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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.


Related in: MedlinePlus

Successful knockdown of HIF-2α in both 786-O and A498 renal cell lines. (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 with retroviruses encoding single HIF-2α shRNAs (HIF2α shRNA 1; HIF2α shRNA 2) or doubly infected with both shRNAs (HIF2α shRNA 1+2) were immunoblotted for HIF-2α (top panel), GLUT1 (upper middle panel) and VHL (lower middle panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Lysates from A498 cells stably transfected with VHL (VHL +), parental A498 cells (A498), and A498 cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were immunoblotted as in (A).
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Figure 1: Successful knockdown of HIF-2α in both 786-O and A498 renal cell lines. (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 with retroviruses encoding single HIF-2α shRNAs (HIF2α shRNA 1; HIF2α shRNA 2) or doubly infected with both shRNAs (HIF2α shRNA 1+2) were immunoblotted for HIF-2α (top panel), GLUT1 (upper middle panel) and VHL (lower middle panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Lysates from A498 cells stably transfected with VHL (VHL +), parental A498 cells (A498), and A498 cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were immunoblotted as in (A).

Mentions: We initially infected 786-O cells with single HIF-2α shRNA retroviruses and selected to obtain a pool of puromycin resistant cells. However, incomplete knockdown of HIF-2α levels was observed with either of the single shRNA constructs (Figure 1a, upper panel). To obtain a more robust downregulation of HIF-2α, 786-O cells were infected in succession with both HIF-2α shRNA-containing retroviruses. HIF-2α inhibition was observed in the doubly infected cells that mimicked the steady state levels seen in the presence of VHL under normoxia. In parallel, A498 cells were infected with the two shRNA retroviruses, resulting in efficient knockdown of HIF-2α (Figure 1b, upper panel). To ensure that the shRNA was not only diminishing levels of HIF-2α, but was also causing downregulation of HIF target genes, immunoblot analysis was performed for the GLUT1 protein. As anticipated, downregulation of GLUT1 coincided with downregulation of HIF-2α (Figure 1a, upper middle panel; Figure 1b, upper middle panel). Thus, in both 786-O and A498 cell lines, shRNA directed at HIF-2α lowered levels of both HIF-2α and a representative HIF-responsive target.


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)

Successful knockdown of HIF-2α in both 786-O and A498 renal cell lines. (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 with retroviruses encoding single HIF-2α shRNAs (HIF2α shRNA 1; HIF2α shRNA 2) or doubly infected with both shRNAs (HIF2α shRNA 1+2) were immunoblotted for HIF-2α (top panel), GLUT1 (upper middle panel) and VHL (lower middle panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Lysates from A498 cells stably transfected with VHL (VHL +), parental A498 cells (A498), and A498 cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were immunoblotted as in (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919349&req=5

Figure 1: Successful knockdown of HIF-2α in both 786-O and A498 renal cell lines. (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 with retroviruses encoding single HIF-2α shRNAs (HIF2α shRNA 1; HIF2α shRNA 2) or doubly infected with both shRNAs (HIF2α shRNA 1+2) were immunoblotted for HIF-2α (top panel), GLUT1 (upper middle panel) and VHL (lower middle panel). Bottom panel is an alpha tubulin immunoblot to demonstrate equal protein loading (25 μg) in each lane. (B) Lysates from A498 cells stably transfected with VHL (VHL +), parental A498 cells (A498), and A498 cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were immunoblotted as in (A).
Mentions: We initially infected 786-O cells with single HIF-2α shRNA retroviruses and selected to obtain a pool of puromycin resistant cells. However, incomplete knockdown of HIF-2α levels was observed with either of the single shRNA constructs (Figure 1a, upper panel). To obtain a more robust downregulation of HIF-2α, 786-O cells were infected in succession with both HIF-2α shRNA-containing retroviruses. HIF-2α inhibition was observed in the doubly infected cells that mimicked the steady state levels seen in the presence of VHL under normoxia. In parallel, A498 cells were infected with the two shRNA retroviruses, resulting in efficient knockdown of HIF-2α (Figure 1b, upper panel). To ensure that the shRNA was not only diminishing levels of HIF-2α, but was also causing downregulation of HIF target genes, immunoblot analysis was performed for the GLUT1 protein. As anticipated, downregulation of GLUT1 coincided with downregulation of HIF-2α (Figure 1a, upper middle panel; Figure 1b, upper middle panel). Thus, in both 786-O and A498 cell lines, shRNA directed at HIF-2α lowered levels of both HIF-2α and a representative HIF-responsive target.

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