Limits...
VHL Type 2B gene mutation moderates HIF dosage in vitro and in vivo.

Lee CM, Hickey MM, Sanford CA, McGuire CG, Cowey CL, Simon MC, Rathmell WK - Oncogene (2009)

Bottom Line: Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma.Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation.Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.

ABSTRACT
Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma. Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation. Murine embryonic stem (ES) cells in which the endogenous wild-type Vhl gene was replaced with the representative Type 2B VHL hotspot mutation R167Q (Vhl(2B/2B)) displayed preserved physiological regulation of both HIF factors with slightly greater normoxic dysregulation of HIF-2alpha. Differentiated Vhl(2B/2B)-derived teratomas overexpressed joint HIF targets Vegf and EglN3 but not the HIF-1alpha-specific target Pfk1. Vhl(2B/2B) teratomas additionally displayed a growth advantage over Vhl(-/-)-derived teratomas, suggestive of a tight connection between perturbations in the degree and ratio of HIF-1alpha and HIF-2alpha stabilization and cell growth. Vhl(2B/2B) mice displayed mid-gestational embryonic lethality, whereas adult Vhl(2B/+) mice exhibited susceptibility to carcinogen-promoted renal neoplasia compared with wild-type littermates at 12 months. Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

Show MeSH

Related in: MedlinePlus

Homozygous Type 2B Vhl placentas display subtle vascular defects consistent with a Vhl- phenotype, and corresponding embryos display HIF target dysregulation despite normal morphology. A.-F. Histological analysis of Vhl2B/+ (A.-C.) and Vhl2B/2B (D.-F.) murine placentas, with H&E stain for morphology (A., C.) and immunohistochemistry for pVhl (B., E.) and for the HIF target Vegfa (C., F.) at 10X magnification. Arcs demarcate the maternal decidua (above) and placental (below) tissues. Note the lack of nucleated fetal RBCs (*) in the chorionic villi in Vhl2B/2B (D.) versus Vhl2B/+ (A.) placentas. Scale bars indicate 100μm. G. Quantitative RT-PCR for transcriptional activation of HIF targets Vegf, Glut1, EglN3, and Pfk1 in E9.5 Vhl2B/2B embryos relative to representative Vhl2B/+ littermate. Cycle thresholds were corrected with 18S ribosomal RNA. Error bars indicate SEM. *p<0.05. **p<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2667565&req=5

Figure 5: Homozygous Type 2B Vhl placentas display subtle vascular defects consistent with a Vhl- phenotype, and corresponding embryos display HIF target dysregulation despite normal morphology. A.-F. Histological analysis of Vhl2B/+ (A.-C.) and Vhl2B/2B (D.-F.) murine placentas, with H&E stain for morphology (A., C.) and immunohistochemistry for pVhl (B., E.) and for the HIF target Vegfa (C., F.) at 10X magnification. Arcs demarcate the maternal decidua (above) and placental (below) tissues. Note the lack of nucleated fetal RBCs (*) in the chorionic villi in Vhl2B/2B (D.) versus Vhl2B/+ (A.) placentas. Scale bars indicate 100μm. G. Quantitative RT-PCR for transcriptional activation of HIF targets Vegf, Glut1, EglN3, and Pfk1 in E9.5 Vhl2B/2B embryos relative to representative Vhl2B/+ littermate. Cycle thresholds were corrected with 18S ribosomal RNA. Error bars indicate SEM. *p<0.05. **p<0.001.

Mentions: Vhl-/- embryos display embryonic lethality at E9.5-E12.5 due to an embryonic-origin defect in placental labyrinth vascularization. While the presumptive Vhl-/- labyrinth is normal in histological appearance at E9.5, Vhl-/- allantoic vessels fail to invade the chorionic plate, preventing induction of chorionic plate trophoblast cell differentiation into syncitiotrophoblast cells and later manifesting as an absence of fetal blood spaces in the labyrinth at E10.5 (Gnarra et al., 1997). To visualize whether the 2B mutant Vhl allele acts similarly to the allele in the placenta, we compared wild-type (not shown), Vhl2B/+, and Vhl2B/2B placentas by H&E for morphology and IHC for pVhl and the HIF target Vegfa. By H&E, representative E9.5 Vhl2B/+ (Figure 5A) and Vhl2B/2B (Figure 5D) placentas displayed comparable chorionic villous fold formation and maternal red blood cell content in the spongiotrophoblast layer, but allantoic vessels, demarcated by the presence of nucleated fetal red blood cells (*), invaded the chorionic villi to a lesser extent in the Vhl2B/2B placenta. Despite the reduced Type 2B pVhl levels observed by immunoblot in Vhl2B/2B ES cells, spongiotrophoblast and giant cell pVhl expression was comparable between representative Vhl2B/2B (Figure 5E) and Vhl2B/+ (Figure 5B) placentas by IHC. Finally, consistent with findings in Vhl-/- placentas at E10.5 (Gnarra et al., 1997), Vhl2B/2B placenta (Figure 5F) displayed absent or greatly reduced spongiotrophoblast and giant cell Vegf expression relative to Vhl2B/+ (Figure 5C) by IHC. While perhaps counter-intuitive, evidence supports decreased Vegf expression as a marker of placental failure rather than an indicator of HIF dysregulation in the murine placenta (Voss et al., 2000).


