HIF-1 alpha is a regulator of galectin-3 expression in the intervertebral disc.
The regulation of galectin-3 expression in skeletal tissues is not completely understood. Our studies indicate that HIF-1 alpha 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.


BACKGROUND
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.


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)-1 alpha with the gal-3 promoter was confirmed by gel shift and site-directed mutagenesis.


RESULTS
There was minimal oxygen-dependent change in HIF-1 alpha 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-1 alpha wildtype but not HIF-1-null mice. To evaluate the importance of HIF-1 in regulation of gal-3 expression, we overexpressed HIF-1 alpha or constitutively active-HIF-1 alpha in null MEF. An increase in gal-3 promoter activity was observed in both normoxia and hypoxia. Similarly, suppression of HIF-1 alpha 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-1 alpha 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.
Zeng
Yan
Y
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA.
Danielson
Keith G
KG
Albert
Todd J
TJ
Shapiro
Irving M
IM
Risbud
Makarand V
MV
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D053222:Fas Ligand Protein / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D037502:Galectin 3 / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010101:Oxygen Consumption / Q000502:physiology; D051381:Rats; D020218:Response Elements / Q000502:physiology*
D053222:Fas Ligand Protein; D037502:Galectin 3; D051795:Hypoxia-Inducible Factor 1, alpha Subunit