Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Numerous bone matrix proteins can interact with alpha(v)-containing integrins including alpha(v)beta3. To elucidate the net effects of the interaction between these proteins and alpha(v)beta3 on osteoblast function, we developed a murine osteoblastic cell line that overexpressed human alpha(v)beta3. Human alpha(v)beta3-integrin was expressed on cell membrane, in which its presence did not alter the surface level of endogenous mouse alpha(v)beta3. The expressed human alpha(v)beta3 was functional because cell adhesion to osteopontin was increased and this increment was abolished by antibody against human alpha(v)beta3. The proliferation rate of cells overexpressing alpha(v)beta3 (alpha(v)beta3-cells) was increased whereas matrix mineralization was decreased. To elucidate the mechanisms leading to inhibition of matrix mineralization, the expression of proteins important for mineralization was analyzed. Alkaline phosphatase activity and the expression of osteocalcin, type I collagen, and bone sialoprotein (BSP) were decreased whereas osteopontin was stimulated in alpha(v)beta3-cells. The regulation of osteopontin, osteocalcin, and BSP expression was mediated via transcriptional mechanism because their promoter activities were altered. Examination of molecules involved in integrin signaling indicated that activator protein-1 (AP-1) and extracellular signal-regulated kinase (Erk) activities were enhanced whereas c-jun N-terminal kinase (JNK) activity was decreased in alpha(v)beta3-cells. The activity of p38 and the levels of focal adhesion kinase (FAK) and vinculin were not altered. Moreover, the adhesions of alpha(v)beta3-cells to type I collagen and fibronectin were inhibited, which was attributed to decreased beta1-integrin levels on cell surface. In conclusion, overexpressing alpha(v)beta3-integrin in osteoblasts stimulated cell proliferation but retarded differentiation, which were derived via altered integrin-matrix interactions, signal transduction, and matrix protein expression.
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PMID:Bone mineralization and osteoblast differentiation are negatively modulated by integrin alpha(v)beta3. 1120 28

We investigated the mechanisms of parathyroid hormone-related peptide (PTHrP)-mediated effects on osteogenic cells in primary rat bone marrow cell (BMC) cultures. We first demonstrated by reverse transcriptase-polymerase chain reaction and immunocytochemistry that BMCs express the type I parathyroid hormone/PTHrP receptor. Treatment with PTHrP increased osteogenic cell proliferation as determined by [(3)H]thymidine and bromodeoxyuridine incorporation and augmented osteogenic colonies. Immunocytochemistry and Western blotting revealed no direct effect on expression of the osteoblast markers, type I collagen, bone sialoprotein, and osteocalcin, indicating that PTHrP did not directly stimulate differentiation in this system. PTHrP increased mitogen-activated protein kinase (MAPK) activity in BMC and MAPK activity, and PTHrP-induced osteogenic cell proliferation could be blocked by the MEK inhibitor PD-098059. PTHrP also increased Ras activity in BMC. Although wortmannin and H8, inhibitors of phosphoinositol 3-kinase and protein kinase A, respectively, did not block PTHrP-stimulated Ras or MAPK activity, chelerythrin chloride, a known protein kinase C inhibitor, did block these PTHrP actions as well as PTHrP-induced osteogenic cell proliferation. These results demonstrate that PTHrP stimulates osteogenic cell proliferation in rat marrow mesenchymal progenitor cells through protein kinase C-dependent activation of the Ras and MAPK signaling pathway.
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PMID:Parathyroid hormone-related peptide stimulates osteogenic cell proliferation through protein kinase C activation of the Ras/mitogen-activated protein kinase signaling pathway. 1140 23

