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Subcellular localization of hypoxia-inducible factors and HIF regulatory hydroxylases in rat liver.

Khan, Zahida (2006) Subcellular localization of hypoxia-inducible factors and HIF regulatory hydroxylases in rat liver. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Many signals involved in pathophysiology are controlled by hypoxia-inducible factors (HIFs), transcription factors that induce expression of hypoxia-responsive genes. HIFs are highly conserved master regulators of oxygen homeostasis. These factors are post-translationally regulated by a family of oxygen-dependent HIF hydroxylases, whose members include four prolyl 4-hydroxylases (PHD1-4) and an asparaginyl hydroxylase (FIH-1). All HIF hydroxylases require molecular oxygen, Fe(II), ascorbate, and 2-oxoglutarate as cofactors. We hypothesized that alterations in subcellular localization may provide an additional point of regulation for the HIF pathway in response to hypoxia. Most of these enzymes are abundant in resting liver, an organ which is itself unique due to its physiologic oxygen gradient, and they can exist in both nuclear and cytoplasmic pools. In this study, we analyzed the localization of endogenous HIFs and their regulatory hydroxylases in primary rat hepatocytes cultured under hypoxia-reoxygenation conditions. We observed an absence of nuclear HIF-1á activation in hypoxic hepatocytes, even though several known HIF target genes were upregulated, suggesting that HIF-2á and HIF-3á are the predominant isoforms in liver. We show that in hepatocytes, hypoxia-reoxygenation targets HIF-1á to the peroxisome rather than the nucleus, where it co-localizes with the von Hippel Lindau protein (VHL) and the HIF hydroxylases. Confocal immunofluorescence microscopy demonstrated that the HIF hydroxylases can translocate from the nucleus to the cytoplasm in response to hypoxia, with increased accumulation in peroxisomes upon reoxygenation. These results were confirmed via immuno-transmission electron microscopy and Western blotting. Surprisingly, in resting liver tissue, peri-venous localization of the HIF hydroxylases was detected, consistent with areas of low oxygenation. This was in contrast to nuclear HIF-1á, which was undetectable in a number of liver injury models. In conclusion, these studies establish the peroxisome as a highly relevant site of subcellular localization and function for the endogenous HIF pathway in hepatocytes.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Khan, Zahidazak1@pitt.eduZAK1
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairStolz, Donna Bdstolz@pitt.eduDSTOLZ
Committee MemberMichalopoulos, George Kmichalopoulosgk@upmc.eduMICHAL
Committee MemberDefrances, Marie Cdefrancesmc@upmc.eduMCD14
Committee MemberCheng,
Committee MemberStrom, Stephen Cstrom@pitt.eduSTROM
Date: 29 June 2006
Date Type: Completion
Defense Date: 2 May 2006
Approval Date: 29 June 2006
Submission Date: 16 May 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cellular and Molecular Pathology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: hydroxylase; liver; hepatocyte; hypoxia
Other ID:, etd-05162006-145231
Date Deposited: 10 Nov 2011 19:44
Last Modified: 19 Dec 2016 14:36


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