Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transforming growth factor-beta (TGF-beta) is a multifunctional regulator of a variety of cellular functions, including proliferation, differentiation, matrix synthesis, and apoptosis. In growth plate chondrocytes, TGF-beta slows the rate of maturation. Because the current paradigm of TGF-beta signaling involves Smad proteins as downstream regulators of target genes, we have characterized their role as mediators of TGF-beta effects on chondrocyte maturation. Both Smad2 and 3 translocated to the nucleus upon TGF-beta1 signaling, but not upon BMP-2 signaling. Cotransfection experiments using the TGF-beta responsive and Smad3 sensitive p3TP-Lux luciferase reporter demonstrated that wild-type Smad3 potentiated, whereas dominant negative Smad3 inhibited TGF-beta1 induced luciferase activity. To confirm the role of Smad2 and 3 as essential mediators of TGF-beta1 effects on chondrocyte maturation, we overexpressed both wild-type and dominant negative Smad2 and 3 in virally infected chondrocyte cultures. Overexpression of both wild-type Smad2 and 3 potentiated the inhibitory effect of TGF-beta on chondrocyte maturation, as determined by colx and alkaline phosphatase activity, whereas dominant negative Smad2 and 3 blocked these effects. Wild-type and dominant negative forms of Smad3 had more pronounced effects than Smad2. Our results define Smad2 and 3 as key mediators of the inhibitory effect of TGF-beta1 signaling on chondrocyte maturation.
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PMID:Smad2 and 3 mediate transforming growth factor-beta1-induced inhibition of chondrocyte maturation. 1110 88

Transforming growth factor (TGF) beta inhibits alkaline phosphatase (ALP) activity and mineralization in mouse osteoblastic MC3T3-E1 cells, whereas local administration of TGF-beta stimulates bone formation in vivo. We recently demonstrated that Smad3, a TGF-beta signaling molecule, promotes ALP activity and mineralization in MC3T3-E1 cells. Moreover, the target disruption of Smad3 in mouse is reported to cause a decrease in bone mineral density. These findings indicate that Smad3 plays an important role in the regulation of bone formation. However, why the effects of TGF-beta and Smad3 on ALP activity and mineralization are different remains unknown. The purpose of the present study is to clarify the role of mitogen-activated protein kinase (MAPK) in TGF-beta and Smad3 pathways in osteoblast. TGF-beta activated extracellular signal-regulated kinases/p42/p44 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in mouse osteoblastic MC3T3-E1 cells. The expression of dominant negative type Smad3, Smad3DeltaC, affected neither TGF-beta-activated MAPKs nor TGF-beta-inhibited ALP activity. Specific inhibitors of ERK1/2 activation (PD98059 and U0126), as well as JNK inhibitors (curcumin and dicumarol) antagonized the inhibitory effects of TGF-beta on ALP activity and mineralization, whereas the specific inhibitor of p38 MAPK (SB203580) did not affect them. PD98059 and curcumin enhanced Smad3-induced ALP activity and mineralization, whereas SB203580 inhibited them. In the luciferase reporter assay using 3TP-lux with the specific Smad3-responsive element, PD98059, and curcumin enhanced TGF-beta- and Smad3-induced transcriptional activity in MC3T3-E1 cells. On the other hand, TGF-beta-induced production of type I collagen was antagonized by curcumin but not by PD98059. The present study indicated that TGF-beta-responsive ERK1/2 and JNK cascades negatively regulate Smad3-induced transcriptional activity as well as ALP activity and mineralization in osteoblasts.
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PMID:Activations of ERK1/2 and JNK by transforming growth factor beta negatively regulate Smad3-induced alkaline phosphatase activity and mineralization in mouse osteoblastic cells. 1213 Jun 49

Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late-stage differentiation.
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PMID:Menin is required for bone morphogenetic protein 2- and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2. 1515 Feb 73

