GGCX and VKORC1 inhibit osteocalcin endocrine functions

Abstract

Osteocalcin (OCN) is an osteoblast-derived hormone favoring glucose homeostasis, energy expenditure, male fertility, brain development, and cognition. Before being secreted by osteoblasts in the bone extracellular matrix, OCN is γ-carboxylated by the γ-carboxylase (GGCX) on three glutamic acid residues, a cellular process requiring reduction of vitamin K (VK) by a second enzyme, a reductase called VKORC1. 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. We further show that VKORC1 is required for OCN γ-carboxylation in osteoblasts, whereas its paralogue, VKORC1L1, is dispensable for this function and cannot compensate for the absence of VKORC1 in osteoblasts. 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.

Figure 1.

Partial reduction of OCN γ-carboxylation in Ggcx^fl/fl;Col1a1-Cre mice. (A) Cre-mediated inactivation of GGCX in Ggcx^fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by QPCR (left; n = 4 per group) or by Western blotting (right). (B) 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). (C) Detection of the deletion allele (ΔPCR) of Ggcx by PCR on genomic DNA isolated from tissues of Ggcx^fl/fl;Col1a1-Cre and Ggcx^fl/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. (D and E) Serum levels of GLA- and total OCN (D) and of GLU-OCN (E) in Ggcx^fl/fl (n = 4) and Ggcx^fl/fl;Col1a1-Cre (n = 4) 2-mo-old mice. Results are given as means ± SEM. **, P < 0.01; ***, P < 0.001.

Figure 2.

Improved glucose tolerance, insulin sensitivity, and energy expenditure in Ggcx^fl/fl;Col1a1-Cre mice. (A) GTTs in Ggcx^fl/fl (n = 11) and Ggcx^fl/fl;Col1a1-Cre (n = 9) 2–3-mo-old male mice. Mice were fasted for 16 h and injected i.p. with 2 g/kg glucose. (B) ITTs in Ggcx^fl/fl (n = 16) and Ggcx^fl/fl;Col1a1-Cre (n = 11) 2–3-mo-old male mice. Mice were fasted for 4 h and injected i.p. with 0.7 U/kg insulin. (C) Body weight (left) and epididymal fat pad weight normalized to body weight (right) in Ggcx^fl/fl (n = 10) and Ggcx^fl/fl;Col1a1-Cre (n = 10) 5-mo-old male mice. (D) Metabolic rates and heat production (energy expenditure) in Ggcx^fl/fl (n = 9) and Ggcx^fl/fl;Col1a1-Cre (n = 8) 3-mo-old male mice during the dark 12-h phases. (E) GTTs in Ggcx^fl/fl (n = 13) and Ggcx^fl/fl;Col1a1-Cre (n = 9) 2–3 mo-old female mice. Mice were fasted for 16 h and injected i.p. with 2 g/kg glucose. (F) ITTs in Ggcx^fl/fl (n = 12) and Ggcx^fl/fl;Col1a1-Cre (n = 8) 2–3-mo-old female mice. Mice were fasted for 4 h and injected i.p. with 0.3 U/kg insulin. Results are given as means ± SEM. *, P < 0.05; **, P < 0.01.

Figure 3.

Ggcx^fl/fl;Col1a1-Cre mice are protected from diet-induced obesity and glucose intolerance. (A) Body weight curves. (B) Blood glucose levels after 16-h fasting. (C) GTTs. Mice were fasted for 16 h and injected i.p. with 1.3 g/kg glucose. (D) ITTs. Mice were fasted for 4 h and injected i.p. with 0.7 U/kg insulin. (B–D) Metabolic analyses were performed in mice fed an ND or HFD for 8 wk. Ggcx^fl/fl ND (n = 14), Ggcx^fl/fl;Col1a1-Cre ND (n = 14), Ggcx^fl/fl HFD (n = 8), Ggcx^fl/fl;Col1a1-Cre HFD (n = 11). Results are given as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 when comparing with Ggcx^fl/fl ND group; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 when comparing with Ggcx^fl/fl;Col1a1-Cre ND group; &, P < 0.05; &&, P < 0.01; &&&, P < 0.001 when comparing with Ggcx^fl/fl;Col1a1-Cre HFD group.

Figure 4.