VHL Type 2B gene mutation moderates HIF dosage in vitro and in vivo.

Lee CM, Hickey MM, Sanford CA, McGuire CG, Cowey CL, Simon MC, Rathmell WK - Oncogene (2009)

Homozygous Type 2B Vhl placentas display subtle vascular defects consistent with a Vhl- phenotype, and corresponding embryos display HIF target dysregulation despite normal morphology. A.-F. Histological analysis of Vhl2B/+ (A.-C.) and Vhl2B/2B (D.-F.) murine placentas, with H&E stain for morphology (A., C.) and immunohistochemistry for pVhl (B., E.) and for the HIF target Vegfa (C., F.) at 10X magnification. Arcs demarcate the maternal decidua (above) and placental (below) tissues. Note the lack of nucleated fetal RBCs (*) in the chorionic villi in Vhl2B/2B (D.) versus Vhl2B/+ (A.) placentas. Scale bars indicate 100μm. G. Quantitative RT-PCR for transcriptional activation of HIF targets Vegf, Glut1, EglN3, and Pfk1 in E9.5 Vhl2B/2B embryos relative to representative Vhl2B/+ littermate. Cycle thresholds were corrected with 18S ribosomal RNA. Error bars indicate SEM. *p<0.05. **p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667565&req=5

Figure 5: Homozygous Type 2B Vhl placentas display subtle vascular defects consistent with a Vhl- phenotype, and corresponding embryos display HIF target dysregulation despite normal morphology. A.-F. Histological analysis of Vhl2B/+ (A.-C.) and Vhl2B/2B (D.-F.) murine placentas, with H&E stain for morphology (A., C.) and immunohistochemistry for pVhl (B., E.) and for the HIF target Vegfa (C., F.) at 10X magnification. Arcs demarcate the maternal decidua (above) and placental (below) tissues. Note the lack of nucleated fetal RBCs (*) in the chorionic villi in Vhl2B/2B (D.) versus Vhl2B/+ (A.) placentas. Scale bars indicate 100μm. G. Quantitative RT-PCR for transcriptional activation of HIF targets Vegf, Glut1, EglN3, and Pfk1 in E9.5 Vhl2B/2B embryos relative to representative Vhl2B/+ littermate. Cycle thresholds were corrected with 18S ribosomal RNA. Error bars indicate SEM. *p<0.05. **p<0.001.
Mentions: Vhl-/- embryos display embryonic lethality at E9.5-E12.5 due to an embryonic-origin defect in placental labyrinth vascularization. While the presumptive Vhl-/- labyrinth is normal in histological appearance at E9.5, Vhl-/- allantoic vessels fail to invade the chorionic plate, preventing induction of chorionic plate trophoblast cell differentiation into syncitiotrophoblast cells and later manifesting as an absence of fetal blood spaces in the labyrinth at E10.5 (Gnarra et al., 1997). To visualize whether the 2B mutant Vhl allele acts similarly to the allele in the placenta, we compared wild-type (not shown), Vhl2B/+, and Vhl2B/2B placentas by H&E for morphology and IHC for pVhl and the HIF target Vegfa. By H&E, representative E9.5 Vhl2B/+ (Figure 5A) and Vhl2B/2B (Figure 5D) placentas displayed comparable chorionic villous fold formation and maternal red blood cell content in the spongiotrophoblast layer, but allantoic vessels, demarcated by the presence of nucleated fetal red blood cells (*), invaded the chorionic villi to a lesser extent in the Vhl2B/2B placenta. Despite the reduced Type 2B pVhl levels observed by immunoblot in Vhl2B/2B ES cells, spongiotrophoblast and giant cell pVhl expression was comparable between representative Vhl2B/2B (Figure 5E) and Vhl2B/+ (Figure 5B) placentas by IHC. Finally, consistent with findings in Vhl-/- placentas at E10.5 (Gnarra et al., 1997), Vhl2B/2B placenta (Figure 5F) displayed absent or greatly reduced spongiotrophoblast and giant cell Vegf expression relative to Vhl2B/+ (Figure 5C) by IHC. While perhaps counter-intuitive, evidence supports decreased Vegf expression as a marker of placental failure rather than an indicator of HIF dysregulation in the murine placenta (Voss et al., 2000).

Bottom Line: Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma.Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation.Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.

ABSTRACT
Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma. Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation. Murine embryonic stem (ES) cells in which the endogenous wild-type Vhl gene was replaced with the representative Type 2B VHL hotspot mutation R167Q (Vhl(2B/2B)) displayed preserved physiological regulation of both HIF factors with slightly greater normoxic dysregulation of HIF-2alpha. Differentiated Vhl(2B/2B)-derived teratomas overexpressed joint HIF targets Vegf and EglN3 but not the HIF-1alpha-specific target Pfk1. Vhl(2B/2B) teratomas additionally displayed a growth advantage over Vhl(-/-)-derived teratomas, suggestive of a tight connection between perturbations in the degree and ratio of HIF-1alpha and HIF-2alpha stabilization and cell growth. Vhl(2B/2B) mice displayed mid-gestational embryonic lethality, whereas adult Vhl(2B/+) mice exhibited susceptibility to carcinogen-promoted renal neoplasia compared with wild-type littermates at 12 months. Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

Show MeSH
Related in: MedlinePlus