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HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma.

ABSTRACT: BACKGROUND:A key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-?, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not clear. METHODS:To gain insight into the functions of high PHD3 expression in ccRCC, we used PHD3 knockdown by siRNA in 786-O cells under normoxic and hypoxic conditions and performed discovery mass spectrometry (LC-MS/MS) of the purified peptide samples. The LC-MS/MS results were analysed by label-free quantification of proteome data using a peptide-level expression-change averaging procedure and subsequent gene ontology enrichment analysis. RESULTS:Our data reveals an intriguingly widespread effect of PHD3 knockdown with 91 significantly regulated proteins. Under hypoxia, the response to PHD3 silencing was wider than under normoxia illustrated by both the number of regulated proteins and by the range of protein expression levels. The main cellular functions regulated by PHD3 expression were glucose metabolism, protein translation and messenger RNA (mRNA) processing. PHD3 silencing led to downregulation of most glycolytic enzymes from glucose transport to lactate production supported by the reduction in extracellular acidification and lactate production and increase in cellular oxygen consumption rate. Moreover, upregulation of mRNA processing-related proteins and alteration in a number of ribosomal proteins was seen as a response to PHD3 silencing. Further studies on upstream effectors of the translational machinery revealed a possible role for PHD3 in regulation of mTOR pathway signalling. CONCLUSIONS:Our findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.

SUBMITTER: Miikkulainen P

PROVIDER: S-EPMC5496173 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma.

Miikkulainen Petra P Högel Heidi H Rantanen Krista K Suomi Tomi T Kouvonen Petri P Elo Laura L LL Jaakkola Panu M PM

Cancer & metabolism 20170704

<h4>Background</h4>A key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-α, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not cl ...[more]

PMID: 28680592

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Project description:Introduction: We previously reported that malignant hypertension is associated with impaired capillary density of target organs. Here, we tested the hypothesis that stabilization of hypoxia-inducible factor (HIF) in a modified "preconditioning" approach prevents the development of malignant hypertension. To stabilize HIF, we employed pharmacological inhibition of HIF prolyl hydroxylases (PHD), that profoundly affect HIF metabolism. Methods: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in rats; controls were sham operated. 2K1C rats received either intermittent injections of the PHD inhibitor ICA (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate) or placebo. Thirty-five days after clipping, the frequency of malignant hypertension was assessed (based on weight loss and the occurrence of characteristic vascular lesions). In addition, kidney injury was compared between all ICA treated versus all placebo treated 2K1C, regardless of the occurrence of malignant hypertension. HIF stabilization was evaluated by immunohistochemistry, and HIF target gene expression by RT-PCR. Results: Blood pressure was elevated to the same degree in ICA- and placebo-treated 2K1C compared to control rats. ICA treatment did not affect the frequency of malignant hypertension or the extent of kidney tissue fibrosis, inflammation, or capillary density. There was a trend towards higher mortality and worse kidney function in ICA-treated 2K1C rats. ICA increased the number of HIF-1α-positive renal tubular cell nuclei and induced several HIF-1 target genes. In contrast, expression of HIF-2α protein as well as HIF-2 target genes were markedly enhanced by 2K1C hypertension, irrespective of ICA treatment. Discussion: We conclude that intermittent PHD inhibition did not ameliorate severe renovascular hypertension in rats. We speculate that the unexpected strong renal accumulation of HIF-2α in renovascular hypertension, which could not be further augmented by ICA, may contribute to the lack of a benefit from PHD inhibition.

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HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma.

Publications

HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma.

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