The human HIF (hypoxia-inducible factor)-3alpha gene is a HIF-1 target gene and may modulate hypoxic gene induction

Abstract

HIF (hypoxia-inducible factor)-3alpha is the third member of the HIF transcription factor family. Whereas HIF-1alpha and -2alpha play critical roles in the cellular and systemic adaptation to hypoxia, little is known about the regulation and function of HIF-3alpha. At least five different splice variants may be expressed from the human HIF-3alpha locus that are suggested to exert primarily negative regulatory effects on hypoxic gene induction. In the present paper, we report that hypoxia induces the human HIF-3alpha gene at the transcriptional level in a HIF-1-dependent manner. HIF-3alpha2 and HIF-3alpha4 transcripts, the HIF-3alpha splice variants expressed in Caki-1 renal carcinoma cells, rapidly increased after exposure to hypoxia or chemical hypoxia mimetics. siRNA (small interfering RNA)-mediated HIF-alpha knockdown demonstrated that HIF-3alpha is a specific target gene of HIF-1alpha, but is not affected by HIF-2alpha knockdown. In contrast with HIF-1alpha and HIF-2alpha, HIF-3alpha is not regulated at the level of protein stability. HIF-3alpha protein could be detected under normoxia in the cytoplasm and nuclei, but increased under hypoxic conditions. Promoter analyses and chromatin immunoprecipitation experiments localized a functional hypoxia-responsive element 5' to the transcriptional start of HIF-3alpha2. siRNA-mediated knockdown of HIF-3alpha increased transactivation of a HIF-driven reporter construct and mRNA expression of lysyl oxidase. Immunohistochemistry revealed an overlap of HIF-1alpha-positive and HIF-3alpha-positive areas in human renal cell carcinomas. These findings shed light on a novel aspect of HIF-3alpha as a HIF-1 target gene and point to a possible role as a modulator of hypoxic gene induction.