Hypoxic regulation of the 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase gene family (PFKFB‐1–4) expression in vivo

Abstract

When oxygen becomes limiting, cells shift primarily to a glycolytic mode for generation of energy. A key regulator of glycolytic £ux is fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric regulator of 6-phosphofructo-1-kinase (PFK-1). The levels of F-2,6-BP are maintained by a family of bifunctional enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB or PFK-2), which have both kinase and phosphatase activities. Each member of the enzyme family is characterized by their phosphatase:kinase activity ratio (K:B) and their tissue-speci¢c expression. Previous work demonstrated that one of the PFK-2 isozyme genes, PFKFB-3, was induced by hypoxia through the hypoxia-inducible factor-1 (HIF-1) pathway. In this study we examined the basal and hypoxic expression of three members of this family in di¡erent organs of mice. Our ¢ndings indicate that all four isozymes (PFKFB-1^4) are responsive to hypoxia in vivo. However, their basal level of expression and hypoxia responsiveness varies in the di¡erent organs studied. Particularly, PFKFB-1 is highly expressed in liver, heart and skeletal muscle, with the highest response to hypoxia found in the testis. PFKFB-2 is mainly expressed in the lungs, brain and heart. However, the highest hypoxia responses are found only in liver and testis. PFKFB-3 has a variable low basal level of expression in all organs, except skeletal muscle, where it is highly expressed. Most importantly, its hypoxia responsiveness is the most ample of all three genes, being strongly induced in the lungs, liver, kidney, brain, heart and testis. Further studies showed that PFKFB-1 and PFKFB-2 were highly responsive to hypoxia mimics such as transition metals, iron chelators and inhibitors of HIF hydroxylases, suggesting that the hypoxia responsiveness of these genes is also regulated by HIF proteins. In summary, our data demonstrate that PFK-2 genes are responsive to hypoxia in vivo, indicating a physiological role in the adaptation of the organism to environmental or localized hypoxia/ischemia.

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase

“…We examined primary lung fibroblasts and the two resultant cell lines from each strain for the presence of PFKFB1-4 mRNA species by multiplex polymerase chain reaction (PCR) and observed simultaneous expression of all four PFKFB mRNAs (PFKFB1-4) in each of the cell populations (Figure 1b and c). This phenomenon has been previously described in certain transformed cells and whole organs (Minchenko et al, 2003) and these data suggest that all four isozymes may be coordinating to set F2,6BP production. We then examined PFKFB3 protein product expression by Western blot analysis and observed an increase in this enzyme with the introduction of LT and a small increase after H-ras V12 introduction (Figure 1d and e).…”

“…Minchenko et al (2003) have demonstrated that hypoxia induces PFKFB3 transcription in various cell types and mouse tissues and that this induction is completely abrogated in mouse embryonic fibroblasts conditionally nullizygous for HIF-1a (Minchenko et al, 2002(Minchenko et al, , 2003. PFKFB3 thus may serve as an essential transcriptional target of HIF-1a and the adaptive response to hypoxia by transformed cells.…”

6-Phosphofructo-2-kinase (pfkfb3) Gene Promoter Contains Hypoxia-inducible Factor-1 Binding Sites Necessary for Transactivation in Response to Hypoxia

“…Sequence analysis revealed the presence of several putative HREs within the Ϫ3566 nucleotides of the human pfkfb3 promoter, which could explain the described effect of hypoxia on the induction of pfkfb3 gene (11,29). Luciferase expression showed that the two longest constructs (PFKFB3/Ϫ3566 and PFKFB3/Ϫ1407) exerted the maximal hypoxic response, pointing out the major contribution of nucleotides over Ϫ1407 bp.…”

Molecular Control of Cardiac Fetal/Neonatal Remodeling

“…The postnatal decrease in cardiac HIF signaling therefore leads to downregulation of cardiac Hand1 expression, in turn leading to upregulation of lipid metabolism and lipid oxidation after birth. Several potential metabolic control targets of HIF signaling have been found in skeletal muscle [23], and the expression of some genes encoding glycolytic enzymes in the heart are directly HIF-regulated [24]. It therefore seems likely that HIF signaling has a pivotal (yet incompletely defined) role in the neonatal cardiac metabolic shift.…”