RESUMO
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.
Assuntos
Acrossomo/metabolismo , Acrossomo/patologia , Astenozoospermia/metabolismo , Astenozoospermia/patologia , N-Acetilgalactosaminiltransferases/deficiência , Oligospermia/metabolismo , Oligospermia/patologia , Animais , Apoptose , Astenozoospermia/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , N-Acetilgalactosaminiltransferases/genética , Oligospermia/genética , Espermatozoides/anormalidades , Espermatozoides/metabolismo , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Neoplasias , Osteogênese , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Biomarcadores/análise , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core , Subunidade beta de Fator de Ligação ao Core , Fatores de Ligação ao Core , Proteínas de Ligação a DNA/genética , Dimerização , Embrião de Mamíferos/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Genes Letais , Hematopoese/genética , Hematopoese/fisiologia , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/fisiologia , Fenótipo , Estrutura Terciária de Proteína , RNA Mensageiro/análise , Crânio/citologia , Fator de Transcrição AP-2 , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Runx2 plays important roles in the regulation of chondrocyte differentiation and proliferation; however, the Runx2 target molecules still remain to be investigated. We searched the genes upregulated by the introduction of Runx2 into Runx2(-/-) chondrocytes using microarray and found that Tcf7 is upregulated by Runx2. Thus, we examined the functions of Runx2 in the regulation of the Tcf/Lef family of transcription factors. Runx2 induced Tcf7 and Lef1 strongly, but Tcf7l1 and Tcf7l2 only slightly in Runx2(-/-) chondrocytes; the expressions of Tcf7 and Tcf7l2 were reduced in Runx2(-/-) cartilaginous skeletons and calvaria, and Tcf7 showed a similar expression pattern to Runx2. In reporter assays, Runx2 mildly activated the 8.6 and 1.8 kb Tcf7 promoter constructs. The reporter assays using the deletion constructs of the 1.8-kb fragment showed that the 0.3-kb promoter region is responsible for the Runx2-dependent transcriptional activation. To investigate the function of Tcf7 in skeletal development, we generated dominant-negative (dn) Tcf7 transgenic mice using the Col2a1 promoter. Dn-Tcf7 transgenic embryos showed dwarfism, and mineralization was retarded in limbs, ribs, and vertebrae in a manner dependent on the expression levels of the transgene. In situ hybridization analysis showed that endochondral ossification is retarded in dn-Tcf7 transgenic embryos due to the decelerated chondrocyte maturation. Further, BrdU labeling showed a reduction in chondrocyte proliferation in the proliferating layer of the growth plate in dn-Tcf7 transgenic embryos. These findings indicate that Runx2 regulates chondrocyte maturation and proliferation at least partly through the induction of Tcf7.
Assuntos
Diferenciação Celular/genética , Condrócitos/metabolismo , Condrócitos/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Fator 1 de Transcrição de Linfócitos T/genética , Animais , Sequência de Bases , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Bromodesoxiuridina/metabolismo , Proliferação de Células , Imunoprecipitação da Cromatina , Extremidades/patologia , Genes Reporter/genética , Fator 1-alfa Nuclear de Hepatócito , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Osteogênese/genética , Regiões Promotoras Genéticas/genética , Fator 1 de Transcrição de Linfócitos T/metabolismoRESUMO
Although Akt plays key roles in various cellular processes, the functions of Akt and Akt downstream signaling pathways in the cellular processes of skeletal development remain to be clarified. By analyzing transgenic embryos that expressed constitutively active Akt (myrAkt) or dominant-negative Akt in chondrocytes, we found that Akt positively regulated the four processes of chondrocyte maturation, chondrocyte proliferation, cartilage matrix production, and cell growth in skeletal development. As phosphorylation of GSK3beta, S6K, and FoxO3a was enhanced in the growth plates of myrAkt transgenic mice, we examined the Akt downstream signaling pathways by organ culture. The Akt-mTOR pathway was responsible for positive regulation of the four cellular processes. The Akt-FoxO pathway enhanced chondrocyte proliferation but inhibited chondrocyte maturation and cartilage matrix production, while the Akt-GSK3 pathway negatively regulated three of the cellular processes in limb skeletons but not in vertebrae due to less GSK3 expression in vertebrae. These findings indicate that Akt positively regulates the cellular processes of skeletal growth and endochondral ossification, that the Akt-mTOR, Akt-FoxO, and Akt-GSK3 pathways positively or negatively regulate the cellular processes, and that Akt exerts its function in skeletal development by tuning the three pathways in a manner dependent on the skeletal part.
