Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans.

Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-D-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2(-/-) cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3(-/-) mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3(-/-) mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation.

Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia.

Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3(-/-)) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3(-/-) mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3(-/-) mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3(-/-) mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3(-/-) mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm-egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm-egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.

Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation by regulating Fgfr2 and Fgfr3.

Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1-3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1-3, we compared osteoblast progenitors in Sp7-/- and Runx2-/- mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7-/- but not of Runx2-/- mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7-/- background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2-/- but not in Sp7-/- calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7-/- osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.

Cbfb2 Isoform Dominates More Potent Cbfb1 and Is Required for Skeletal Development.

Cbfb is a cotranscription factor that forms a heterodimer with Runx proteins Runx1, Runx2, and Runx3. It is required for fetal liver hematopoiesis and skeletal development. Cbfb has two functional isoforms, Cbfb1 and Cbfb2, which are formed by alternative splicing. To address the biological functions of these isoforms in skeletal development, we examined Cbfb1(-/-) and Cbfb2(-/-) mouse embryos. Intramembranous and endochondral ossification was retarded and chondrocyte and osteoblast differentiation was inhibited in Cbfb2(-/-) embryos but not in Cbfb1(-/-) embryos. Cbfb2 mRNA was upregulated in calvariae, limbs, livers, thymuses, and hearts of Cbfb1(-/-) embryos but Cbfb1 mRNA was not in those of Cbfb2(-/-) embryos, and the total amount of Cbfb1 and Cbfb2 mRNA in Cbfb1(-/-) embryos was similar to that in wild-type embryos but was severely reduced in Cbfb2(-/-) embryos. The absolute numbers of Cbfb2 mRNA in calvariae, limbs, livers, thymuses, and brains in wild-type embryos were about three times higher than those of Cbfb1 in the respective tissue. The levels of Runx proteins were reduced in calvariae, limbs, and primary osteoblasts from Cbfb2(-/-) embryos, but the reduction in Runx2 protein was very mild. Furthermore, the amounts of Runx proteins and Cbfb in Cbfb2(-/-) embryos differed similarly among skeletal tissues, livers, and thymuses, suggesting that Runx proteins and Cbfb are mutually required for their stability. Although Cbfb1(-/-) embryos developed normally, Cbfb1 induced chondrocyte and osteoblast differentiation and enhanced DNA binding of Runx2 more efficiently than Cbfb2. Our results indicate that modulations in the relative levels of the isoforms may adjust transcriptional activation by Runx2 to appropriate physiological levels. Cbfb2 was more abundant, but Cbfb1 was more potent for enhancing Runx2 activity. Although only Cbfb2 loss generated overt skeletal phenotypes, both may play major roles in skeletal development with functional redundancy. © 2016 American Society for Bone and Mineral Research.

Cbfb regulates bone development by stabilizing Runx family proteins.

Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA-binding capacity. Cbfb-deficient (Cbfb(-/-) ) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb(-/-) mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb(fl/fl) mice with Dermo1 Cre knock-in mice. Cbfb(fl/fl/Cre) mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb(fl/fl/Cre) chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb(fl/fl/Cre) embryos compared with the respective protein in the respective tissue of Cbfb(fl/fl) embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb(fl/fl/Cre) primary osteoblasts, and Runx2 protein was less stable in Cbfb(fl/fl/Cre) osteoblasts than Cbfb(fl/fl) osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons.

Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation.

Statins such as simvastatin are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that inhibit cholesterol synthesis. We presently investigated statin effects on vascular endothelial growth factor (VEGF) expression in osteoblastic cells. Hydrophobic statins including simvastatin, atorvastatin, and cerivastatin-but not a hydrophilic statin, pravastatin-markedly increased VEGF mRNA abundance in nontransformed osteoblastic cells (MC3T3-E1). Simvastatin (10(-6) M) time-dependently augmented VEGF mRNA expression in MC3T3-E1 cells, mouse stromal cells (ST2), and rat osteosarcoma cells (UMR-106). According to heterogeneous nuclear RNA and Northern analyses, 10(-6) M simvastatin stimulated gene expression for VEGF in MC3T3-E1 cells without altering mRNA stability. Transcriptional activation of a VEGF promoter-luciferase construct (-1128 to +827), significantly increased by simvastatin administration. As demonstrated by gel mobility shift assay, simvastatin markedly enhanced the binding of hypoxia-responsive element-protein complexes. These results indicate that the stimulation of the VEGF gene by simvastatin in MC3T3-E1 cells is transcriptional in nature. VEGF secretion into medium was increased in MC3T3-E1 by 10(-6) M simvastatin. Pretreating MC3T3-E1 cells with mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced VEGF mRNA expression; manumycin A, a protein prenylation inhibitor, mimicked statin effects on VEGF expression. The effect of simvastatin was blocked by pretreatment with wortmannin and LY294002, specific phosphatidylinositide-3 kinase inhibitors. Simvastatin enhanced mineralized nodule formation in culture, whereas coincubation with mevalonate, geranylgeranyl pyrophosphate, LY294002, or VEGF receptor 2 inhibitor (SU1498) abrogated statin-induced mineralization. Thus, statins stimulate VEGF expression in osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation.

