Abstract:Background: Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene has been linked to hereditary as well as sporadic clear cell renal carcinomas. The product of the VHL gene, pVHL, acts to target hypoxia-inducible factor alpha (HIF-α) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2α in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2α toward cellular pVHL activities.
“…In particular, an analysis of VHL mutants with and without destabilizing effects on HIF regulation demonstrated that pVHL appears to play a role in fibronectin deposition independent of functions regulating HIF stability [39,59]. In support of this prediction, HIF-directed shRNA, which does abrogate cell growth in xenograft form has minimal impact on the development of the extra cellular matrix [60]. In addition to fibronectin matrix formation, pVHL has been found to have a role in actin and vinculin assembly and functions to restrict cellular motility [61].…”
Clear cell renal cell carcinoma (ccRCC) provides a tumor paradigm for the integration of genetics, molecular biology, therapeutic target validation, and the introduction of high impact treatment strategies. Most cases of sporadic as well as familial ccRCC acquire somatic inactivating mutations of the von Hippel-Lindau tumor suppressor gene, VHL. pVHL, VHL gene product and a protein member of the E3 ubiquitin ligase family, acts in normal cells to direct the degradation and clearance of the hypoxia inducible factor (HIFα) transcription factor family, such that in its absence, as in ccRCC, the HIF proteins stabilize, accumulate to supraphysiologic levels, and activate the transcription of genes such as vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF), which contributes substantially to the physiology of the tumor, and has been assessed indirectly as a prognostic factor. Molecularly targeted therapy blocking components of this pathway has been successfully introduced to the clinic with a substantive impact on clinical parameters of RCC. This review will examine the regulation of these molecular pathways in RCC and discuss the impact on the clinical management of patients with RCC.
“…In particular, an analysis of VHL mutants with and without destabilizing effects on HIF regulation demonstrated that pVHL appears to play a role in fibronectin deposition independent of functions regulating HIF stability [39,59]. In support of this prediction, HIF-directed shRNA, which does abrogate cell growth in xenograft form has minimal impact on the development of the extra cellular matrix [60]. In addition to fibronectin matrix formation, pVHL has been found to have a role in actin and vinculin assembly and functions to restrict cellular motility [61].…”
Clear cell renal cell carcinoma (ccRCC) provides a tumor paradigm for the integration of genetics, molecular biology, therapeutic target validation, and the introduction of high impact treatment strategies. Most cases of sporadic as well as familial ccRCC acquire somatic inactivating mutations of the von Hippel-Lindau tumor suppressor gene, VHL. pVHL, VHL gene product and a protein member of the E3 ubiquitin ligase family, acts in normal cells to direct the degradation and clearance of the hypoxia inducible factor (HIFα) transcription factor family, such that in its absence, as in ccRCC, the HIF proteins stabilize, accumulate to supraphysiologic levels, and activate the transcription of genes such as vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF), which contributes substantially to the physiology of the tumor, and has been assessed indirectly as a prognostic factor. Molecularly targeted therapy blocking components of this pathway has been successfully introduced to the clinic with a substantive impact on clinical parameters of RCC. This review will examine the regulation of these molecular pathways in RCC and discuss the impact on the clinical management of patients with RCC.
