerebral I/R injury. Furthermore, FOXO1 overexpression reversed the neuroprotective effects of Gadd45α silencing, confirming that FOXO1 acts as a critical downstream mediator. These findings demonstrate that Gadd45α silencing alleviates cerebral I/R injury by suppressing FOXO1 signaling, suggesting the Gadd45α/FOXO1 axis as a promising therapeutic target for ischemic stroke.
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