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GGCX and VKORC1 inhibit osteocalcin endocrine functions.

Ferron M, Lacombe J, Germain A, Oury F, Karsenty G - J. Cell Biol. (2015)

Bottom Line: Although circumstantial evidence suggests that γ-carboxylation may inhibit OCN endocrine functions, genetic evidence that it is the case is still lacking.Here we show using cell-specific gene inactivation models that γ-carboxylation of OCN by GGCX inhibits its endocrine function.This study genetically and biochemically delineates the functions of the enzymes required for OCN modification and demonstrates that it is the uncarboxylated form of OCN that acts as a hormone.

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Affiliation: Unité de recherche en physiologie intégrative et moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada Département de médecine, Département de biochimie et médecine moléculaire, and Programmes de biologie moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada Département de médecine, Département de biochimie et médecine moléculaire, and Programmes de biologie moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada Département de médecine, Département de biochimie et médecine moléculaire, and Programmes de biologie moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, Québec H3A 1A3, Canada mathieu.ferron@ircm.qc.ca gk2172@columbia.edu.

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VKORC1L1 does not compensate for VKORC1 absence in osteoblasts. (A) Serum levels of GLU-OCN in control (n = 12), Vkorc1fl/fl;OC-Cre (n = 4), Vkorc1l1fl/fl;OC-Cre (n = 5), Vkorc1fl/fl;Vkorc1l1+/fl;OC-Cre (n = 5), and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre (n = 6) 2-mo-old mice. (B) Bone OCN content in 2-mo-old mice was assessed from whole bone extracts by Western blotting. Each lane represents an individual animal. (C) Cre-mediated inactivation of VKORC1 and VKORC1L1 in Vkorc1fl/fl;Vkorc1l1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by Western blotting. (D) Percentage of GLA- over total OCN measured in the supernatant of osteoblasts of the indicated genotype cultured in the absence of VK (−VK) or in the presence of VKO (+VKO) or VK1 (+VK1; n = 4 for each condition). (E) Immunofluorescence analyses of mouse osteoblasts. Cells were stained with rabbit antibodies against GGCX (top), VKORC1 (middle), or VKORC1L1 (bottom) and with a mouse anti-KDEL antibody to label the ER. The areas boxed on the merge panels are zoomed in the panels labeled “merge (zoom).” The graphs displayed on the right show the intensity of each of the fluorescent signals (green, red, and blue) in a 50-µm cross-section of each cell (dashed lines). At least 20 individual cells were analyzed for each staining condition, and comparable results were obtained in all cases. Results are given as means ± SEM. ***, P < 0.001.
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fig7: VKORC1L1 does not compensate for VKORC1 absence in osteoblasts. (A) Serum levels of GLU-OCN in control (n = 12), Vkorc1fl/fl;OC-Cre (n = 4), Vkorc1l1fl/fl;OC-Cre (n = 5), Vkorc1fl/fl;Vkorc1l1+/fl;OC-Cre (n = 5), and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre (n = 6) 2-mo-old mice. (B) Bone OCN content in 2-mo-old mice was assessed from whole bone extracts by Western blotting. Each lane represents an individual animal. (C) Cre-mediated inactivation of VKORC1 and VKORC1L1 in Vkorc1fl/fl;Vkorc1l1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by Western blotting. (D) Percentage of GLA- over total OCN measured in the supernatant of osteoblasts of the indicated genotype cultured in the absence of VK (−VK) or in the presence of VKO (+VKO) or VK1 (+VK1; n = 4 for each condition). (E) Immunofluorescence analyses of mouse osteoblasts. Cells were stained with rabbit antibodies against GGCX (top), VKORC1 (middle), or VKORC1L1 (bottom) and with a mouse anti-KDEL antibody to label the ER. The areas boxed on the merge panels are zoomed in the panels labeled “merge (zoom).” The graphs displayed on the right show the intensity of each of the fluorescent signals (green, red, and blue) in a 50-µm cross-section of each cell (dashed lines). At least 20 individual cells were analyzed for each staining condition, and comparable results were obtained in all cases. Results are given as means ± SEM. ***, P < 0.001.

Mentions: Even though VKORC1L1 is not required in vivo or ex vivo for γ-carboxylation of OCN, the possibility remains that there was a functional redundancy between VKORC1 and VKORC1L1 and that VCORC1L1 could have supported VKO or VK1 reduction in the absence of VKORC1 (Fig. 6 A). To determine whether it was indeed the case, we generated mice lacking both Vkorc1 and Vkorc1l1 in osteoblasts and compared circulating OCN levels in those double mutant mice with those of either mice lacking Vkorc1 or Vkorc1l1 in osteoblasts. As shown in Fig. 7 A, deletion of Vkorc1 in combination with either one or two alleles of Vkorc1l1 (i.e., Vkorc1fl/fl;Vkorc1l1fl/+;OC-Cre and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre mice) did not cause any further increase of the circulating levels of GLU-OCN when compared with Vkorc1fl/fl;OC-Cre littermates. Similarly, the bone content of OCN was equally decreased in Vkorc1fl/fl;OC-Cre and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre mice when compared with control or Vkorc1l1fl/fl;OC-Cre mice (Fig. 7 B).


