HIF-1α Is a Regulator of Galectin-3 Expression in the Intervertebral Disc

Abstract

The regulation of galectin-3 expression in skeletal tissues is not completely understood. Our studies indicate that HIF-1α regulates galectin-3 expression by interacting with hypoxia regulatory elements in the promoter region. Finally, we show that galectin-3 serves a prosurvival role in the intervertebral disc. Introduction: Earlier reports indicated that galectin-3 (gal-3) is highly expressed in the epiphyseal growth plate cartilage and the intervertebral disc. Because these skeletal tissues have a limited vascular supply and the cells reside in a low O₂ environment, we determined if the oxemic status modulates gal-3 expression. Materials and Methods: Cells were cultured in normoxia (21% O₂) or hypoxia (2% O₂), and gal-3 expression and promoter activity were evaluated. Interaction of hypoxia inducible factor (HIF)-1α with the gal-3 promoter was confirmed by gel shift and site-directed mutagenesis. Results: There was minimal oxygen-dependent change in HIF-1α levels and no change in gal-3 expression and promoter activity in nucleus pulposus cells. In contrast, hypoxia induced gal-3 mRNA, protein, and promoter activity in HeLa cells and mouse embryonic fibroblasts (MEFs) from HIF-1α wildtype but not HIF-1–null mice. To evaluate the importance of HIF-1 in regulation of gal-3 expression, we overexpressed HIF-1α or constitutively active-HIF-1α in null MEF. An increase in gal-3 promoter activity was observed in both normoxia and hypoxia. Similarly, suppression of HIF-1α in nucleus pulposus cells, and wildtype MEF, using siRNA and pharmacological inhibitors resulted in suppression of gal-3 promoter activity and mRNA levels. Analysis of the gal-3 promoter indicated that it contained two hypoxia response elements (HREs). Gel-shift and chromatin immunoprecipitation analysis confirmed that there was binding of HIF-1α to the gal-3 HRE. Furthermore, site-directed mutagenesis of HRE completely blocked hypoxic induction of gal-3 promoter activity. In nucleus pulposus cells, suppression of gal-3 expression promoted FasL-mediated apoptosis. Conclusions: Together, these studies showed that gal-3 is a HIF-1–regulated lectin that plays an important role in nucleus pulposus cell survival.