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 XBP1u (unspliced XBP1) and XBP1s (spliced XBP1) 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-induced calcified vascular smooth muscle cells, calcified aortas from mice with adenine diet-induced chronic renal failure, and calcified radial arteries from patients with chronic renal failure. RESULTS: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers Runx2 (runt-related transcription factor 2) and Msx2 (msh homeobox 2), and exacerbated high phosphate-induced vascular smooth muscle cell calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high phosphate-induced vascular smooth muscle cells significantly inhibited osteogenic differentiation and calcification. Consistently, smooth muscle cell-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 is bound directly to β-catenin, a key regulator of vascular calcification, via amino acid (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/TCF (T-cell factor)-mediated Runx2 and Msx2 transcription. Knockdown of β-catenin abolished the effect of XBP1u deficiency on vascular smooth muscle cell calcification, suggesting a β-catenin-mediated mechanism. Moreover, the degradation of β-catenin promoted by XBP1u was independent of GSK-3β (glycogen synthase kinase 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.