RESUMO
Mice carrying homozygous disruption of the c-src proto-oncogene (Src-/-) develop osteopetrosis due to an impaired ability of osteoclasts to adhere to the bone surface and/or to form bone-resorbing ruffled border. It has also been reported that osteopontin (OPN), a secreted phosphoprotein, mediates osteoclast adherence to the bone matrix. We report here that cells from Src-/- mice, both in vitro and in vivo, express OPN mRNA and protein at a significantly reduced level as compared to cells from Src+/- and +/+ animals, suggesting a potential role for the proto-oncogene c-src in the regulation of OPN gene expression. Our data also show that OPN gene expression can be induced by treatment of SR-/- cells with epidermal growth factor (EGF) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Results obtained from studies using inhibitors of receptor tyrosine kinases (RTKs) and protein kinase C (PKC) suggest that PKC and RTK are positioned in a pathway with PKC as the downstream effector for the EGF-induced OPN gene expression in SRC-/- cells, and that pp60c-src and EGF may regulate OPN gene expression through a common signalling pathway. Furthermore, contrary to published reports, our study shows that EGF-mediated cell signalling does not require functional interaction between the EGF-receptor and pp60c-src.
Assuntos
Homozigoto , Osteopetrose/patologia , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Sialoglicoproteínas/biossíntese , Células 3T3 , Animais , Sequência de Bases , Benzoquinonas , Osso e Ossos/ultraestrutura , Adesão Celular/genética , Linhagem Celular , Primers do DNA , Replicação do DNA/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Lactamas Macrocíclicas , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Osteopetrose/genética , Osteopontina , Proteína Quinase C/antagonistas & inibidores , Quinonas/farmacologia , Rifabutina/análogos & derivados , Sialoglicoproteínas/genética , Acetato de Tetradecanoilforbol/antagonistas & inibidoresRESUMO
Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types. Stem cell-derived, differentiated cells are of increasing importance as a potential source for non-proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications. Differentiation of ESC is initiated by the formation of embryoid bodies (EB). Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state. To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies. Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas. However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula-like stage, so that no cystic EB can be formed within the beads. ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes. Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.
Assuntos
Divisão Celular , Embrião de Mamíferos/citologia , Microesferas , Células-Tronco/citologia , Animais , Diferenciação Celular , Linhagem Celular , Imunofluorescência , CamundongosAssuntos
Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Adesão Celular , Eletroforese em Gel Bidimensional , Peso Molecular , Osteopontina , Fosfoproteínas/fisiologia , Fosforilação , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
We have reported previously that the 69-kDa major phosphoprotein, secreted by normal rat kidney (NRK) cells, is osteopontin, a glycosylated bone matrix protein. Here we show that this 69-kDa osteopontin is secreted by NRK cells in both phosphorylated (pp69) and nonphosphorylated (np69) forms, with estimated isoelectric points of 3.8 and 4.5, respectively. Electrophoretic analysis of radioiodinated cell surface proteins immunoprecipitated with an anti-69-kDa osteopontin serum, demonstrates that the 69-kDa osteopontin is also present on the cell surface, but only its phosphorylated form (pp69) shows such cell surface association. Because osteopontin mediates cell adhesion and spreading, and contains an Arg-Gly-Asp-Ser cell-binding sequence, our observations strongly suggest that the cell surface localization of pp69 osteopontin is receptor-mediated, and the modification by phosphorylation may be crucial for its receptor binding activity. We also report that antisera directed against either fibronectin or 69-kDa osteopontin co-immunoprecipitate both np69 osteopontin and fibronectin as a heat-dissociable complex. In contrast, pp69 osteopontin does not co-precipitate with fibronectin. These observations demonstrate an interactive relationship between np69 and soluble fibronectin. Furthermore, compared to NRK cells, vanadyl sulfate-treated NRK cells which acquire a reversible transformed phenotype, including anchorage-independent growth, show increased levels of pp69 on the cell surface, concomitant with significantly decreased levels of pp69 and elevated levels of np69 in the conditioned media. The data presented here establish transformation sensitivity of NRK cell-secreted osteopontin with respect to its secretion and cell surface localization, and demonstrate that phosphorylated and nonphosphorylated forms of osteopontin have different physiological properties, which may regulate the functional roles of this extracellular matrix protein.
Assuntos
Fosfoproteínas/biossíntese , Sialoglicoproteínas/biossíntese , Animais , Linhagem Celular , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Rim , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Peso Molecular , Osteopontina , Fosfatos/metabolismo , Radioisótopos de Fósforo , Fosforilação , Ratos , Sialoglicoproteínas/isolamento & purificação , Sialoglicoproteínas/fisiologiaRESUMO
Osteopontin (OPN) is a sialic acid-rich, adhesive, extracellular matrix (ECM) protein with Arg-Gly-Asp cell-binding sequence that interacts with several integrins, including alpha(v)beta(3). Since the ECM is a key regulator of mammary gland morphogenesis, and mammary epithelial cells express OPN at elevated levels, we sought to determine whether this protein plays a role in the postnatal mammary gland development. By generating transgenic mice that express OPN antisense-RNA (AS-OPN mice) in the mammary epithelia we achieved suppression of OPN production in this organ. The pregnant AS-OPN mice displayed a lack of mammary alveolar structures, a drastic reduction in the synthesis of beta-casein, whey acidic milk protein, and lactation deficiency. In agreement with these findings, we uncovered that a mammary cell line, NMuMG, which undergoes both structural and functional differentiation on ECM-coated plates, when transfected with an antisense OPN-cDNA construct, failed to undergo such differentiation. Furthermore, the results of gel-invasion assays demonstrated that these cells manifest elevated matrix metalloproteinase (MMP) activity when OPN expression is significantly reduced. The identity of this proteinase as MMP-2 is confirmed by Western blotting, zymography, and inhibition of its activity by a specific inhibitor, TIMP-2. Taken together, our results demonstrate, for the first time, an essential role of OPN in mammary gland differentiation and that the molecular mechanism(s) of its action, at least in part, involves down-regulation of MMP-2.
Assuntos
Lactação/genética , Glândulas Mamárias Animais/anormalidades , Glândulas Mamárias Animais/fisiologia , RNA Antissenso/genética , Sialoglicoproteínas/genética , Animais , Animais Recém-Nascidos , Caseínas/genética , Citocinas/genética , Morte , Células Epiteliais/fisiologia , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Glândulas Mamárias Animais/embriologia , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Morfogênese , Osteopontina , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/deficiênciaRESUMO
/ar, a tumor promoter-inducible protein secreted by mouse JB6 epidermal cells, is the murine homolog of rat osteopontin, or 44 kD bone phosphoprotein. We report here that 2ar is also related to pp69, a major phosphoprotein secreted by normal rat kidney cells. Antisera raised against pp69 and against beta-galactosidase-2ar fusion proteins are able to immunoprecipitate the same major phosphoproteins, of apparent Mr 55-69 kD, secreted by several rat and mouse cell lines. The levels of secreted protein and cytoplasmic mRNA are dramatically elevated in NIH 3T3 cells transformed with the human bladder cancer T24 (H-ras) oncogene. These results and the work of Senger and colleagues (Cancer Res., 45, 5818-5823, 1985) imply that enhanced secretion of 2ar/pp69/osteopontin by transformation of a wide variety of mammalian fibroblasts and epithelial cells is often correlated with tumorigenicity.