Follicle-stimulating hormone (FSH), a glycoprotein consisting of an alpha subunit and a unique beta subunit, is essential for gonadal development and function in vertebrates including teleosts. FSH is regulated by a variety of neuroendocrine and endocrine factors, and its biosynthesis is primarily determined by the expression of the beta subunit. Although the regulation of FSH biosynthesis has been well documented in mammals, the molecular mechanisms underlying the regulation are poorly understood. Our previous studies demonstrated that activin stimulated goldfish FSHbeta expression in the primary pituitary cell culture and enhanced its promoter activity in the mouse gonadotrope cell line LbetaT-2 cells. However, little is known about the signal transduction pathway involved in the transcriptional activation of this gene by activin. To assess the involvement of intracellular signaling protein Smads in regulating goldfish FSHbeta promoter, we first cloned full-length cDNAs for goldfish Smad2, Smad3, Smad4, and Smad7 from the pituitary. All Smads cloned show high sequence conservation with their mammalian counterparts. The spatial expression of these Smads overlapped with that of activin subunits and its receptors in various tissues examined. In addition, we demonstrated that activin induced Smad3 and Smad7 expression, but not Smad2 and Smad4. Co-transfection of Smad2 or Smad3 cDNA into the LbetaT-2 cells with the reporter construct of goldfish FSHbeta promoter significantly enhanced basal and activin-stimulated reporter (SEAP, secreted alkaline phosphatase) expression, while Smad7 completely blocked basal and Smad2/3-stimulated FSHbeta activity. Interestingly, the effect of Smad3 was much higher than that of Smad2, suggesting that Smad3 is likely the principal signal transducing molecule involved in activin stimulation of FSHbeta expression in the goldfish. This work lays a foundation for further analysis of goldfish FSHbeta promoter for the cis-regulatory elements involved in activin signaling.
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PMID:Cloning of Smad2, Smad3, Smad4, and Smad7 from the goldfish pituitary and evidence for their involvement in activin regulation of goldfish FSHbeta promoter activity. 1570

Central in the pathogenesis of glucocorticoid (GC)-induced osteoporosis is the effects of GC on bone formation. However, the mechanism of GC-inhibited bone formation is not well known. Transforming growth factor (TGF)-beta is most abundant in bone matrix compared with other tissues, and we have recently proposed that Smad3, a TGF-beta signaling molecule, is important for promoting bone formation. However, no reports have been available about the effects of GC on Smad3 in osteoblasts. In the present study, we investigated whether dexamethasone (Dex), an active GC analog, would affect the expression and activity of Smad3 in mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Dex significantly suppressed Smad3-stimulated alkaline phosphatase (ALP) activity, although it did not affect TGF-beta-inhibited ALP activity in MC3T3-E1 cells. Moreover, pretreatment with Dex suppressed TGF-beta-enhanced expression of type I collagen in MC3T3-E1 and UMR-106 cells. In the luciferase assay using p3TP-Lux with a Smad3-specific response element, Dex significantly suppressed the transcriptional activity induced by TGF-beta as well as Smad3. However, Dex did not affect the expression of Smad3 in these cells at both mRNA and protein levels. In conclusion, the present study indicates that Dex inhibits ALP activity and type I collagen expression, presumably by suppressing Smad3-induced transcriptional activity but not by modulating Smad3 expression in osteoblastic cells.
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PMID:Dexamethasone suppresses Smad3 pathway in osteoblastic cells. 1581 34

Chondrogenesis is a critical step in palatogenesis. All-trans retinoic acid (atRA), a vitamin A derivative, is a known teratogenic effector of cleft palate. Here, we evaluated the effects of atRA on the osteo-/chondrogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells. MEPM cells, in a high-density micromass environment, undergo active chondrogenesis in a manner analogous to that of limb-derived mesenchymal cells, and served as a valid model system to investigate the mechanisms regulating chondrogenesis during palatogenesis. atRA-treated MEPM micromass expressed relatively higher levels of osteoblastic gene markers (alkaline phosphatase and collagen type I) and lower levels of chondrocytic gene markers (collagen type II and aggrecan). As transforming growth factor-beta3 (TGF-beta3) is an essential growth factor for chondrogenesis of embryonic mesenchymal cells both in in vivo and in vitro conditions, we thereby explored the effects of atRA on TGF-beta3 signaling pathway. atRA led to an increase in mRNA expression of TGF-beta3 and an instantaneous decrease in TGF-beta type II receptor (TbetaRII) as determined by real-time RT-PCR. Further study showed that atRA inhibited phosphorylation of Smad2 and Smad3 and increased Smad7 expression. Activation of the Smad pathways by transfection with Smad7deltaC mutant or constitutively active TbetaRII retroviral vector abolished atRA-induced inhibition of chondrogenesis as indicated by Alcian blue staining, indicating that Smad signaling is essential for this response. Taken together, these data for the first time demonstrated a role for RA-induced hypochondrogenesis through regulation of the TGF-beta3 pathway and suggested a role for TbetaRII /Smad in retinoid-induced cleft palate.
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PMID:All-trans retinoic acid inhibited chondrogenesis of mouse embryonic palate mesenchymal cells by down-regulation of TGF-beta/Smad signaling. 1641 76

