Peroxisomal Localization of Hypoxia-Inducible Factors and Hypoxia-Inducible Factor Regulatory Hydroxylases in Primary Rat Hepatocytes Exposed to Hypoxia-Reoxygenation

Abstract

Many signals involved in pathophysiology are controlled by hypoxia-inducible factors (HIFs), transcription factors that induce expression of hypoxiaresponsive genes. HIFs are post-translationally regulated by a family of O 2 -dependent HIF hydroxylases: four prolyl 4-hydroxylases and an asparaginyl hydroxylase. Most of these enzymes are abundant in resting liver, which is itself unique because of its physiological O 2 gradient, and they can exist in both nuclear and cytoplasmic pools. In this study, we analyzed the cellular localization of endogenous HIFs and their regulatory hydroxylases in primary rat hepatocytes cultured under hypoxia-reoxygenation conditions. In hepatocytes, hypoxia targeted HIF-1␣ to the peroxisome, rather than the nucleus, where it co-localized with von Hippel-Lindau tumor suppressor protein and the HIF hydroxylases. Confocal immunofluorescence microscopy demonstrated that the HIF hydroxylases translocated from the nucleus to the cytoplasm in response to hypoxia, with increased accumulation in peroxisomes on reoxygenation. These results were confirmed via immunotransmission electron microscopy and Western blotting. Surprisingly, in resting liver tissue, perivenous localization of the HIF hydroxylases was observed, consistent with areas of low pO 2 . In conclusion, these studies establish the peroxisome as a highly relevant site of subcellular localization and function for the endogenous HIF pathway in

Perinecrotic Hypoxia Contributes to Ischemia/Reperfusion-Accelerated Outgrowth of Colorectal Micrometastases

“…43,46,47 It was recently suggested that in the liver, HIF-1␣ targets to the peroxisome rather than the nucleus after hepatic hypoxia/reoxygenation. 28 We found that HIF-1␣ staining was sustained up to 5 days, which is consistent with other reports. 51 We used the geldanamycin analogue 17-DMAG to promote a von Hippel-Lindau-independent degradation of HIF-1␣.…”

Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

“…hypoxia (15). On the other hand, we did observe a nuclear induction of HIF-2␣ in hepatocytes, but this was only following reoxygenation experiments (15). Interestingly, and in contrast to HIF-1␣, the HIF-2␣ isoforms have been reported to negatively regulate BNIP3 expression (33), raising the intriguing possibility that hypoxia-reoxygenation-induced nuclear HIF-2␣ also contributes to the dramatic decrease of BNIP3 levels following reoxygenation.…”

“…Representative Western blot (top) shows BNIP3 protein expression under the same experimental conditions as described above. hypoxia (15). On the other hand, we did observe a nuclear induction of HIF-2␣ in hepatocytes, but this was only following reoxygenation experiments (15).…”

“…Freshly isolated mouse hepatocytes (wild type and iNOS null) plated on coverslips (2 ϫ 10 5 cells/22-mm glass coverslip; BD Biocoat, Bedford, MA) were cultured under normoxic (control) or hypoxic (1% O2) conditions for 6 h. Parallel hypoxic cultures were returned to the normoxic incubator for 18 h of reoxygenation. The cells were then fixed in 2% paraformaldehyde in PBS for 1 h and processed for imaging as described elsewhere (15). For fluorescence labeling, we used a rabbit polyclonal anti-BNIP3 from Abgent (San Diego, CA), a mouse monoclonal anti-ATP synthase from Affinity Bioreagents (Golden, CO; catalog no.…”

“…To investigate the effects of hypoxia and hypoxia-reoxygenation on endogenous BNIP3 protein in hepatocytes, we used an in vitro culture system as previously reported (15). Freshly isolated hepatocytes were plated and allowed to adhere and equilibrate overnight (18 h).…”