Transcriptional Up-regulation of Inhibitory PAS Domain Protein Gene Expression by Hypoxia-inducible Factor 1 (HIF-1)

Abstract

The inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a dominant negative regulator of hypoxia-inducible transcription factors (HIFs), is potentially implicated in negative regulation of angiogenesis in such tissues as the avascular cornea of the eye. We have previously shown IPAS mRNA expression is up-regulated in hypoxic tissues, which at least in part involves hypoxiadependent alternative splicing of the transcripts from the IPAS/ HIF-3␣ locus. In the present study, we demonstrate that a hypoxia-driven transcriptional mechanism also plays a role in augmentation of IPAS gene expression. Isolation and analyses of the promoter region flanking to the first exon of IPAS gene revealed a functional hypoxia response element at position ؊834 to ؊799, whereas the sequence upstream of the HIF-3␣ first exon scarcely responded to hypoxic stimuli. A transient transfection experiment demonstrated that HIF-1␣ mediates IPAS promoter activation via the functional hypoxia response element under hypoxic conditions and that a constitutively active form of HIF-1␣ is sufficient for induction of the promoter in normoxic cells. Moreover, chromatin immunoprecipitation and electrophoretic mobility shift assays showed binding of the HIF-1 complex to the element in a hypoxia-dependent manner. Taken together, HIF-1 directly up-regulates IPAS gene expression through a mechanism distinct from RNA splicing, providing a further level of negative feedback gene regulation in adaptive responses to hypoxic/ischemic conditions.

Progress on hypoxia-inducible factor-3: Its structure, gene regulation and biological function (Review)

“…Pasanen et al (21) demonstrated that HIF-3α2 and HIF-3α4 are inducible during hypoxia and the inductions require HIF-1α. A similar previous study revealed that HIF-1α binds to the hypoxia response element (HRE) in the IPAS promoter and induces the expression of IPAS (36). However, the stabilized form of HIF-1α does not affect the mRNA expression levels of HIF-3α in zebrafish embryos (35) and 3T3-L1 cells (37).…”

“…At first, HIF-1α binds to the HRE in the IPAS promoter and induces the expression of IPAS (36). Increased levels of IPAS dimerize with HIF-1α protein and inhibits further induction of IPAS (36). In hepatoma cells, ectopic expression of IPAS decreases the expression of vascular endothelial growth factor (VEGF), resulting in reduced tumor growth and decreased tumor vascular density in vivo (27).…”

“…Splice variant IPAS dimerizes with HIF-1α protein and disrupts the interaction between HIF-1α and the HRE of its target genes (36). Makino et al (36) demonstrated that there is a negative feedback mechanism between HIF-1α and IPAS. At first, HIF-1α binds to the HRE in the IPAS promoter and induces the expression of IPAS (36).…”

“…Makino et al (36) demonstrated that there is a negative feedback mechanism between HIF-1α and IPAS. At first, HIF-1α binds to the HRE in the IPAS promoter and induces the expression of IPAS (36). Increased levels of IPAS dimerize with HIF-1α protein and inhibits further induction of IPAS (36).…”

Understanding complexity in the HIF signaling pathway using systems biology and mathematical modeling

“…The IPAS-specific splicing product of the HIF-3 locus is hypoxia-inducible and 8 HIF-1 binds to the hypoxia-responsive cis-element of the IPAS promoter representing a classic negative feedback that restricts HIF-mediated gene expression in hypoxia (Figure 2A) [39,41]. Interestingly, however, the IPAS-specific mRNA splicing was still observed in the absence of the HIF-1-binding site of the IPAS promoter indicating that HIF-independent factors may also be involved in the production of the dominant negative isoform [41]. The uncoupled nature of IPAS expression and splicing suggest additional control mechanisms in this regulation.…”