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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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Efficient reduction of OCN γ-carboxylation in Vkorc1fl/fl;OC-Cre mice. (A) Hypothetical role of VKORC1 in regulating OCN γ-carboxylation osteoblasts. (B) Detection of the deletion allele (ΔPCR) of Vkorc1 by PCR on genomic DNA isolated from tissues of Vkorc1fl/fl;OC-Cre and Vkorc1fl/fl mice. PCR for the floxed allele (3′lox PCR) was used as a loading control. WAT and BAT, white and brown adipose tissue, respectively. (C) Western blotting analyses of VKORC1 expression in Vkorc1fl/fl and Vkorc1fl/fl;OC-Cre bone marrow–derived osteoblasts. (D and E) Serum levels of GLA- and total OCN (D) and of GLU-OCN (E) in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 5) 2-mo-old mice. (F) Bone OCN content in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 4) 3-mo-old mice was assessed from whole bone extracts by Western blotting (left) or by ELISA and normalized to the total protein content (right). (G) Cre-mediated inactivation of VKORC1 in Vkorc1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by QPCR (left; n = 4 per group) or by Western blotting (right). (H) 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). (I) GTTs in Vkorc1fl/fl (n = 7) and Vkorc1fl/fl;OC-Cre (n = 7) 3-mo-old mice fed an ND. Mice were fasted for 16 h and injected i.p. with 2 g/kg glucose. Results are given as means ± SEM. ***, P < 0.001.
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fig5: Efficient reduction of OCN γ-carboxylation in Vkorc1fl/fl;OC-Cre mice. (A) Hypothetical role of VKORC1 in regulating OCN γ-carboxylation osteoblasts. (B) Detection of the deletion allele (ΔPCR) of Vkorc1 by PCR on genomic DNA isolated from tissues of Vkorc1fl/fl;OC-Cre and Vkorc1fl/fl mice. PCR for the floxed allele (3′lox PCR) was used as a loading control. WAT and BAT, white and brown adipose tissue, respectively. (C) Western blotting analyses of VKORC1 expression in Vkorc1fl/fl and Vkorc1fl/fl;OC-Cre bone marrow–derived osteoblasts. (D and E) Serum levels of GLA- and total OCN (D) and of GLU-OCN (E) in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 5) 2-mo-old mice. (F) Bone OCN content in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 4) 3-mo-old mice was assessed from whole bone extracts by Western blotting (left) or by ELISA and normalized to the total protein content (right). (G) Cre-mediated inactivation of VKORC1 in Vkorc1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by QPCR (left; n = 4 per group) or by Western blotting (right). (H) 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). (I) GTTs in Vkorc1fl/fl (n = 7) and Vkorc1fl/fl;OC-Cre (n = 7) 3-mo-old mice fed an ND. Mice were fasted for 16 h and injected i.p. with 2 g/kg glucose. Results are given as means ± SEM. ***, P < 0.001.

Mentions: VKORC1 is the enzyme responsible for reducing VKO in hepatocytes, thereby allowing the γ-carboxylation of various coagulation factors (Li et al., 2004; Rost et al., 2004). To test whether VKORC1 contributes to OCN γ-carboxylation (Fig. 5 A), mice harboring a floxed allele of Vkorc1 (Fig. S3, A–C) were first bred with Cmv-Cre mice (Schwenk et al., 1995) to inactivate Vkorc1 in germ cells and to obtain mice carrying a allele of this gene (Vkorc1+/−; Fig. S3 D). When Vkorc1+/− mice were intercrossed, no Vkorc1−/− pups were retrieved at postnatal day 14 (P14; Fig. S3 E). A careful monitoring revealed that Vkorc1−/− mice were born at the expected Mendelian ratio but died within 2–5 d of intra-abdominal hemorrhages (Fig. S3, E and F). These results, which confirm a previous study on global Vkorc1 deletion (Spohn et al., 2009), established that an efficient ablation of VKORC1 activity occurred in Vkorc1−/− mice in vivo. Western blot analysis also confirmed the absence of Vkorc1 protein in Vkorc1−/− liver (Fig. S3 G).


GGCX and VKORC1 inhibit osteocalcin endocrine functions.

