OTUB2 contributes to vascular calcification in chronic kidney disease via the YAP-mediated transcription of PFKFB3

Rationale: Chronic kidney disease (CKD) is a global public health issue, with vascular calcification (VC) being a common and deadly complication.Despite its prevalence, the underlying mechanisms of VC remain unclear.In this study, we aimed to investigate whether and how Otubain-2 (OTUB2) contributes to VC. Methods: The relationship between OTUB2 and VC was examined via immunohistochemical and immunofluorescence staining of discarded calcified radial arteries from uremic patients who underwent arteriovenous fistula operations.Additionally, mice were fed a 0.2% adenine diet supplemented with 1.2% phosphorus to establish a model of CKD-related VC.Vascular smooth muscle cell (VSMC)-specific OTUB2 knockout and overexpression were performed in vivo via the delivery of adeno-associated virus 9 vectors to manipulate the expression of OTUB2.Additionally, a calcified VSMC model was established to explore the roles of OTUB2 in VC by evaluating changes in osteogenic marker expression and calcium deposition.Results: Our results revealed a significant upregulation of OTUB2 expression during VC progression.OTUB2 overexpression upregulated the expression of osteogenic markers and exacerbated VSMC calcification, as verified by Von Kossa and Alizarin red staining.Conversely, VSMC-specific OTUB2 deficiency significantly mitigated adenine diet-induced VC in CKD mice.OTUB2 knockdown or inhibition decreased Yes-associated protein (YAP) abundance.Mechanistically, OTUB2 bound to YAP, decreasing its K48-linked polyubiquitination and inhibiting its subsequent degradation.Knockdown or inhibition of YAP abolished the effect of OTUB2 overexpression on VSMC calcification, indicating a YAP-mediated mechanism.Furthermore, the YAP/TEAD1 complex bound to the promoter of PFKFB3, increasing its transcriptional activity, as determined by CUT&RUN-qPCR.The knockdown or inhibition of PFKFB3 alleviated the procalcific effects of OTUB2.Conclusions: Our findings indicate that OTUB2 promotes VC at least partially by activating the YAP-PFKFB3 signaling pathway.Targeting OTUB2 may be an appealing therapeutic strategy for VC.