Assuntos
Desenvolvimento Ósseo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Técnicas de Cultura de Células , Embrião de Mamíferos , Fatores de Transcrição Forkhead/genética , Genes Reporter , Quinase 3 da Glicogênio Sintase/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Luciferases de Renilla/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Técnicas de Cultura de Órgãos , Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TORRESUMO
The mammalian RUNX protein family comprises three transcription factorsRUNX1, RUNX2, and RUNX3. RUNX1 is involved in hematopoiesis, RUNX2 has multiple roles in osteogenesis and RUNX3 is associated with neural and gut development. In addition, all RUNX proteins are expressed during chondrogenesis, the process by which cartilage is formed. This review describes the involvement of Runx proteins in chondrogenesis, delineating their expression pattern and emphasizing their active roles in mesenchymal condensation, chondrocyte proliferation, and chondrocyte maturation. It also highlights how Runx proteins regulate transcription of target genes and how Runx proteins are regulated in the cartilaginous skeleton.
Assuntos
Condrócitos/citologia , Condrogênese/genética , Subunidades alfa de Fatores de Ligação ao Core/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Sequência de Aminoácidos , Animais , Proliferação de Células , Condrócitos/metabolismo , Condrócitos/patologia , Subunidades alfa de Fatores de Ligação ao Core/química , Subunidades alfa de Fatores de Ligação ao Core/genética , Humanos , Hipertrofia/metabolismo , Mesoderma , Camundongos , Ativação Transcricional/fisiologiaRESUMO
Disuse osteoporosis, which occurs commonly in prolonged bed rest and immobilization, is becoming a major problem in modern societies; however, the molecular mechanisms underlying unloading-driven bone loss have not been fully elucidated. The osteocyte network is considered to be an ideal mechanosensor and mechanotransduction system. We searched for the molecules responsible for disuse osteoporosis using BCL2 transgenic mice, in which the osteocyte network was disrupted. Pyruvate dehydrogenase kinase 4 (Pdk4), which inactivates pyruvate dehydrogenase complex (PDC), was upregulated in femurs and tibiae of wild-type mice but not of BCL2 transgenic mice after tail suspension. Bone in Pdk4(-/-) mice developed normally and was maintained. At unloading, however, bone mass was reduced due to enhanced osteoclastogenesis and Rankl expression in wild-type mice but not in Pdk4(-/-) mice. Osteoclast differentiation of Pdk4(-/-) bone marrow-derived monocyte/macrophage lineage cells (BMMs) in the presence of M-CSF and RANKL was suppressed, and osteoclastogenesis was impaired in the coculture of wild-type BMMs and Pdk4(-/-) osteoblasts, in which Rankl expression and promoter activity were reduced. Further, introduction of Pdk4 into Pdk4(-/-) BMMs and osteoblasts enhanced osteoclastogenesis and Rankl expression and activated Rankl promoter. These findings indicate that Pdk4 plays an important role in bone loss at unloading by promoting osteoclastogenesis.
Assuntos
Reabsorção Óssea/metabolismo , Osteoclastos/fisiologia , Proteínas Quinases/metabolismo , Animais , Diferenciação Celular , Expressão Gênica , Elevação dos Membros Posteriores , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Análise em Microsséries , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoporose/metabolismo , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismoRESUMO
RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2(-/-) calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Osteoblastos/citologia , Fatores de Transcrição/metabolismo , Animais , Osso e Ossos/metabolismo , Imunoprecipitação da Cromatina , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Genes Reporter , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Osteoblastos/metabolismo , Osteócitos/citologia , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fator de Transcrição Sp7 , Regulação para CimaRESUMO
Phenotypic plasticity and the switching of vascular smooth muscle cells (SMCs) play a critical role in atherosclerosis. Although Runx2, a key osteogenic transcription factor, is expressed in atherosclerotic plaques, the molecular mechanisms by which Runx2 regulates SMC differentiation remain unclear. Here we demonstrated that Runx2 repressed SMC differentiation induced by myocardin, which acts as a coactivator for serum response factor (SRF). Myocardin-mediated induction of SMC gene expression was enhanced in mouse embryonic fibroblasts derived from Runx2 null mice compared to wild-type mice. Forced expression of Runx2 decreased the expression of SMC genes and promoted osteogenic gene expression, whereas the reduction of Runx2 expression by small interfering RNA enhanced SMC differentiation in human aortic SMCs. Runx2 interacted with SRF and interfered with the formation of the SRF/myocardin ternary complex. Thus, this study provides the first evidence that Runx2 inhibits SRF-dependent transcription, as a corepressor independent of its DNA binding. We propose that Runx2 plays a pivotal role in osteogenic conversion tightly coupled with repression of the SMC phenotype in atherosclerotic lesions.
Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Proteínas Nucleares/fisiologia , Osteogênese , Fator de Resposta Sérica/fisiologia , Transativadores/fisiologia , Animais , Aorta/citologia , Aterosclerose/etiologia , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/farmacologia , Transcrição Gênica , TransfecçãoRESUMO
Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant-negative (dn)-Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells. In transgenic mice that expressed dn-Runx2 in osteoblasts, the trabecular bone had increased mineralization, increased volume, and features of compact bone, and the expression of major bone matrix protein genes was relatively maintained. After ovariectomy, neither osteolysis nor bone formation was enhanced and bone was relatively conserved. In wild-type mice, Runx2 was strongly expressed in immature osteoblasts but downregulated during osteoblast maturation. These findings indicate that the maturity and turnover rate of bone are determined by the level of functional Runx2 and Runx2 is responsible for bone loss in estrogen deficiency, but that Runx2 is not essential for maintenance of the expression of major bone matrix protein genes in postnatal bone development and maintenance.
Assuntos
Desenvolvimento Ósseo/fisiologia , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Estrogênios/deficiência , Animais , Linhagem Celular , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismoRESUMO
Runx2 and Cbfbeta are essential for skeletal development during the embryonic stage. Runx2 has two isoforms with different N-termini. We examined the functions of the Runx2 isoforms and Cbfbeta in postnatal bone development. On luciferase and electrophoretic mobility shift assays, Runx2-I was less active than Runx2-II in the absence of Cbfb, but the two Runx2 isoforms had similar activity levels in the presence of Cbfb. We generated Runx2-I transgenic mice under the control of Col1a1 promoter and Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice. Runx2-I transgenic mice showed less severe osteopenia and fragility than Runx2-II transgenic mice due to milder inhibition of both osteoblast maturation and transition to osteocytes, even though the former mice showed higher transgene expression. However, Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice had enhanced inhibition of osteoblast maturation, resulting in similar severity of osteopenia and fragility, although the latter mice had less osteocytes. These findings indicate that (1) Runx2-II more strongly inhibits osteoblast maturation and transition to osteocytes than Runx2-I; (2) Cbfbeta regulates Runx2 function isoform-dependently; and (3) Runx2-I activity is highly dependent on Cbfbeta. These findings demonstrate that Runx2 isoforms exert their functions through at least partly different mechanisms and Cbfbeta regulates bone development by regulating Runx2 function isoform-dependently.
Assuntos
Desenvolvimento Ósseo/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/fisiologia , Isoformas de Proteínas/metabolismo , Animais , Animais Recém-Nascidos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Subunidade beta de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/fisiologia , Camundongos , Camundongos Transgênicos , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Crânio/metabolismoRESUMO
The differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed in Runx2-deficient (Runx2(-/-)) mice, terminal differentiation of chondrocytes does occur, indicating that additional factors are involved in chondrocyte maturation. We investigated the involvement of Runx3 in chondrocyte differentiation by generating Runx2-and-Runx3-deficient (Runx2(-/-)3(-/-)) mice. We found that chondrocyte differentiation was inhibited depending on the dosages of Runx2 and Runx3, and Runx2(-/-)3(-/-) mice showed a complete absence of chondrocyte maturation. Further, the length of the limbs was reduced depending on the dosages of Runx2 and Runx3, due to reduced and disorganized chondrocyte proliferation and reduced cell size in the diaphyses. Runx2(-/-)3(-/-) mice did not express Ihh, which regulates chondrocyte proliferation and maturation. Adenoviral introduction of Runx2 in Runx2(-/-) chondrocyte cultures strongly induced Ihh expression. Moreover, Runx2 directly bound to the promoter region of the Ihh gene and strongly induced expression of the reporter gene driven by the Ihh promoter. These findings demonstrate that Runx2 and Runx3 are essential for chondrocyte maturation and that Runx2 regulates limb growth by organizing chondrocyte maturation and proliferation through the induction of Ihh expression.
Assuntos
Condrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Extremidades/embriologia , Proteínas de Neoplasias/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core , Subunidade alfa 3 de Fator de Ligação ao Core , Proteínas Hedgehog , Camundongos , Regiões Promotoras GenéticasRESUMO
Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2-/- calvaria-derived cell line (CA120-4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120-4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2-/- livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2-/- mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.