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

Mechanical unloading causes detrimental effects on the skeleton, but the underlying mechanisms are still unclear. We investigated the effect of microgravity on osteoblast ability to regulate osteoclastogenesis. Mouse osteoblast primary cultures were grown for 24 h at unit gravity or under simulated microgravity, using the NASA-developed Rotating Wall Vessel bioreactor. Conditioned media (CM) from osteoblasts subjected to microgravity increased osteoclastogenesis and bone resorption in mouse bone marrow cultures. In these osteoblasts, the RANKL/OPG ratio was higher relative to 1g. Consistently, treatment with high concentrations of OPG-inhibited osteoclastogenesis and bone resorption in the presence of CM arising from osteoblasts cultured under microgravity. Microgravity failed to affect osteoblast differentiation and function in the time frame of the experiment, as we found no effect on alkaline phosphatase mRNA and activity, nor on Runx2, osteocalcin, osteopontin, and collagen1A2 mRNA expression. In contrast, microgravity induced a time dependent increase of ERK-1/2 phosphorylation, while phospho-p38 and phospho-JNK remained unchanged. Apoptosis, revealed by bis-benzimide staining, was similar among the various gravity conditions, while it was increased under microgravity after treatment with the MEK-1/2 inhibitor, PD98059, suggesting a protection role by ERK-1/2 against cell death. In conclusion, microgravity is capable to indirectly stimulate osteoclast formation and activity by regulating osteoblast secretion of crucial regulatory factors such as RANKL and OPG. We hypothesize that this mechanism could contribute to bone loss in individuals subjected to weightlessness and other unloading conditions.
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PMID:Modeled microgravity stimulates osteoclastogenesis and bone resorption by increasing osteoblast RANKL/OPG ratio. 1692 71

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.
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PMID:Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells. 1697 47

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. In conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.
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PMID:Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells. 1697 71

The aim of this study was to identify the signal transduction pathways and mechano-transducers that play critical roles in the processes induced by changes in cyclic hydrostatic pressure and fluid shear in 3-dimensional (3D) culture systems. Mesenchymal stem cells were loaded into a polymeric scaffold and divided into three groups according to the stress treatment: static, fluid shear, and hydrostatic pressure with fluid shear. Cells were exposed daily to a hydrostatic pressure of 0.2 MPa for 1 min followed by 14 min rest with fluid flow at 30 rpm. Protein extracts were analyzed by Western blot for extracellular signal-regulated kinase 1/2 (ERK1/2). The complexes were cultured under the mechanical stimuli for 21 days with or without phospho-ERK1/2 inhibitor (U0126) and evaluated by RT-PCR, calcium contents, and immunohistochemistry. Under conditions of mechanical stimulation, the activation of ERK1/2 was sustained or increased with time. U0126 suppressed mechanical stimuli-induced expression of osteocalcin. In addition, calcium contents and the degrees of osteocalcin and osteopontin staining were decreased by this inhibitor. These results demonstrate that mechanical stimuli, particularly hydrostatic pressure with fluid shear, enhance osteogenesis in 3D culture systems via ERK1/2 activation.
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PMID:ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure. 1706 Dec 51

Impairment of the complex regulatory network of cell death and survival is frequently the reason for therapy resistance of breast cancer cells and a major cause of tumor progression. We established two independent cell lines from a fast growing mouse breast tumor (WAP-SVT/t transgenic animal). Cells from one line (ME-A cells) are sensitive to apoptotic stimuli such as growth factor depletion or treatment with antitumor agents (e.g. doxorubicin). Cells from the second line (ME-C cells), which carry a missense mutation at the p53 codon 242, are very insensitive to apoptotic stimuli. Co-cultivation experiments revealed that the ME-C cells mediate cell death resistance to the ME-A cells. Microarray and Western blot analysis showed that osteopontin (OPN) is selectively overexpressed by the ME-C cells. This glycoprotein is the most abundant protein secreted by the ME-C cells and we obtained strong indications that OPN is the main antiapoptotic factor. However, the OPN containing ME-C cell medium does not alter the expression level of pro- or antiapoptotic genes or known inhibitors of apoptosis (IAPs). Its signaling involves mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)1/2 as the kinase inhibitor PD98059 restores apoptosis but not the Akt inhibitor. In the ME-A cells, mitochondrial cytochrome c release occurs with and without external apoptotic stimuli. OPN containing ME-C cell medium does not prevent the mitochondrial cytochrome c release and caspase-9 processing. In serum starved ME-A cells, the OPN containing ME-C cell medium prevents caspase-3 activation. However, in doxorubicin-treated cells, although apoptosis is blocked, it does not inhibit caspase-3. This indicates that the ME-A cells distinguish between the initial apoptotic stimuli and that the cells possess a further uncharacterized control element acting downstream from caspase-3.
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PMID:Chemotherapy resistance of mouse WAP-SVT/t breast cancer cells is mediated by osteopontin, inhibiting apoptosis downstream of caspase-3. 1716 24