Efficient reduction of OCN γ-carboxylation in Ggcx^fl/fl;OC-Cre mice. (A) Detection of the deletion allele (ΔPCR) of Ggcx by PCR on genomic DNA isolated from tissues of Ggcx^fl/fl;OC-Cre and Ggcx^fl/fl mice. PCR for the floxed allele (3′lox PCR) was used as a loading control. (B) Western blotting analyses of GGCX expression in Ggcx^fl/fl and Ggcx^fl/fl;OC-Cre bone marrow–derived osteoblasts. WAT and BAT, white and brown adipose tissue, respectively. (C and D) Serum levels of GLA- and total OCN (C) and of GLU-OCN (D) in Ggcx^fl/fl (n = 4) and Ggcx^fl/fl;OC-Cre (n = 4) 2-mo-old mice. (E) Bone OCN content in Ggcx^fl/fl (n = 6) and Ggcx^fl/fl;OC-Cre (n = 6) 3-mo-old mice was assessed from whole bone extracts by Western blotting (left) or ELISA and normalized to the total protein content (right). (F) GTTs in Ggcx^fl/fl (n = 13) and Ggcx^fl/fl;OC-Cre (n = 16) 3-mo-old mice fed an ND. Mice were fasted for 16 h and injected i.p. with 2 g/kg glucose. (G) GTTs in Ggcx^fl/fl (n = 7), Ggcx^fl/fl;Ocn^+/− (n = 9), Ggcx^fl/fl;OC-Cre;Ocn^+/− (n = 9), and Ggcx^fl/fl;OC-Cre (n = 9) mice fed an ND. Mice were fasted for 16 h and injected i.p. with 1.3 g/kg glucose. Results are given as means ± SEM. (C–F) **, P < 0.01; ***, P < 0.001. (G) #, P < 0.05 when comparing with Ggcx^fl/fl;Ocn^+/− and Ggcx^fl/fl;OC-Cre;Ocn^+/− mice; &, P < 0.05 when comparing with Ggcx^fl/fl, Ggcx^fl/fl;Ocn^+/−, and Ggcx^fl/fl;OC-Cre;Ocn^+/− mice.

Figure 5.

Efficient reduction of OCN γ-carboxylation in Vkorc1^fl/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 Vkorc1^fl/fl;OC-Cre and Vkorc1^fl/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 Vkorc1^fl/fl and Vkorc1^fl/fl;OC-Cre bone marrow–derived osteoblasts. (D and E) Serum levels of GLA- and total OCN (D) and of GLU-OCN (E) in Vkorc1^fl/fl (n = 5) and Vkorc1^fl/fl;OC-Cre (n = 5) 2-mo-old mice. (F) Bone OCN content in Vkorc1^fl/fl (n = 5) and Vkorc1^fl/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 Vkorc1^fl/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 Vkorc1^fl/fl (n = 7) and Vkorc1^fl/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.

Figure 6.

Deletion of VKORC1L1 in osteoblasts does not impact OCN γ-carboxylation. (A) Hypothetical role of VKORC1L1 in regulating OCN γ-carboxylation osteoblasts. (B) Detection of the deletion allele (ΔPCR) of Vkorc1l1 by PCR on genomic DNA isolated from tissues of Vkorc1l1^fl/fl;OC-Cre and Vkorc1l1^fl/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 and D) Serum levels of GLA- and total OCN (C) and of GLU-OCN (D) in Vkorc1l1^fl/fl (n = 5) and Vkorc1l1^fl/fl;OC-Cre (n = 5) 2-mo-old mice. (E) Bone OCN content in Vkorc1l1^fl/fl (n = 7) and Vkorc1l1^fl/fl;OC-Cre (n = 6) 3-mo-old mice was assessed from whole bone extracts by ELISA and normalized to the total protein content (left) or by Western blotting (right). (F) Cre-mediated inactivation of VKORC1L1 in Vkorc1l1^fl/fl osteoblasts infected with either Ad-GFP or Ad-Cre was assessed by QPCR (left; n = 4 per group) or by Western blotting (right). (G) 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). Results are given as means ± SEM. ***, P < 0.001.

Figure 7.

VKORC1L1 does not compensate for VKORC1 absence in osteoblasts. (A) Serum levels of GLU-OCN in control (n = 12), Vkorc1^fl/fl;OC-Cre (n = 4), Vkorc1l1^fl/fl;OC-Cre (n = 5), Vkorc1^fl/fl;Vkorc1l1^+/fl;OC-Cre (n = 5), and Vkorc1^fl/fl;Vkorc1l1^fl/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 Vkorc1^fl/fl;Vkorc1l1^fl/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.

Figure 8.

Model of the regulation of OCN function by GGCX and VKORC1. In WT mice (top left), OCN is γ-carboxylated in osteoblasts before it is secreted. Most of the secreted GLA-OCN is stored in the bone ECM, but some GLA-OCN escapes bone ECM embedding and reaches the circulation. Bone resorption by osteoclasts was previously shown to decarboxylate GLA-OCN, generating active GLU-OCN, which favors glucose tolerance (Ferron et al., 2010a). In Ggcx^fl/fl;Col1a1-Cre mice (top right), there is an increase in serum GLU-OCN, leading to improved glucose tolerance. Some GLA-OCN remains in the bone ECM and in the serum because of the partial inactivation of Ggcx. In Ggcx^fl/fl;OC-Cre mice (bottom left), OCN is poorly carboxylated and therefore escapes ECM embedding. Most of the serum OCN is undercarboxylated. In Vkorc1^fl/fl;OC-Cre mice (bottom right), some GLA-OCN is secreted by osteoblasts, most likely because of the exogenous contribution of VK1, which is converted to VKH₂ in osteoblasts by an unknown enzyme. Our data suggest that this enzyme is not VKORC1L1. It is possible that the high level of serum GLU-OCN in Ggcx^fl/fl;OC-Cre and in Vkorc1^fl/fl;OC-Cre mice leads to desensitization of the OCN receptor.