Hypoxia-inducible factor - Essay Example

Hypoxia-inducible factor (HIF) induces the expression of prolyl domain proteins, PHD2 and PHD3, during hypoxic conditions Response to low oxygen conditions involves the action of hypoxia inducible factor (HIF). Under normal conditions, HIF is inactive and is regulated by binding of the prolyl hydroxylase containing domain proteins (PHD). Results of this study showed that PHD gene expression during hypoxia was significantly increased, and appears to be regulated by the presence of active HIF. The mechanism appeared not to be due to histone acetylation but by binding of HIF to conserved DNA sequences of the hypoxia response element binding sites on a 5-kb genomic DNA strand that codes for PHD. More studies that measure the direct regulatory effect of HIF on PHD gene expression are recommended. Introduction Oxygen is an essential component of life. Hypoxia is a condition where physiologic oxygen levels fall lower than the normal, which can result in stroke, brain injury, spinal cord injury, other neurodegenerative diseases, and cancer. Because of the importance of oxygen for life, organisms have developed mechanisms to cope and survive low oxygen levels (hypoxia). During hypoxia, cells adapt by altering the expression of many genes: those involved in maintaining oxygen homeostasis, coping with reactive oxygen species and other effects of low oxygen stress. Many of these genes are directly regulated by the hypoxia-inducible transcription factor (HIF; with common isoforms: HIF-1 and HIF-2). When oxygen levels are normal (normoxia), HIF is barely discernible; under hypoxia the HIF concentration increases dramatically. The active form of HIF is composed of two sub-units, HIF α, and HIF β. The latter is constitutively expressed regardless of physiologic oxygen concentration, while HIF α concentration is very low under normoxic conditions but increases with hypoxia. In normoxia, HIF α is hydroxylated by through the action of prolyl hydroxylases domain proteins or PHD. Hydroxylation allows HIF α to associate with a protein complex that makes it a target for proteolytic degradation. The proof of the inhibitory effect of PHD on HIF was established when silencing of PHD2 gene increased normoxic HIF levels (Berraet al., 2003). Silencing of other identified PHD, 1 and 3, did not affect normoxic levels of HIF, leading to the conclusion that the PHDs have different roles in vivo (Berra et al., 2003). Furthermore, hypoxia alters the induced PHD expression thereby affecting their relative abundances and inhibitory effects on HIF (Appelhoff et al., 2004). Under hypoxic conditions; HIF α is not hydroxylated, escapes proteolysis, and readily forms the HIF α/β heterodimer. HIF recruits the p300/CBP co-activator to form a transcriptionally active complex that binds the hypoxia response elements (HRE) in the promoter region of HIF-regulated genes. Studies have shown that HIF-1 may induce the expression of PHD genes using a negative feedback loop mechanism (Marxsen et al., 2004; Qutub & Popel, 2007). The dysfunction of HIF-regulated pathways has significant implications in the development of diseases including strokes and cancer progression. Hence it is important to characterize the HIF-regulating pathway to understand and treat resulting diseases. Therefore the aim of this study is to look at the role of HIF-1 in regulating PHD2 and PHD3 gene expression under hypoxic conditions in SH-SY5Y neuroblastoma cells. Furthermore, the study also investigates the role of acetylation in regulating gene expression. To show evidence that HIF protein binds PHD3 gene regions, a structural analysis was conducted on the DNA sequence coding for the PHD3 gene. Results The results of the Western blot analysis for HIF levels of SH-SY5Y cells under normoxic and hypoxic conditions are shown in Figure 1. HIF level under normal physiologic oxygen concentration was very low, while the large blot of protein obtained from hypoxic cells shows that the concentration of HIF was very highly increased. Hypoxia also increased the gene expression of PHD2 and PHD3 relative to the control level at zero time after hypoxia was induced (Figure 2). PHD2 gene expression already showed an indication of an increase at 0.5 hours, although this appears to be statistically similar to the increase at 0 and 1 hour after cobalt chloride treatment. The high deviation is typical for gene expression studies using real-time PCR analysis. Significantly higher gene expression occurred at 2 hours, which further increased at 4 hours of hypoxia (Figure 2A). The increase in PHD3 gene expression in hypoxic cells was also observed at 2 hours after induction. However, compared to PHD2, the increase of PHD3 gene expression was very high, averaging at 1200 % over the control (Figure 2B). This is similar to results of similar studies where PHD3 expression was higher than that of PHD2 (Marxsen et al., 2004) (DAngelo et al., 2003). Differential PHD expression has been observed in the different tissues (Stiehl et al., 2006). Figure 2. Time induction of PHD2(A) and PHD3 (B)gene expression after induced hypoxia in SH-SY5Y cells. Trichostatin A (TSA) is a potent inhibitor