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The function of hypoxia-inducible factor (HIF) is independent of the endoplasmic reticulum protein OS-9.

ABSTRACT: The protein "amplified in osteosarcoma-9" (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the ?-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-?. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET) showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization.

SUBMITTER: Brockmeier U

PROVIDER: S-EPMC3084761 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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The function of hypoxia-inducible factor (HIF) is independent of the endoplasmic reticulum protein OS-9.

Brockmeier Ulf U Platzek Corinna C Schneider Kirsten K Patak Pauline P Bernardini André A Fandrey Joachim J Metzen Eric E

PloS one 20110429 4

The protein "amplified in osteosarcoma-9" (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of P ...[more]

PMID: 21559462

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Project description:Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor, plays a critical role in adaption to hypoxia, which is a major feature of diseases, including cancer. Protein disulfide isomerase (PDI) is up-regulated in numerous cancers and leads to cancer progression. PDI, a member of the TRX superfamily, regulates the transcriptional activities of several transcription factors. To investigate the mechanisms by which PDI affects the function of HIF-1alpha, the overexpression or knockdown of PDI was performed. The overexpression of PDI decreased HIF-1alpha expression in the human hepatocarcinoma cell line, Hep3B, whereas the knockdown of endogenous PDI increased its expression. NH4Cl inhibited the decrease in HIF-1alpha expression by PDI overexpression, suggesting that HIF-1alpha was degraded by the lysosomal pathway. HIF-1alpha is transferred to lysosomal membranes by heat shock cognate 70 kDa protein (HSC70). The knockdown of HSC70 abolished the decrease, and PDI facilitated the interaction between HIF-1alpha and HSC70. HIF-1alpha directly interacted with PDI. PDI exists not only in the endoplasmic reticulum (ER), but also in the cytosol. Hypoxia increased cytosolic PDI. We also investigated changes in the redox state of HIF-1alpha using PEG-maleimide, which binds to thiols synthesized from disulfide bonds by reduction. An up-shift in the HIF-1alpha band by the overexpression of PDI was detected, suggesting that PDI formed disulfide bond in HIF-1alpha. HIF-1alpha oxidized by PDI was not degraded in HSC70-knockdown cells, indicating that the formation of disulfide bond in HIF-1alpha was important for decreases in HIF-1alpha expression. To the best of our knowledge, this is the first study to show the regulation of the expression and redox state of HIF-1alpha by PDI. We also demonstrated that PDI formed disulfide bonds in HIF-1alpha 1-245 aa and decreased its expression. In conclusion, the present results showed that PDI is a novel factor regulating HIF-1alpha through lysosome-dependent degradation by changes in its redox state.

Dataset Information

The function of hypoxia-inducible factor (HIF) is independent of the endoplasmic reticulum protein OS-9.

Publications

The function of hypoxia-inducible factor (HIF) is independent of the endoplasmic reticulum protein OS-9.

Similar Datasets

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