IGF-I stimulation of smooth muscle cell (SMC) migration and proliferation requires alphaVbeta3 ligand occupancy. We hypothesized that changes in the levels of extracellular matrix proteins induced by alterations in glucose concentrations may regulate the ability of SMCs to respond to IGF-I. IGF-I stimulated migration and proliferation of SMCs that had been maintained in 25 mM glucose containing media, but it had no stimulatory effect when tested using SMCs that had been grown in 5 mM glucose. IGF-I stimulated an increase in Shc phosphorylation and enhanced activation of the MAPK pathway in SMCs grown in 25 mM glucose, whereas in cells maintained in 5 mM glucose, IGF-I had no effect on Shc phosphorylation, and the MAPK response to IGF-I was markedly reduced. In cells grown in 25 mM glucose, the levels of alphaVbeta3 ligands, e.g. osteopontin, vitronectin, and thrombospondin, were all significantly increased, compared with cells grown in 5 mM glucose. The addition of these alphaVbeta3 ligands to SMCs grown in 5 mM glucose was sufficient to permit IGF-I-stimulated Shc phosphorylation and downstream signaling. Because we have shown previously that alphaVbeta3 ligand occupancy is required for IGF-I-stimulated Shc phosphorylation and stimulation of SMC growth, our data are consistent with a model in which 25 mM glucose stimulates increases in the concentrations of these extracellular matrix proteins, thus enhancing alphaVbeta3 ligand occupancy, which leads to increased Shc phosphorylation and enhanced cell migration and proliferation in response to IGF-I.
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PMID:Hyperglycemia alters the responsiveness of smooth muscle cells to insulin-like growth factor-I. 1725 2

Inorganic pyrophosphate (PP(i)) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P(i) in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP(i) are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP(i) on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP(i). This inhibition could be reversed by Tnap, but not if PP(i) was bound to mineral. PP(i) also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP(i) was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP(i), hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP(i) inhibits Tnap-mediated P(i) release from beta-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP(i) prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.
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PMID:Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity. 1738 65

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Since tumor metastasis shortens patients' lifetime, establishment of therapy for anti-metastasis is very important. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, extracellular matrix (ECM) invasion and cell proliferation via interaction with its receptor, that is, alphavbeta3 integrin. OPN is believed to be a positive regulator of tumor metastasis in vivo. However, how OPN regulates metastasis is largely unknown. Here, we explore the role of OPN in cell migration. Serum from wild-type mice induced cell migration of B16 melanoma cells, while serum from OPN-deficient mouse suppressed this event. The presence of recombinant OPN significantly enhanced cell migration compared to albumin containing medium. OPN-induced cell migration was suppressed by inhibiting the ERK/MAPK pathway indicating that OPN-induced cell migration depends on this pathway. Overexpression of OPN in these cancer cells per se promoted cell proliferation and tended to increase B16 cell migration suggesting that OPN promotes bone metastasis by playing dual roles both in host microenvironment and in tumor cell itself. In conclusion, the elevated OPN expression in host tissue and tumor cell itself promotes tumor cell migration reading to tumor metastasis, suggesting that neutralization of OPN-induced signal might be effective in suppression of tumor metastasis.
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PMID:Serum osteopontin, an enhancer of tumor metastasis to bone, promotes B16 melanoma cell migration. 1739 Mar 43

The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression correlates with end-organ protection regardless of overt antihypertensive action.
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PMID:P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1. 1743 56

The extracellular matrix protein osteopontin (OPN) plays a nonredundant role in atherosclerosis and restenosis. Here we investigated the impact of OPN up-regulation in an in vitro model of re-endothelialization after mechanical injury of the endothelial cell monolayer. Murine aortic endothelial (MAE) cells interact via alpha(v) integrins with the integrin-binding Arg-Gly-Asp OPN sequence and adhere to immobilized OPN. On this basis, MAE cells were stably transfected with a wild-type OPN cDNA (OPN-MAE cells), with an OPN mutant lacking the Arg-Gly-Asp sequence (DeltaRGD-OPN-MAE cells), or with vector alone (mock-MAE cells). When compared with mock-MAE and DeltaRGD-OPN-MAE cells, OPN-MAE cells showed a reduced sprouting activity in fibrin gel, a reduced motility in a Boyden chamber assay, and a reduced capacity to repair the wounded monolayer. Accordingly, OPN-MAE cells at the edge of the wound were unable to form membrane ruffles, to reorganize their cytoskeleton, and to activate the focal adhesion kinase and the small GTPase Rac1, key regulators of the cell entry into the first phase of the cell migration cycle. Accordingly, wounded OPN-MAE cells failed to activate the intracellular signals RhoA and ERK1/2, involved in the later phases of the cell migration cycle. Also, parental MAE cells showed reduced re-endothelialization after wounding when seeded on immobilized OPN and exhibited increased adhesiveness to OPN-enriched extracellular matrix. In conclusion, OPN up-regulation impairs re-endothelialization by inhibiting the first phase of the cell migration cycle via alpha(v) integrin engagement by the extracellular matrix-immobilized protein. This may contribute to the adverse effects exerted by OPN in restenosis and atherosclerosis.
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PMID:Osteopontin overexpression inhibits in vitro re-endothelialization via integrin engagement. 1745 74


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