of histone deacetylases. In its presence, acetylated structures are maintained. Cells that were previously exposed to CoCl2 showed increase in both PHD2 and PHD3 gene expression, but the expression decreased starting at 12 hours after addition of either methanol or 300 nM TSA. Similarly, cells under normoxic conditions did not exhibit changes in PHD gene expression levels (Figure 3). Figure 3. Expressionof PHD2 and PHD3 induction in hypoxia and normoxia in response to Trichostatin A (TSA) treatment. An analysis of the gene sequence of the DNA that encodes for PHD3 showed that the presence of conserved HRE sequences at +12588 and +17379 bases downstream of the 5’ terminal of the 5 kb nucleotide stretch (Figure 4). Figure 4: Potential HRE sites in human PHD3 gene Discussion HIF-1 protein levels were shown to increase under hypoxic conditions. The results are similar to those obtained by previous research (Marxsen et al., 2004). Under normoxic conditions, HIF-1α has two hydroxylated proline residues, Pro-564 and Pro-402, in its oxygen-dependent degradation domain. This structure associates freely with the pVHL E3 ligase complex, which signals HIF-1α’s degradation to the ubiquitin-proteasome pathway. Reduction in the oxygen levels inhibits hydroxylation, and stabilizes the HIF-1α subunit concentration. This leads to the dimerization of the HIF protein sub-units to form the functionally active HIF-1 transcription factor (reviewed by Ke & Costa, 2006).Three autocrine feedback loops, prolyl hydroxylase, succinate levels and HIF-1α, were shown to regulate the concentration of HIF-1α and determine the response to hypoxia (Qutub & Popel, 2007). Hypoxia was shown to induce large increases in PHD2 and PHD3 gene expression similar to results obtained by Marxsen et al. (2004) and Qutub and Popel (2007). The increase in PHD levels under hypoxia was first observed in assays for the three PHD isoforms in rats (DAngelo et al., 2003). Hypoxia initially reduced PHD levels but after a certain period there was selective increase in PHD2 mRNA. This increase in PHD was associated with the rapid hydroxylation of Pro-564 and degradation of HIF α during reoxygenation, proposing a role for hypoxic preconditioning of the cells. PHD expression can also be induced in vivo in a tissue specific manner. PHDs have high Km values for oxygen, making them optimal sensors for physiological oxygen levels. PHDs function even under oxygen deprived conditions which allow for HIF to adapt. The autoregulatory oxygen-sensing system of PHDs explained how one mechanism works in differently oxygenated tissues (Stiehl et al., 2006). PHD gene expression levels were lost in cells where HIF-1α expression was silenced by RNA interference (Marxsen et al., 2004) suggesting the dependence of hypoxic PHD expression on HIF α. The increase in PHD gene expression limits HIF α accumulation during hypoxia and accelerates its rapid degradation upon reoxygenation (Marxsen, et al., 2004). This response is critical in controlling the levels of HIF which has been implicated in the progression of diseases like cancer and stroke (Greijer & van der Wall, 2004; Erler et al., 2004). The role of acetylation in increasing PHD gene expression during hypoxia was studied. The results show that acetylation was not the mechanism underlying high PHD levels because the addition of the deacetylase inhibitor TSA was not effective in retaining the achieved expression after the cells were reoxygenated. Acetylation has also been proposed as a mechanism for increased HIF α level during hypoxia. This process was mediated by ARD1, a mammalian acetyltransferase that directly binds and stabilizes HIF-1 (Jeong et al., 2002). Results of this study were refuted in another study which showed that ARD1 mRNA and protein levels were not affected by hypoxia, are expressed in a number of cells, and has no impact on HIF signaling (Bilton et al., 2005). Furthermore, the role of acetylation in the degradation of HIF-1α has been questioned after a review of the current literature (Arnesen, 2006). Sequence analysis for the genomic DNA sequence coding for PHD3 shows the presence of conserved sequences for HRE. This result presents the mechanism that binding by HIF on the PHD gene regulates its expression during hypoxia (Fong & Takeda, 2008). Increased abundance of PHD2 and PHD3 during hypoxia, in turn, prevents the accumulation of HIF α (Marxsen, et al., 2004; DAngelo et al., 2003). Conclusion and Recommendations This study was able to prove that hypoxia induces increased HIF levels and PHD gene expression. It also showed that the possible mechanism for the HIF-mediated increase in PHD expression was due to HIF binding to the HRE sites on the PHD gene. This shows that a HIF regulates PHD expression via a feedback regulatory loop. However, the data only provided baseline information on the effects of hypoxia on HIF and PHD, and sequence for the PHD gene-coding region of the DNA. No measurement was performed on the direct action of HIF on PHD gene expression, and protein-DNA interactions. It is recommended that succeeding studies should be conducted on these aspects. 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