Protein Kinase C Binding Protein 1 Modulates Hypoxia Inducible Factor 1 Activity

Abstract

During ischemia, the heart elicits robust physiological and molecular changes in response to oxygen deprivation (i.e. hypoxia). Hypoxia-inducible factor 1 (HIF-1) is a conserved transcription factor that directs the cellular response to hypoxia by regulating genes important in glycolysis, angiogenesis, and contractility. Recently, our lab observed that overexpression of the chromatin factor, PRKCBP1, inhibits HIF-1 activity and that Prkcbp1 is upregulated in the hypoxic heart. Previous studies have shown PRKCBP1 recruits histone demethylases to specific enhancer elements, leading to gene repression. The function of PRKCBP1 in the heart is unknown. During ischemia, PRKCBP1 may act in a negative-feedback loop to reduce HIF-1- target gene expression. We hypothesized that decreasing PRKCBP1 would lead to upregulation of HIF-1 target genes. To explore this, we simultaneously induced HIF-1 activity by expressing the active form of HIF-1a and depleted Prkcbp1 mRNA using siRNA in mouse embryonic fibroblasts. Using semi-quantitative PCR, co-transfection of siPRKCBP1 and the HIF-1a construct showed increased expression of two HIF-1 target genes, Ldha and Pfkp, relative to treatment with the HIF-1a construct alone. To determine endogenous PRKCBP1 levels in neonatal hearts, we performed western blotting using a PRKCBP1-specific antibody and detected a faint band at the predicted molecular weight of PRKCBP1. We also tested if ischemia causes upregulation of PRKCBP1 levels in adult heart tissue and found an increased abundance of PRKCBP1 in ischemic regions relative to unaffected regions. Taken together, our results support a potential HIF-1/PRKCBP1 regulatory circuit in the heart and suggests that PRKCBP1 regulates HIF-1 activity through chromatin remodeling.