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

Efficient reduction of OCN γ-carboxylation in Vkorc1fl/fl;OC-Cre mice. (A) Hypothetical role of VKORC1 in regulating OCN γ-carboxylation osteoblasts. (B) Detection of the deletion allele (ΔPCR) of Vkorc1 by PCR on genomic DNA isolated from tissues of Vkorc1fl/fl;OC-Cre and Vkorc1fl/fl mice. PCR for the floxed allele (3′lox PCR) was used as a loading control. WAT and BAT, white and brown adipose tissue, respectively. (C) Western blotting analyses of VKORC1 expression in Vkorc1fl/fl and Vkorc1fl/fl;OC-Cre bone marrow–derived osteoblasts. (D and E) Serum levels of GLA- and total OCN (D) and of GLU-OCN (E) in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 5) 2-mo-old mice. (F) Bone OCN content in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 4) 3-mo-old mice was assessed from whole bone extracts by Western blotting (left) or by ELISA and normalized to the total protein content (right). (G) Cre-mediated inactivation of VKORC1 in Vkorc1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by QPCR (left; n = 4 per group) or by Western blotting (right). (H) 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). (I) GTTs in Vkorc1fl/fl (n = 7) and Vkorc1fl/fl;OC-Cre (n = 7) 3-mo-old mice fed an ND. Mice were fasted for 16 h and injected i.p. with 2 g/kg glucose. Results are given as means ± SEM. ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Efficient reduction of OCN γ-carboxylation in Vkorc1fl/fl;OC-Cre mice. (A) Hypothetical role of VKORC1 in regulating OCN γ-carboxylation osteoblasts. (B) Detection of the deletion allele (ΔPCR) of Vkorc1 by PCR on genomic DNA isolated from tissues of Vkorc1fl/fl;OC-Cre and Vkorc1fl/fl mice. PCR for the floxed allele (3′lox PCR) was used as a loading control. WAT and BAT, white and brown adipose tissue, respectively. (C) Western blotting analyses of VKORC1 expression in Vkorc1fl/fl and Vkorc1fl/fl;OC-Cre bone marrow–derived osteoblasts. (D and E) Serum levels of GLA- and total OCN (D) and of GLU-OCN (E) in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 5) 2-mo-old mice. (F) Bone OCN content in Vkorc1fl/fl (n = 5) and Vkorc1fl/fl;OC-Cre (n = 4) 3-mo-old mice was assessed from whole bone extracts by Western blotting (left) or by ELISA and normalized to the total protein content (right). (G) Cre-mediated inactivation of VKORC1 in Vkorc1fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by QPCR (left; n = 4 per group) or by Western blotting (right). (H) 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). (I) GTTs in Vkorc1fl/fl (n = 7) and Vkorc1fl/fl;OC-Cre (n = 7) 3-mo-old mice fed an ND. Mice were fasted for 16 h and injected i.p. with 2 g/kg glucose. Results are given as means ± SEM. ***, P < 0.001.
Mentions: VKORC1 is the enzyme responsible for reducing VKO in hepatocytes, thereby allowing the γ-carboxylation of various coagulation factors (Li et al., 2004; Rost et al., 2004). To test whether VKORC1 contributes to OCN γ-carboxylation (Fig. 5 A), mice harboring a floxed allele of Vkorc1 (Fig. S3, A–C) were first bred with Cmv-Cre mice (Schwenk et al., 1995) to inactivate Vkorc1 in germ cells and to obtain mice carrying a allele of this gene (Vkorc1+/−; Fig. S3 D). When Vkorc1+/− mice were intercrossed, no Vkorc1−/− pups were retrieved at postnatal day 14 (P14; Fig. S3 E). A careful monitoring revealed that Vkorc1−/− mice were born at the expected Mendelian ratio but died within 2–5 d of intra-abdominal hemorrhages (Fig. S3, E and F). These results, which confirm a previous study on global Vkorc1 deletion (Spohn et al., 2009), established that an efficient ablation of VKORC1 activity occurred in Vkorc1−/− mice in vivo. Western blot analysis also confirmed the absence of Vkorc1 protein in Vkorc1−/− liver (Fig. S3 G).

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