u-0126 and herbimycin

Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.

Topics: Animals; Benzoquinones; Binding Sites; Bone Marrow Cells; Butadienes; Cell Culture Techniques; Cell Line, Tumor; Clone Cells; Enzyme Inhibitors; Fibroblast Growth Factor 2; Homeodomain Proteins; Humans; Insulin-Like Growth Factor I; Integrin-Binding Sialoprotein; Lactams, Macrocyclic; Mutation; Nitriles; Osteosarcoma; Promoter Regions, Genetic; Quinones; Rats; Response Elements; Rifabutin; RNA, Messenger; Sialoglycoproteins; Transcription, Genetic

Matrix metalloproteinases (MMPs) play an important role in pulp tissue destruction. However, the mechanisms and signal transduction pathways involved in the production of MMPs in human pulp cells are not fully understood. The purpose of this study was to investigate the gelatinolytic activity in human pulp cells stimulated with various pharmacological agents.. Human dental pulp cells were cultured using an explant technique obtained from impacted third molars with informed consent of the patients. The effects of p38 inhibitor SB203580, MEK inhibitor U0126, extracellular signal-regulated kinase (ERK) inhibitor PD098059, phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002, cyclooxygenase-2 (COX-2) inhibitor NS-398, nuclear factor kappa B (NF-kappaB) inhibitor dexamethasone, and tyrosine kinase inhibitor herbimycin A on the production and secretion of MMPs by human pulp cells were determined by gelatin zymography.. The main gelatinase secreted by human pulp cells migrated at 72 kd and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kd regions that correspond to MMP-9. After a 4-day culture period, NS-398, dexamethasone, and herbimycin A were found to depress MMP-2 production (P<.05). The inhibition decreased in an order of dexamethasone >NS-398>herbimycin A. Human pulp cells, however, treated with various pharmacological agents had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions (P>.05).. These observations suggest that NS-398, dexamethasone, and herbimycin A can regulate MMP-2 produced by human pulp cells. The signal transduction pathways COX-2, NF-kappaB, and tyrosine kinase may be involved in the production of MMPs.

Topics: Benzoquinones; Butadienes; Cells, Cultured; Chromones; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dental Pulp; Dexamethasone; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Isoenzymes; Lactams, Macrocyclic; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Morpholines; NF-kappa B; Nitriles; Nitrobenzenes; p38 Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Pyridines; Quinones; Rifabutin; Signal Transduction; Sulfonamides

Tissue inhibitor metalloproteinases 1 (TIMP-1) and 2 have been reported to inhibit bone resorption. However, here, we report the direct action of both TIMP-1 and TIMP-2 on isolated rabbit mature osteoclasts to stimulate their bone-resorbing activity at significantly lower concentrations (approximately ng/ml) than those (approximately microg/ml) required for the inhibition of bone resorption. The cell population used in this study consisted of a mature osteoclast population with >95% purity. TIMP-1 (approximately 50 ng/ml) and TIMP-2 (approximately 8-10 ng/ml) increased the pit area excavated by the isolated mature osteoclasts. The stimulatory effects of TIMPs were abolished by simultaneous addition of anti-TIMP antibodies. At higher concentrations, the stimulation of bone resorption decreased reversely to the control level. The magnitude of the stimulatory effect of TIMP-2 was more than that of TIMP-1. Metalloproteinase inhibitors such as BE16627B and R94138 could not replace TIMPs with respect to the bone-resorbing activity, suggesting that the osteoclast-stimulating activity of TIMPs was independent of the inhibitory activity on matrix metalloproteinases (MMPs). TIMPs stimulated tyrosine phosphorylation of cellular proteins in the isolated mature osteoclasts. Both herbimycin A, an inhibitor of tyrosine kinases, and PD98059 and U0126, inhibitors of mitogen-activated protein kinase (MAPK), completely blocked the TIMP-induced stimulation of osteoclastic bone-resorbing activity. On the plasma membrane of osteoclasts, some TIMP-2-binding proteins were detected by a cross-linking experiment. These findings show that TIMPs directly stimulate the bone-resorbing activity of isolated mature osteoclasts at their physiological concentrations and that the stimulatory action of TIMPs is likely to be independent of their activities as inhibitors of MMPs.

Topics: Acetamides; Animals; Benzoquinones; Bone Resorption; Butadienes; Cells, Cultured; Dipeptides; Flavonoids; Humans; Lactams, Macrocyclic; Metalloendopeptidases; Mitogen-Activated Protein Kinases; Nitriles; Osteoclasts; Protein-Tyrosine Kinases; Quinones; Rabbits; Rifabutin; Succinates; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

Expression of the PRL gene is regulated by many factors, including cAMP, estradiol (E2), phorbol esters, epidermal growth factor (EGF), and TRH. The promoter region of the rat PRL gene has been shown to contain DNA sequences that are thought to support the direct interaction of estrogen receptors (ERs) with DNA. It is by this direct ER/DNA interaction that estrogen is thought to modulate expression of PRL. We report here that estrogeninduced PRL expression requires an intact mitogen-activated protein kinase (MAPK) signal transduction pathway in cultured rat pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines). Interfering with the MAPK signaling cascade by inhibiting the activity of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA and protein. In these cell lines, estrogen activates extracellular regulated protein kinases ERK-1 and ERK-2 enzyme activities maximally within 10 min of 1 nM E2 treatment. This activity is blocked by pretreatment of the cells with the MEK inhibitors PD98059 and UO126. The mechanism by which ERKs-1 and -2 are activated by estrogen appears to be independent of c-Src since the effects of estrogen on PRL gene expression are not affected by herbimycin A or PP1 administration. c-Raf-1 may be involved in the effects of E2 because estrogen causes the rapid and transient tyrosine phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both ERK-1 and ERK-2 activation in addition to PRL protein and mRNA, implying a central role for the classical ER in the activation of the MAPK pathway resulting in PRL gene expression.

Topics: Animals; Benzoquinones; Butadienes; Cells, Cultured; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Estrogens; Female; Flavonoids; Gene Expression Regulation; Lactams, Macrocyclic; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Pituitary Gland; Prolactin; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; Pyrazoles; Pyrimidines; Quinones; Rats; Rats, Inbred F344; Rifabutin; RNA, Messenger; Signal Transduction; src-Family Kinases; Steroids; Transcription, Genetic; Tyrosine