Osteoblasts secrete a complex extracellular matrix (ECM) containing collagenous and noncollagenous proteins, bone morphogenetic proteins (BMPs), and growth factors. Osteoblast-specific gene expression requires ascorbic acid (AA)-dependent assembly of a collagenous ECM. Matrix responsiveness requires an alpha2beta1 integrin-collagen interaction and mitogen-activated protein kinase (MAPK) activity, which phosphorylates and activates the osteoblast-specific transcription factor Cbfa1. This study examines interactions between this integrin/MAPK-mediated pathway and signals initiated by BMPs contained in the osteoblast matrix. MC3T3-E1 cells were shown to constitutively express BMP-2, BMP-4, and BMP-7. Noggin, a specific BMP inhibitor, reversibly blocked AA-induced gene expression, indicating that BMP production by MC3T3-E1 cells was necessary for differentiation. The ability of exogenously added BMP-2, BMP-4, or BMP-7 to stimulate osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs or OCN promoter activity was synergistically increased in cells that were actively synthesizing an ECM (i.e., were grown in the presence of AA). A minimum of 4 days of ECM accumulation was required for this synergistic response to be observed. Neither BMP-7, AA, nor a combination of these two treatments had major effects on Cbfa1 messenger RNA (mRNA) or protein levels, as would be expected if regulation was mainly at the posttranscriptional level. U0126, a specific inhibitor of MAPK/extracellular signal-regulated kinase (MEK), blocked AA- or BMP-7/AA-dependent gene expression in a time- and dose-dependent manner that was closely correlated with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. This work establishes that autocrine BMP production as well as integrin-mediated cell-collagen interactions are both required for osteoblast differentiation, and both these pathways require MAP kinase activity.
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PMID:Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. 1177 55

When triggered appropriately, dental follicle cells are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. However, factors and mechanisms regulating follicle cell differentiation remain undefined. This study focused on determining the ability of bone morphogenetic protein (BMP) 2 to promote the differentiation of follicle cells and periodontal ligament (PDL) cells along a cementoblast/ osteoblast pathway. Follicle cells and PDL cells were isolated from the first molar region of CD-1 mice and immortalized with SV40. Both cell types expressed BMP-4 and BMP receptors (BMPR) IA and II, but only follicle cells expressed BMP-2 mRNA. Cells were exposed to recombinant human BMP (rhBMP)-2 (0-100 ng/ml) and Northern blots were used to determine the expression of mineral-associated markers. BMP-2, in a dose- and time-dependent manner, induced cementoblast/osteoblast differentiation of follicle cells, as reflected by enhanced core binding factor alpha (Cbfal), bone sialoprotein (BSP), and osteocalcin (OCN) mRNA expression and enhanced mineral formation. U0126, a specific inhibitor of MEK-1/2 members of the MAPK family, abolished BMP-2-mediated expression of BSP and OCN. In contrast, exposure of PDL cells to BMP-2 resulted in modest expression of OCN and minimal promotion of mineralization. These results suggest that BMP-2 triggers follicle cells to differentiate toward a cementoblast/osteoblast phenotype and that the MAPK pathway is involved.
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PMID:Bone morphogenetic protein 2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype. 1216 98

Parathyroid hormone-related protein (PTHrP) regulates proliferation and differentiation of osteoblastic cells via binding to the parathyroid hormone receptor (PTH-1R). The cAMP-dependent protein kinase A pathway governs the majority of these effects, but recent evidence also implicates the MAPK pathway. MC3T3-E1 subclone 4 cells (MC4) were treated with the MAPK inhibitor U0126 and PTHrP. In differentiated MC4 cells, osteocalcin and bone sialoprotein gene expression were both down-regulated by PTHrP and also by inhibition of the MAPK pathway. PTHrP-mediated down-regulation of PTH-1R mRNA and up-regulation of c-fos mRNA were MAPK-independent, whereas PTHrP stimulation of fra-2 and interleukin-6 (IL-6) mRNA was MAPK-dependent. Luciferase promoter assays revealed that regulation of IL-6 involved the cAMP-dependent protein kinase A and MAPK pathways with a potential minor role of the protein kinase C pathway, and a promoter region containing an activator protein-1 site was necessary for PTHrP-induced IL-6 gene transcription. An alternative pathway, through cAMP/Epac/Rap1/MAPK, mediated ERK phosphorylation but was not sufficient for IL-6 promoter activation. Phosphorylation of the transcription factor CREB was also necessary but not sufficient for PTHrP-mediated IL-6 promoter activity. Most interesting, a bidirectional effect was found with PTHrP increasing phosphorylated ERK in undifferentiated MC4 cells but decreasing phosphorylated ERK in differentiated cells. These data indicate that inactivation of the MAPK pathway shows differential regulation of PTHrP-stimulated activator protein-1 members, blocks PTHrP-stimulated IL-6, and synergistically down-regulates certain osteoblastic markers associated with differentiation. These novel findings indicate that the MAPK pathway plays a selective but important role in the actions of PTHrP.
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PMID:Impact of the mitogen-activated protein kinase pathway on parathyroid hormone-related protein actions in osteoblasts. 1512 46

