Unspliced XBP1 Counteracts β-catenin to Inhibit Vascular Calcification.

Background: Vascular calcification is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. XBP1 (X-box binding protein 1), existing as the unspliced (XBP1u) and spliced (XBP1s) forms, is a key component of the endoplasmic reticulum stress involved in vascular diseases. However, whether XBP1u participates in the development of vascular calcification remains unclear. Methods: We aim to investigate the role of XBP1u in vascular calcification.XBP1u protein levels were reduced in high phosphate (Pi)-induced calcified vascular smooth muscle cells (VSMCs), calcified aortas from mice with adenine diet-induced chronic renal failure (CRF) and calcified radial arteries from CRF patients. Results: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers runt-related transcription factor 2 (Runx2) and msh homeobox2 (Msx2), and exacerbated high Pi-induced VSMC calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high Pi-induced VSMCs significantly inhibited osteogenic differentiation and calcification. Consistently, SMC-specific XBP1 deficiency in mice markedly aggravated the adenine diet- and 5/6 nephrectomy-induced vascular calcification compared with that in the control littermates. Further interactome analysis revealed that XBP1u bound directly to β-catenin, a key regulator of vascular calcification, via aa 205-230 in its C-terminal degradation domain. XBP1u interacted with β-catenin to promote its ubiquitin-proteasomal degradation and thus inhibited β-catenin/T-cell factor (TCF)-mediated Runx2 and Msx2 transcription. Knockdown of β-catenin abolished the effect of XBP1u deficiency on VSMC calcification, suggesting a β-catenin-mediated mechanism. Moreover, the degradation of β-catenin promoted by XBP1u was independent of glycogen synthase kinase 3β (GSK-3β)-involved destruction complex. Conclusions: Our study identified XBP1u as a novel endogenous inhibitor of vascular calcification by counteracting β-catenin and promoting its ubiquitin-proteasomal degradation, which represents a new regulatory pathway of β-catenin and a promising target for vascular calcification treatment.