Abstract
OASIS/CREB3L1, an endoplasmic reticulum (ER)-resident transcription factor, plays important roles in osteoblast differentiation. In this study, we identified new crosstalk between OASIS and the hypoxia signaling pathway, which regulates vascularization during bone development. RT-PCR and real-time PCR analyses revealed significant decreases in the expression levels of hypoxia-inducible factor-1α (HIF-1α) target genes such as vascular endothelial growth factor A (VEGFA) in OASIS-deficient (Oasis−/−) mouse embryonic fibroblasts. In coimmunoprecipitation experiments, the N-terminal fragment of OASIS (OASIS-N; activated form of OASIS) bound to HIF-1α through the bZIP domain. Luciferase assays showed that OASIS-N promoted the transcription activities of a reporter gene via a hypoxia-response element (HRE). Furthermore, the expression levels of an angiogenic factor Vegfa was decreased in Oasis−/− osteoblasts. Immunostaining and metatarsal angiogenesis assay showed retarded vascularization in bone tissue of Oasis−/− mice. These results suggest that OASIS affects the expression of HIF-1α target genes through the protein interaction with HIF-1α, and that OASIS-HIF-1α complexes may play essential roles in angiogenesis during bone development.
Highlights
IntroductionOASIS is preferentially expressed in osteoblasts and astrocytes[13,21], BBF2H7 in chondrocytes[22], CREBH in liver cells[23,24], and AIbZIP in the testis and prostate[25,26]
We found that OASIS is upregulated in a time-dependent manner during hypoxia, and that OASIS binds to Hypoxia-inducible factors (HIFs)-1α through its bZIP domain and promotes the transcription of hypoxia-inducible genes including vascular endothelial growth factor A (VEGFA) under hypoxic conditions
Western blot analyses of cell lysates from WT and Oasis−/− mouse embryonic fibroblasts (MEFs) cultured under normoxia or hypoxia showed no significant difference in the protein levels of HIF-1α between WT and Oasis−/− MEFs (Fig. 1e,f)
Summary
OASIS is preferentially expressed in osteoblasts and astrocytes[13,21], BBF2H7 in chondrocytes[22], CREBH in liver cells[23,24], and AIbZIP in the testis and prostate[25,26] These observations suggest that the functions of the individual members may be involved in regulation of the UPR in specific organs and tissues. OASIS is constitutively degraded under normal conditions, but ER stress leads to inhibition of ubiquitination and proteasome-mediated degradation of OASIS by HRD1, which is an ER-resident E3 ubiquitin ligase Both full-length and cleaved OASIS are significantly stabilized by ER stress, with cleaved OASIS enhancing transcriptional activation of its target genes[27]. HIF-1α dimerizes with HIF-1β through its bHLH-PAS domain[49], and binds to the promoter region of target genes such as vascular endothelial growth factor A (VEGFA) to facilitate their expression[40]. It was reported that ER stress potentiated HIF-1α activity to promote transcription of VEGFA35
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