Matrix extracellular phosphoglycoprotein (MEPE)/ osteoblast/osteocyte factor 45 (OF45) is a recently isolated RGD-containing matrix protein that acts as the tumor-derived phosphaturic factor in oncogenic hypophosphatemic osteomalacia. It is also highly expressed by osteoblasts and osteocytes. We examined the regulation of MEPE/OF45 mRNA expression in osteoblastic cells derived from high-density cultures of primary rat bone marrow stromal cells incubated with dexamethasone, beta-glycerophosphate, and ascorbic acid. The level of MEPE/OF45 mRNA in these cells was down-regulated by the addition of fibroblast growth factor 2 (FGF2) for 48 h. These effects were observed in a dose-dependent manner between 2 and 10 ng/mL. FGF2 also reduced the expression of osteocalcin mRNA in these cells. In contrast, bone sialoprotein mRNA expression was increased by FGF2, while alpha1(I) procollagen mRNA expression was not altered. Additionally, neither Runx2 and osterix mRNA expression nor cell proliferation were affected by the addition of FGF2 in these high-density cultures, indicating that regulation by FGF2 may not be dependent on these transcription factors or on the proliferation of cells. Experiments using actinomycin D indicated that FGF2 decreased the stability of the MEPE/OF45 mRNA. Moreover, inhibition of a specific mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) by PD98059 blocked FGF2-regulated MEPE/OF45 expressions, indicating that this regulation requires the MAPK pathway. These results suggest that MEPE/OF45 gene is one of the targets of FGF2 and may play an important role during bone formation and calcification.
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PMID:Regulation of mRNA expression of matrix extracellular phosphoglycoprotein (MEPE)/ osteoblast/osteocyte factor 45 (OF45) by fibroblast growth factor 2 in cultures of rat bone marrow-derived osteoblastic cells. 1524 99

Extracellular matrix proteins (ECMs) serve as both a structural support for cells and a dynamic biochemical network that directs cellular activities. ECM proteins such as those of the SIBLING family (small integrin-binding ligand glycoprotein) could possess inherent growth factor activity. In this study, we demonstrate that exon 5 of dentin matrix protein 3 (phosphophoryn (PP)), a non-collagenous dentin ECM protein and SIBLING protein family member, up-regulates osteoblast marker genes in primary human adult mesenchymal stem cells (hMSCs), a mouse osteoblastic cell line (MC3T3-E1), and a mouse fibroblastic cell line (NIH3T3). Quantitative real-time PCR technology was used to quantify gene expression levels of bone markers such as Runx2, Osx (Osterix), bone/liver/kidney Alp (alkaline phosphatase), Ocn (osteocalcin), and Bsp (bone sialoprotein) in response to recombinant PP and stably transfected PP. PP up-regulated Runx2, Osx, and Ocn gene expression. PP increased OCN protein production in hMSCs and MC3T3-E1. ALP activity and calcium deposition was increased by PP in hMSC. Furthermore, an alpha(v)beta(3) integrin-blocking antibody significantly inhibited recombinant PP-induced expression of Runx2 in hMSCs, suggesting that signaling by PP is mediated through the integrin pathway. PP was also shown to activate p38, ERK1/2, and JNK, three components of the MAPK pathway. These data demonstrate a novel signaling function for PP in cell differentiation beyond the hypothesized role of PP in biomineralization.
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PMID:Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway. 1537 33