Leptin corrects increased gene expression of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase and -24-hydroxylase in leptin-deficient, ob/ob mice.

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We investigated leptin effects on bone metabolism using male leptin-deficient obese (ob/ob) mice, which had lower bone mineral density (BMD) and shorter femurs than lean (?/+) controls. Serum concentrations of calcium, phosphate, tartrate-resistant acid phosphatase (a bone resorption marker) and alkaline phosphatase, and urinary calcium and phosphate excretion were significantly elevated in ob/ob mice, whereas urinary concentrations of deoxypyridinoline did not differed between ob/ob and control mice. Because ob/ob mice develop severe hypogonadism, testosterone was administered to these mice for 10 wk (5 mg/kg, sc, twice weekly); this did not affect femoral BMD. Control and ob/ob mice showed similar vitamin D-receptor densities in bone and kidney; the obese mice had marked increases in serum 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] and in mRNA expression and activities of renal 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (CYP27B1) and -24-hydroxylase (CYP24) compared with control mice. Leptin administration to ob/ob mice (4 mg/kg body weight, ip, every 12 h for 2 d) greatly reduced mRNAs and activities of 1 alpha-hydroxylase and 24-hydroxylase. Elevated concentrations of serum calcium, phosphate, and 1,25-(OH)(2)D(3) were normalized by leptin treatment. Thus, leptin suppresses renal gene overexpression for 1 alpha-hydroxylase and 24-hydroxylase and corrects increased serum concentrations of calcium and phosphate in ob/ob mice. Therefore, low BMD in leptin-deficient mice appears to be related to stimulation of bone resorption by 1,25-(OH)(2)D(3).

Dlx5 and mef2 regulate a novel runx2 enhancer for osteoblast-specific expression.

Runx2 is essential for osteoblast differentiation and chondrocyte maturation. The expression of Runx2 is the first requisite step for the lineage determination from mesenchymal stem cells to osteoblasts. Although the transcript from Runx2 distal promoter is majorly expressed in osteoblasts, the promoter failed to direct green fluorescent protein (GFP) expression to osteoblasts. To find the regulatory region, we generated GFP reporter mice driven by a bacterial artificial chromosome (BAC) of Runx2 locus, and succeeded in the reproduction of endogenous Runx2 expression. By serially deleting it, we identified a 343-bp enhancer, which directed GFP expression specifically to osteoblasts, about 30 kb upstream of the distal promoter. The sequence of the 343-bp enhancer was highly conserved among mouse, human, dog, horse, opossum, and chicken. Dlx5, Mef2c, Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, which localized on the enhancer region in primary osteoblasts, synergistically upregulated the enhancer activity, whereas Msx2 downregulated the activity in mouse osteoblastic MC3T3-E1 cells. Msx2 was predominantly bound to the enhancer in mouse multipotent mesenchymal C3H10T1/2 cells, whereas Dlx5 was predominantly bound to the enhancer in MC3T3-E1 cells. Dlx5 and Mef2 directly bound to the enhancer, and the binding sites were required for the osteoblast-specific expression in mice, whereas the other factors bound to the enhancer by protein-protein interaction. The enhancer was characterized by the presence of the histone variant H2A.Z, the enrichment of histone H3 mono- and dimethylated at Lys4 and acetylated at Lys18 and Lys27, but the depletion of histone H3 trimethylated at Lys4 in primary osteoblasts. These findings indicated that the enhancer, which had typical histone modifications for enhancers, contains sufficient elements to direct Runx2 expression to osteoblasts, and that Dlx5 and Mef2, which formed an enhanceosome with Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, play an essential role in the osteoblast-specific activation of the enhancer. © 2014 American Society for Bone and Mineral Research.

Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1alpha,25-dihydroxyvitamin D3 synthesis in leptin-deficient mice.

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone-derived fibroblast growth factor 23 (FGF-23) as a mediator of the influence of leptin on renal 1alpha-hydroxylase mRNA expression in leptin-deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF-23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12-hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF-23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Administration of FGF-23 (5 microg i.p. at 12-hour intervals for 2 days) to ob/ob mice suppressed renal 1alpha-hydroxylase mRNA expression. The main site of FGF-23 mRNA expression was the bone, and leptin markedly increased the FGF-23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1alpha-hydroxylase and sodium-phosphate cotransporters (NaP(i)-IIa and NaP(i)-IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF-23 level and renal expression of 1alpha-hydroxylase mRNA were not influenced by administration of leptin to leptin receptor-deficient (db/db) mice. These results indicate that leptin directly stimulates FGF-23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)(2)D(3) synthesis in these mice is at least partly due to elevated bone production of FGF-23.

Identification of the promoter region of the parathyroid hormone receptor gene responsible for transcriptional suppression by insulin-like growth factor-I.

We investigated parathyroid hormone (PTH)/PTH-related protein receptor (PTH1R) gene suppression induced by insulin-like growth factor (IGF)-I using a rat osteoblast-like cell line (UMR-106). Observations were made with PD98059, a specific ERK signaling pathway inhibitor, and UMR-106 cells transfected with dominant negative or constitutively active forms of MAP kinase kinase. IGF-I inhibited PTH1R gene expression via an ERK1/2 MAP kinase pathway. We cloned the 8-kb promoter region of the rat PTH1R gene and characterized the U3 promoter, a major IGF-I-responsive promoter among the two present in rat osteoblasts. The IGF-I-suppressive region was between +1 and +25, identical to the previously described PTH-suppressive region (PTHSR). Gel mobility-shift detected a specific DNA-protein complex decreased by IGF-I. Mutation involving a three base sequence (+1 to +3) among more than 3.5 kb constituting the PTH1R promoter region completely abolished IGF-I action. Thus, IGF-I signaling may act at the osteoblast exon U3 transcription initiation site to repress the transcriptional activity.

Dexamethasone enhances vitamin D-24-hydroxylase expression in osteoblastic (UMR-106) and renal (LLC-PK1) cells treated with 1alpha,25-dihydroxyvitamin D3.

Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK1 kidney cells. To induce 24-hydroxylase expression, 1,25(OH)2D3 (10(-7)M) and dexamethasone were added simultaneously to the medium of LLC-PK1 cells, and 24 h before dexamethasone treatment, 1,25(OH)2D3 was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells. Maximal stimulation was observed with 10(-6) M dexamethasone in both cell lines. The addition of 10(-6) M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10(-6) M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)2D3-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)2D3-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)2D3 enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.

Atorvastatin enhances bone density in ovariectomized rats given 17beta-estradiol or human parathyroid hormone(1-34).

We investigated the in vivo effect of atorvastatin on bone mineral density (BMD) in ovariectomized (OVX) rats. Eight-week-old female rats underwent either a sham operation or ovariectomy, and treatments with vehicle, atorvastatin, 17beta-estradiol (E2) and human parathyroid hormone(1-34) [hPTH(1-34)] were initiated 6 wk after the surgery. E2 (10 microg/kg) treatment for 12 wk significantly increased lumbar BMD (L2-L4), whereas atorvastatin did not affect lumbar BMD. The combination of atorvastatin (2 mg/kg) and E2 significantly enhanced the BMD of lumbar vertebrae (L2-L4) and femoral metaphyseal area (2/10-4/10 segments from the most proximal point) compared to that of either E2 or atorvastatin alone. A low dose 1 micro g/kg of hPTH (1-34) did not alter lumbar or femoral BMD, whereas a high dose 17.5 micro g/kg of the peptide significantly increased BMD. Concomitant injections of atorvastatin (2 mg/kg) with hPTH(1-34) (1 microg/kg) for 8 wk significantly enhanced the BMD of lumbar vertebrae and the metaphyseal area of the femur in OVX rats. These findings demonstrate that chronic administration of atorvastatin appears to modestly enhance the BMD of the lumbar vertebrae and femoral metaphysis of OVX rats treated with submaximal doses of E2 and hPTH(1-34).