This study examined the effect of wild-type Smad3 gene on the osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. Bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the complexes of pcDNA3. 0-Myc-Smad3 or pcDNA3. 0-Myc-Smad3deltaC and Lipofectamine reagent. Immunofluorescence staining was performed to evaluate the c-Myc signal in MSCs. The cell proliferation was detected by MTT method. To clarify the osteoblastic characteristics in stably transfected MSCs, alkaline phosphatase (ALP) mRNA and core binding factor alpha1 (Cbfa1) mRNA were investigated by RT-PCR, and ALP activity and mineralization were examined by p-nitrophenolphosphate method and alizarin red staining respectively. PD98059, a specific inhibitor of the ERK signaling pathway, was used to determine the role of ERK in Smad3-MSCs osteoblastic differentiation. c-Myc signal was detected in Smad3-MSCs and Smad3 deltaC-MSCs. The proliferation of Smad3-MSCs was slower than that of Smad3deltaC-MSCs or V-MSCs. The relative levels of ALP mRNA and Cbfal mRNA in Smad3-MSCs, as well as ALP activity and mineralization, were markedly higher than those in Smad3deltaC-MSCs or V-MSCs. Although ALP activity and mineralization were slightly lower in Smad3-MSCs treated with PD98059 than in those without PD98059 treatment, no significant difference was found between them (P > 0.05). It is concluded that the wild-type Smad3 gene, which is a crucial component promoting bone formation, can inhibit the proliferation of MSCs and enhance the osteoblastic differentiation of uncommitted MSCs and the maturation of committed MSCs independent of the ERK signaling pathway.
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PMID:Wild-type Smad3 gene enhances the osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. 1669 23

Hepatitis C-associated osteosclerosis (HCAO) is a rare syndrome characterized by severe, acquired, generalized osteosclerosis and hyperostosis in adults who are infected with the hepatitis C virus. However, the detail of the pathogenesis of HCAO is still unknown. We examined the effects of serum of the HCAO patient on the proliferation, alkaline phosphatase (ALP) activity and transforming growth factor (TGF)-beta-Smad signaling in mouse osteoblastic cells. The patient was compatible with HCAO, characterized by high bone mass, bone thickening and bone pain with normal lamelar bone. The serum from the HCAO patient increased the levels of TGF-beta and Smad3 expression in osteoblastic MC3T3-E1 cells, compared with the control subject. Moreover, the serum from the HCAO patient significantly augmented TGF-beta-induced transcriptional activity with luciferase assay using 3TP-Lux with a Smad3-specific responsive element. In addition, the serum from the HCAO patient significantly stimulated the MTT intensity, the level of proliferating cell nuclear antigen expression, a proliferation marker, and ALP activity in MC3T3-E1 cells, compared with that from the control subject. In conclusion, the present study indicated that the serum from the HCAO patient stimulated TGF-beta-Smad signaling, as well as the proliferation and ALP activity in osteoblastic cells. Some soluble factors other than parathyroid hormone might be related to the pathogenesis of HCAO.
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PMID:Serum soluble factors induce the proliferation, alkaline phosphatase activity and transforming growth factor-beta signal in osteoblastic cells in the patient with hepatitis C-associated osteosclerosis. 1717 44

Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV-family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up-regulated by exogenous TGF-beta. In this study TGF-beta-dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF-beta-free conditions was analysed by Western-blots, metabolic labelling, and CTGF-reporter gene assays. In alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP)-staining of cultured hepatocytes it was demonstrated that latent TGF-beta within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF-beta antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p-Smad2, whereas p-Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF-beta, but calpain inhibitors partially suppressed intracellular TGF-beta activation and subsequently CTGF up-regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF-beta signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4-/Alk 5-specific inhibitor SB-431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF-beta is activated by calpains during culture-induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF-beta. In conclusion, these data are the first to suggest the possibility of intracrine TGF-beta signalling due to calpain-dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF-beta-sensitive reporter gene. This mechanism might be deleterious for keeping long-term hepatocyte cultures due to TGF-beta-induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial-mesenchymal transition of hepatocytes in injured liver.
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PMID:Activation of TGF-beta within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor. 1826 73

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt-beta-catenin signaling pathway via the transforming growth factor (TGF)-beta signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF-beta-independent in stimulating the osteoblast phenotype and PTH-induced Wnt-beta-catenin signaling. For this, the TGF-beta receptor type 1 [activin receptor-like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total beta-catenin and reduced phosphorylated beta-catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3-E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total beta-catenin and reduction of phosphorylated beta-catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF-beta receptor signaling. On the other hand, MC3T3-E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated beta-catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3-E1 cell clones in which wild-type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the beta-catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast beta-catenin levels via Smad3 independently of, and dependently on, TGF-beta in the early and later induction phases, respectively.
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PMID:Role of Smad3, acting independently of transforming growth factor-beta, in the early induction of Wnt-beta-catenin signaling by parathyroid hormone in mouse osteoblastic cells. 1958 75


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