Glucocorticoid hormones have complex stimulatory and inhibitory effects on skeletal metabolism. Endogenous glucocorticoid signaling is required for normal bone formation in vivo, and synthetic glucocorticoids, such as dexamethasone, promote osteoblastic differentiation in several in vitro model systems. The mechanism by which these hormones induce osteogenesis remains poorly understood. We demonstrate here that the coordinate action of dexamethasone and the osteogenic transcription factor Runx2/Cbfa1 synergistically induces osteocalcin and bone sialoprotein gene expression, alkaline phosphatase activity, and biological mineral deposition in primary dermal fibroblasts. Dexamethasone decreased Runx2 phosphoserine levels, particularly on Ser125, in parallel with the upregulation of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) through a glucocorticoid-receptor-mediated mechanism. Inhibition of MKP-1 abrogated the dexamethasone-induced decrease in Runx2 serine phosphorylation, suggesting that glucocorticoids modulate Runx2 phosphorylation via MKP-1. Mutation of Ser125 to glutamic acid, mimicking constitutive phosphorylation, inhibited Runx2-mediated osteoblastic differentiation, which was not rescued by dexamethasone treatment. Conversely, mutation of Ser125 to glycine, mimicking constitutive dephosphorylation, markedly increased osteoblastic differentiation, which was enhanced by, but did not require, additional dexamethasone supplementation. Collectively, these results demonstrate that dexamethasone induces osteogenesis, at least in part, by modulating the phosphorylation state of a negative-regulatory serine residue (Ser125) on Runx2. This work identifies a novel mechanism for glucocorticoid-induced osteogenic differentiation and provides insights into the role of Runx2 phosphorylation during skeletal development.
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PMID:Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation. 1644 55

Protein kinase C (PKC) is a family of kinases whose isoforms show subtle differences in physiological and biochemical responses, with their expression being cell- specific. We hypothesize that there may be a specific profile of expression of PKC isoforms in differentiating osteoblastic cells (OBC) with individual isoforms having specific functions. Herein, the MC3T3-E1 cell line was used as a differentiating model, which was induced from the pre-osteoblast stage to mature osteoblast and characterized with several phenotypic markers, including alkaline phosphatase activity, osteocalcin and bone sialoprotein. The expression of PKC isoforms was monitored using Western blot analysis. Upon induction of osteogenesis, the intracellular localization of PKC eta and theta was determined using immunofluorescence. Lastly, the effect of P38 MAP kinase inhibition was determined using SB203580. Results show 1) PKC alpha, delta, lambda were all highly expressed in MC3T3-E1 osteoblastic cells, 2) the expression of PKC theta was significantly down-regulated upon induction of osteoblastic differentiation; 3) PKC eta was non-detectable at certain cell culture days; however, was up-regulated as the cells transit from each differentiation phase. The increased expression of PKC eta correlated with increases in OC, BSP levels and alkaline phosphatase activity. Immunofluorescence procedure confirmed the Western blot results with an increase in PKC eta and a decrease in PKC theta upon osteogenic stimulation. The inhibition of p38 resulted in a marked down-regulation of PKC eta. The data demonstrate that there is a specific profile of expression of PKC isoforms in differentiating osteoblasts; the different expression pattern of individual isoforms may be either a consequence of the differentiation itself or plays a role in the regulatory mechanism of osteoblastic differentiation. This study has provided primary information on the temporal pattern of expression of PKC isoforms in the differentiating osteoblast and further insight into their possible role in osteoblastic cell maturation.
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PMID:The expression profile of PKC isoforms during MC3T3-E1 differentiation. 1668 25

The intracellular signaling events controlling human mesenchymal stem cells (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERK-dependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECM-induced osteogenic differentiation of hMSC.
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PMID:Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. 1708 17


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