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HIF-2alpha downregulation in the absence of functional VHL is not sufficient for renal cell differentiation.

Hughes MD, Kapllani E, Alexander AE, Burk RD, Schoenfeld AR - Cancer Cell Int. (2007)

Bottom Line: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation.These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation.We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Adelphi University, Garden City, NY 11530-0701, USA. hughes@adelphi.edu

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-alpha) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2alpha in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2alpha toward cellular pVHL activities.

Results: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation. However, cellular morphological changes and downregulation of integrins alpha5 and beta1, which were seen upon pVHL replacement, were not faithfully phenocopied by HIF-2alpha reduction. Moreover, fibronectin deposition and expression of renal cell differentiation markers were observed in cells containing replaced pVHL, but not in HIF-2alpha knockdown cells, indicating that these pVHL functions may occur independently of HIF-2alpha downregulation.

Conclusion: These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation. We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

No MeSH data available.


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Knockdown of HIF-2α does not cause renal cell differentiation. (A) Lysates from 786-O cells stably transfected with VHL (VHL +), parental 786-O cells (786-O), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF-2α shRNA 1+2) were immunoblotted for the renal marker leucine aminopeptidase (top panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Leucine aminopeptidase immunoblotting was performed as in (A) using A498 cells lines. (C) HNF-1α immunoblotting was performed on 786-O cell lines, as in (A).
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Figure 6: Knockdown of HIF-2α does not cause renal cell differentiation. (A) Lysates from 786-O cells stably transfected with VHL (VHL +), parental 786-O cells (786-O), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF-2α shRNA 1+2) were immunoblotted for the renal marker leucine aminopeptidase (top panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Leucine aminopeptidase immunoblotting was performed as in (A) using A498 cells lines. (C) HNF-1α immunoblotting was performed on 786-O cell lines, as in (A).

Mentions: VHL expression has been previously shown to direct the expression of cell surface markers of renal cell differentiation in 786-O cells [26]. Prior studies have shown that pVHL-mediated differentiation of 786-O renal cells is dependent on high cell density [26]. Accordingly, 786-O and A498 cell lines were grown for fourteen days post-confluency on plastic tissue culture dishes to allow for differentiation, and immunoblot analyses were performed using antibodies to the renal cell surface marker leucine aminopeptidase, a brush border enzyme found in proximal tubule epithelium [35]. Expression of leucine aminopeptidase was detected in both the 786-O and A498 cells containing ectopically expressed VHL (Figure 6a and 6b). As expected, expression of this differentiation marker was not detected in the parental or control cell lines. Notably, HIF-2α shRNA cell lines also showed no detectable expression of leucine aminopeptidase. Expression of the renal marker HNF-1α was also observed in 786-O cells expressing pVHL, but not in cells with reduced HIF-2α levels (Figure 6c). These results indicate that HIF-2α regulation alone does not cause renal cell differentiation and that the differentiation process in renal cells is dependent on other VHL activities.


HIF-2alpha downregulation in the absence of functional VHL is not sufficient for renal cell differentiation.

Hughes MD, Kapllani E, Alexander AE, Burk RD, Schoenfeld AR - Cancer Cell Int. (2007)

Knockdown of HIF-2α does not cause renal cell differentiation. (A) Lysates from 786-O cells stably transfected with VHL (VHL +), parental 786-O cells (786-O), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF-2α shRNA 1+2) were immunoblotted for the renal marker leucine aminopeptidase (top panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Leucine aminopeptidase immunoblotting was performed as in (A) using A498 cells lines. (C) HNF-1α immunoblotting was performed on 786-O cell lines, as in (A).
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Figure 6: Knockdown of HIF-2α does not cause renal cell differentiation. (A) Lysates from 786-O cells stably transfected with VHL (VHL +), parental 786-O cells (786-O), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF-2α shRNA 1+2) were immunoblotted for the renal marker leucine aminopeptidase (top panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Leucine aminopeptidase immunoblotting was performed as in (A) using A498 cells lines. (C) HNF-1α immunoblotting was performed on 786-O cell lines, as in (A).
Mentions: VHL expression has been previously shown to direct the expression of cell surface markers of renal cell differentiation in 786-O cells [26]. Prior studies have shown that pVHL-mediated differentiation of 786-O renal cells is dependent on high cell density [26]. Accordingly, 786-O and A498 cell lines were grown for fourteen days post-confluency on plastic tissue culture dishes to allow for differentiation, and immunoblot analyses were performed using antibodies to the renal cell surface marker leucine aminopeptidase, a brush border enzyme found in proximal tubule epithelium [35]. Expression of leucine aminopeptidase was detected in both the 786-O and A498 cells containing ectopically expressed VHL (Figure 6a and 6b). As expected, expression of this differentiation marker was not detected in the parental or control cell lines. Notably, HIF-2α shRNA cell lines also showed no detectable expression of leucine aminopeptidase. Expression of the renal marker HNF-1α was also observed in 786-O cells expressing pVHL, but not in cells with reduced HIF-2α levels (Figure 6c). These results indicate that HIF-2α regulation alone does not cause renal cell differentiation and that the differentiation process in renal cells is dependent on other VHL activities.

Bottom Line: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation.These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation.We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Adelphi University, Garden City, NY 11530-0701, USA. hughes@adelphi.edu

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-alpha) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2alpha in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2alpha toward cellular pVHL activities.

Results: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation. However, cellular morphological changes and downregulation of integrins alpha5 and beta1, which were seen upon pVHL replacement, were not faithfully phenocopied by HIF-2alpha reduction. Moreover, fibronectin deposition and expression of renal cell differentiation markers were observed in cells containing replaced pVHL, but not in HIF-2alpha knockdown cells, indicating that these pVHL functions may occur independently of HIF-2alpha downregulation.

Conclusion: These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation. We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

No MeSH data available.


Related in: MedlinePlus