“…Furthermore, mutation or downregulation of B-Myb results in genome instability (27,48,49). In contrast, pVHL induces cell cycle arrest at the G 0 /G 1 phase (50), and knockdown of HIF-2␣ is sufficient to suppress aneuploidy (50). Based on these data, we hypothesized that pVHL might regulate the cell cycle and genomic instability by inducing the degradation of B-Myb.…”
U biquitin-mediated proteolysis by the 26S proteasome plays an important role in the elimination of short-lived proteins (1), including those involved in cell cycle progression, cellular signaling in response to environmental stress or extracellular ligands, morphogenesis, secretion, DNA repair, and organelle biogenesis (2, 3). The pathway consists of two key steps, namely, the covalent attachment of multiple ubiquitin molecules to a target protein and the degradation of the ubiquitinated protein by the 26S proteasome complex. Several components act in concert to attach ubiquitin to a target protein, including a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein isopeptide ligase (E3). E3 is directly responsible for substrate recognition. On the basis of structural similarity, E3 enzymes are classified into three families: the HECT (homologous to E6-AP COOH terminus) family, the U-box family, and the RING finger-containing protein family.The elongin B and C-Cul2 or Cul5-SOCS box protein (ECS) family belongs to the cullin RING ligase (CRL) superfamily (4). pVHL, the protein product of the von Hippel-Lindau (VHL) tumor suppressor gene, is a member of the ECS family. pVHL forms a complex with elongins B and C, Cul2, and the RING finger protein Rbx1 (5, 6). The CRL2 pVHL complex has ubiquitin ligase activity and targets the hypoxia-inducible factor ␣ (HIF-␣) family of transcription factors (HIF-1 to -3␣) for proteasomal degradation (7). At normal oxygen levels, proline residues in the LXXLAP sequence motif of HIF-␣ proteins are hydroxylated by three prolyl hydroxylases (PHD1 to -3), and an in-depth study revealed that PHD2 is a critical enzyme for the hydroxylation of HIF-1␣ (8, 9). Hydroxylated HIF-␣ is targeted by pVHL for polyubiquitination and proteasomal degradation (10-12). Under conditions of hypoxia (low oxygen level), HIF-␣ is not hydroxylated by PHDs and is therefore not recognized or targeted for degradation by pVHL. The unhydroxylated HIF-␣ dimerizes with constitutively expressed HIF-1, also known as an aryl hydrocarbon receptor nuclear translocator (ARNT), and translocates to the nucleus, where it induces the transcription of downstream target genes, including the genes coding for vascular endothelial growth factor A (VEGFA), solute carrier family 2 member 1 (SLC2A1; also known as GLUT1), and platelet-derived growth factor (PDGF) (13). Loss of functional pVHL protein prevents the O 2 -dependent degradation of HIF-␣, resulting in constitutive expression of HIF-dependent genes and VHL disease, which is characterized by a variety of lesions, including hemangioblastomas, renal clear cell carcinomas, pheochromocytomas, pancreatic islet cell tumors, endolymphatic sac tumors, and papillary cystadenomas of the broad ligament (females) and epididymis (males) (13).Studies showing that heterozygous pVHL ϩ/Ϫ mice are phenotypically normal and VHL Ϫ/Ϫ mice die at embryonic day 10.5 (E10.5) to E12.5 (14), together with the existence of many pVHLinteracting proteins (13), a...
“…Thus, reduction in HIF-2α provided partial protection from UV-mediated apoptosis, indicating that its expression may either promote or be necessary for apoptosis after UV treatment.Figure 3
Reduction of HIF-2α levels leads to protection in UV-triggered apoptosis, but not for apoptosis caused by glucose and serum starvation in 786-O cells. (A) Parental 786-O or those either stably expressing wild-type VHLp19 or stably infected with a control vector (pSuperRetro) or a pool of two HIF-2α shRNAs vectors [21] were grown to confluence and lysed. Cell lysates were equally loaded and separated by SDS-PAGE.…”
BackgroundGermline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to clear cell renal carcinomas, hemangioblastomas, and pheochromocytomas. The VHL gene product forms an ubiquitin E3 ligase complex, with regulation of hypoxia-inducible factor alpha (HIF-α) as its best known function. Lack of VHL expression has been shown previously to sensitize renal cells to apoptosis caused by certain cellular stresses. In this report, the role of HIF-α in apoptosis was investigated using two parent VHL-null renal carcinoma cell lines.Methods786-O and RCC10 renal carcinoma cell lines with manipulated levels of VHL, HIF-1α, or HIF-2α were subjected to cellular stresses and analyzed by western blotting for the abundance of apoptotic markers.ResultsCell lines expressing mutant VHL proteins that were unable to regulate HIF-α had increased levels of apoptosis when irradiated with ultraviolet (UV) light. The influences of the two major isoforms of HIF-α, HIF-1α and HIF-2α, on apoptosis, were compared by creating cell lines in which levels of each isoform were modulated via short hairpin RNA interference. In UV-irradiated cells, HIF-2α expression was determined to promote apoptosis, whereas HIF-1α was anti-apoptotic. In cells deprived of either glucose or serum, HIF-1α expression was generally anti-apoptotic, while HIF-2α expression was observed to either promote apoptosis or have less of an influence on apoptosis, depending on the cell line used.ConclusionsHIF-1α and HIF-2α exerted distinct effects in each of the conditions tested, with expression of HIF-1α largely blocking apoptosis and HIF-2α generally promoting apoptosis. These results reinforce that HIF-1α and HIF-2α have distinct biological roles and that their relative expression levels may influence some therapeutic interventions that are dependent on apoptosis.
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