Involvement of HOXA3 and chaperone-mediated autophagy in DEHP-induced ferroptosis of mouse Leydig cells via regulating GPX4 expression

Abstract Impaired Leydig cell function leads to low testosterone levels, which is associated with male reproductive developmental defects and functional disorders. Exposure to di(2-ethylhexyl) phthalate (DEHP), an endocrine-disrupting chemical, can cause damage to Leydig cells, yet the underlying mechanism remains unclear. In this study, we found that DEHP exposure decreased serum testosterone levels and triggered ferroptosis of mouse Leydig cells by inhibiting GPX4 expression. Subsequently, mono(2-ethylhexyl) phthalate (MEHP), the primary active metabolite of DEHP, was shown to suppress the transcription of Gpx4 gene in TM3 cells by downregulation of HOXA3, which bound to the −264 to −258 region (RE3) of Gpx4 gene promoter. Concurrently, MEHP promoted HSC70 to recognize and degradate GPX4 protein via chaperone-mediated autophagy (CMA). Furthermore, both overexpression of HOXA3 and inhibition of CMA significantly abrogated MEHP-induced ferroptosis of TM3 cells through upregulating the expression of GPX4. Ultimately, tannic acid (TA), a natural polyphenol, was identified to increase the expression of GPX4 and inhibit MEHP-induced ferroptosis of TM3 cells, thereby alleviating DEHP-caused testicular damage in mice through the upregulation of HOXA3 and inhibition of CMA.