Attenuation of HIF-1 DNA-binding activity limits hypoxia-inducible endothelin-1 expression

Hypoxia-inducible factors (HIFs) locate to HIF-binding sites (HBSs) within the hypoxia-response elements (HREs) of oxygen-regulated genes. Whereas HIF -1 alpha is expressed ubiquitously, HIF-2 alpha is found primarily in the e ndothelium, similar to endothelin-1 (ET-1) and fms-like tyrosine kinase 1 ( Flt-1), the expression of which is controlled by HREs. We identified an uni que sequence alteration in both ET-1 and Flt-1 HBSs not found in other HIF- 1 target genes. implying that these HBSs might cause binding of HIF-2 rathe r than HIF-1. However, electrophoretic mobility shift assays showed HIF-1 a nd HIF-2 DNA complex formation with the unique ET-1 HBS to be about equal. Both DNA-binding and hypoxic activation of reporter genes using the ET-1 HB S was decreased compared with transferrin and erythropoietin HBSs. The Flt- 1 HBS was non-functional when assayed in isolation, suggesting that additio nal factors are required for hypoxic up-regulation via the reported Flt-1 H RE. Interestingly, HIF-1 activity could be restored fully by point-mutating the ET-1 (but not the Flt-1) HBS, suggesting that the wild-type ET-1 HBS a ttenuated the full hypoxic response known from other oxygen-regulated genes . Such a mechanism might serve to limit the expression of this potent vasoc onstrictor in hypoxia.