Abstract
Most clear cell renal cell carcinomas (ccRCCs) have inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL), resulting in the accumulation of hypoxia-inducible factor α-subunits (HIF-α) and their downstream targets. HIF-2α expression is particularly high in ccRCC and is associated with increased ccRCC growth and aggressiveness. In the canonical HIF signaling pathway, HIF-prolyl hydroxylase 3 (PHD3) suppresses HIF-2α protein by post-translational hydroxylation under sufficient oxygen availability. Here, using immunoblotting and immunofluorescence staining, qRT-PCR, and siRNA-mediated gene silencing, we show that unlike in the canonical pathway, PHD3 silencing in ccRCC cells leads to down-regulation of HIF-2α protein and mRNA. Depletion of other PHD family members had no effect on HIF-2α expression, and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2α protein expression. Accordingly, PHD3 knockdown decreased HIF-2α target gene expression in ccRCC cells and expression was restored upon forced HIF-2α expression. The effect of PHD3 depletion was pinpointed to HIF2A mRNA stability. In line with these in vitro results, a strong positive correlation of PHD3 and HIF2A mRNA expression in ccRCC tumors was detected. Our results suggest that in contrast to the known negative regulation of HIF-2α in most cell types, high PHD3 expression in ccRCC cells maintains elevated HIF-2α expression and that of its target genes, which may enhance kidney cancer aggressiveness.
Highlights
Most clear cell renal cell carcinomas have inactivation of the von Hippel–Lindau tumor suppressor protein, resulting in the accumulation of hypoxia-inducible factor ␣-subunits (HIF-␣) and their downstream targets
Characteristic for clear cell renal cell carcinomas (ccRCCs) is the inactivation of von Hippel–Lindau tumor suppressor protein that occurs in the majority of both familial and sporadic ccRCC cases [1]. pVHL functions as an ubiquitin E3 ligase in the proteasomal degradation pathway and the best-known targets of pVHL are the ␣-subunits of the hypoxia-inducible factors (HIFs) that are directed to degradation in oxygen-dependent manner
We have previously shown that prolyl hydroxylase 3 (PHD3) silencing down-regulates crucial glucose metabolism–related proteins such as lactate dehydrogenase A (LDHA) and glucose transporter 1 (GLUT1) in ccRCC cells [14]
Summary
We have previously shown that PHD3 silencing down-regulates crucial glucose metabolism–related proteins such as lactate dehydrogenase A (LDHA) and glucose transporter 1 (GLUT1) in ccRCC cells [14]. No effect on HIF-2␣ protein expression was detected with 8-h exposure to panhydroxylase inhibitor dimethyloxalylglysine (DMOG) in VHL mutated 786-O nor RCC4 cells, suggesting a hydroxylase-independent down-regulation of HIF-2␣ by PHD3 siRNA (Fig. 3B). In line with the metabolic enzymes, a significant down-regulation in well-characterized HIF-2␣ target gene VEGFA mRNA level in both 786-O and RCC4 cell lines with PHD3 knockdown was seen (Fig. 4, D and E). The data show that silencing of PHD3, can lead to down-regulation of HIF2A mRNA expression and to reduction of HIF-2␣ target gene expression in ccRCC cells. PHD3 silencing alone had no or increasing effect on the expression level of cyclin-dependent kinase inhibitors p27, p21 or an apoptosis regulator BAX genes Most importantly, both the control and PHD3-silenced cells responded to actinomycin D treatment. Did not significantly correlate with HIF1A or HIF2A expression (Fig. S4B)
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