GGCX and VKORC1 inhibit osteocalcin endocrine functions.

Ferron M, Lacombe J, Germain A, Oury F, Karsenty G - J. Cell Biol. (2015)

VKORC1L1 does not compensate for VKORC1 absence in osteoblasts. (A) Serum levels of GLU-OCN in control (n = 12), Vkorc1fl/fl;OC-Cre (n = 4), Vkorc1l1fl/fl;OC-Cre (n = 5), Vkorc1fl/fl;Vkorc1l1+/fl;OC-Cre (n = 5), and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre (n = 6) 2-mo-old mice. (B) Bone OCN content in 2-mo-old mice was assessed from whole bone extracts by Western blotting. Each lane represents an individual animal. (C) Cre-mediated inactivation of VKORC1 and VKORC1L1 in Vkorc1fl/fl;Vkorc1l1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by Western blotting. (D) Percentage of GLA- over total OCN measured in the supernatant of osteoblasts of the indicated genotype cultured in the absence of VK (−VK) or in the presence of VKO (+VKO) or VK1 (+VK1; n = 4 for each condition). (E) Immunofluorescence analyses of mouse osteoblasts. Cells were stained with rabbit antibodies against GGCX (top), VKORC1 (middle), or VKORC1L1 (bottom) and with a mouse anti-KDEL antibody to label the ER. The areas boxed on the merge panels are zoomed in the panels labeled “merge (zoom).” The graphs displayed on the right show the intensity of each of the fluorescent signals (green, red, and blue) in a 50-µm cross-section of each cell (dashed lines). At least 20 individual cells were analyzed for each staining condition, and comparable results were obtained in all cases. Results are given as means ± SEM. ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig7: VKORC1L1 does not compensate for VKORC1 absence in osteoblasts. (A) Serum levels of GLU-OCN in control (n = 12), Vkorc1fl/fl;OC-Cre (n = 4), Vkorc1l1fl/fl;OC-Cre (n = 5), Vkorc1fl/fl;Vkorc1l1+/fl;OC-Cre (n = 5), and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre (n = 6) 2-mo-old mice. (B) Bone OCN content in 2-mo-old mice was assessed from whole bone extracts by Western blotting. Each lane represents an individual animal. (C) Cre-mediated inactivation of VKORC1 and VKORC1L1 in Vkorc1fl/fl;Vkorc1l1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by Western blotting. (D) Percentage of GLA- over total OCN measured in the supernatant of osteoblasts of the indicated genotype cultured in the absence of VK (−VK) or in the presence of VKO (+VKO) or VK1 (+VK1; n = 4 for each condition). (E) Immunofluorescence analyses of mouse osteoblasts. Cells were stained with rabbit antibodies against GGCX (top), VKORC1 (middle), or VKORC1L1 (bottom) and with a mouse anti-KDEL antibody to label the ER. The areas boxed on the merge panels are zoomed in the panels labeled “merge (zoom).” The graphs displayed on the right show the intensity of each of the fluorescent signals (green, red, and blue) in a 50-µm cross-section of each cell (dashed lines). At least 20 individual cells were analyzed for each staining condition, and comparable results were obtained in all cases. Results are given as means ± SEM. ***, P < 0.001.
Mentions: Even though VKORC1L1 is not required in vivo or ex vivo for γ-carboxylation of OCN, the possibility remains that there was a functional redundancy between VKORC1 and VKORC1L1 and that VCORC1L1 could have supported VKO or VK1 reduction in the absence of VKORC1 (Fig. 6 A). To determine whether it was indeed the case, we generated mice lacking both Vkorc1 and Vkorc1l1 in osteoblasts and compared circulating OCN levels in those double mutant mice with those of either mice lacking Vkorc1 or Vkorc1l1 in osteoblasts. As shown in Fig. 7 A, deletion of Vkorc1 in combination with either one or two alleles of Vkorc1l1 (i.e., Vkorc1fl/fl;Vkorc1l1fl/+;OC-Cre and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre mice) did not cause any further increase of the circulating levels of GLU-OCN when compared with Vkorc1fl/fl;OC-Cre littermates. Similarly, the bone content of OCN was equally decreased in Vkorc1fl/fl;OC-Cre and Vkorc1fl/fl;Vkorc1l1fl/fl;OC-Cre mice when compared with control or Vkorc1l1fl/fl;OC-Cre mice (Fig. 7 B).

Bottom Line: Although circumstantial evidence suggests that γ-carboxylation may inhibit OCN endocrine functions, genetic evidence that it is the case is still lacking.Here we show using cell-specific gene inactivation models that γ-carboxylation of OCN by GGCX inhibits its endocrine function.This study genetically and biochemically delineates the functions of the enzymes required for OCN modification and demonstrates that it is the uncarboxylated form of OCN that acts as a hormone.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité de recherche en physiologie intégrative et moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada Département de médecine, Département de biochimie et médecine moléculaire, and Programmes de biologie moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada Département de médecine, Département de biochimie et médecine moléculaire, and Programmes de biologie moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada Département de médecine, Département de biochimie et médecine moléculaire, and Programmes de biologie moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, Québec H3A 1A3, Canada mathieu.ferron@ircm.qc.ca gk2172@columbia.edu.

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Related in: MedlinePlus