Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P05109 (S100A8)
 1,212 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TGFbeta is of crucial importance during transdifferentiation of resting retinoid-storing hepatic stellate cells (HSC) to extracellular matrix producing myofibroblasts (MFB) and consequently, inhibition of TGFbeta signal transduction is an effective means for preventing experimental fibrosis. We have shown that isolated HSC lose TGFbeta-dependent growth control during in vitro activation and that alpha2 (I) collagen production in transdifferentiated MFB is TGFbeta-independent. Furthermore, Smad complexes with SBE binding activity were only detected in early cultures of HSC, although TGFbeta receptor types I and II were significantly expressed in HSC and MFB. In the present report, we compared the expression pattern of TGFbeta downstream mediators, i.e., the Smads, in TGFbeta responsive HSC versus nonresponding MFB. The transdifferentiation process was monitored by morphology and increasing expression of TGFbeta and alpha-smooth muscle actin, and TGFbeta signaling was investigated by (CAGA)(9)-MLP-Luc. The expression level of all Smads remained essentially unchanged both during the activation process and after TGFbeta-treatment. Smad7 was transiently upregulated upon TGFbeta stimulation in quiescent HSC, indicating a negative feed back loop in responsive cells. In contrast, MFB neither displayed TGFbeta-inducible nor constitutively upregulated Smad7 expression. Instead, Smad3 mRNA was increased in MFB. Our data indicate that abrogation of the TGFbeta response in MFB versus HSC is not based on different regulation of Smad expression.
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PMID:Expression of Smads during in vitro transdifferentiation of hepatic stellate cells to myofibroblasts. 1134 60

Smads, signal transducers of the transforming growth factor-beta (TGF-beta) superfamily proteins, directly bind to DNA and regulate transcription of target genes. Smad3 binds to CAGA box, whereas Smad1 and Smad5 preferentially bind to GC-rich sequences. The beta-hairpin loop in the amino-terminal Mad homology 1 (MH1) domain is the direct DNA-binding site of Smad3; however, the amino acid sequences of the beta-hairpin loop of Smad3 and Smad1/5 are identical, suggesting that other regions may be responsible for the differential DNA binding of Smad3 and Smad1/5. To identify regions other than the beta-hairpin loop responsible for specific DNA binding of Smad3, we generated chimeras containing various regions of Smad3 and Smad1. Luciferase assays using a TGF-beta-responsive reporter (CAGA)9-MLP-Luc and gel-mobility shift assays using 3xCAGA as a probe revealed that alpha-helix 2 (H2) in the amino-terminal part of the MH1 domain plays an important role in specific DNA binding and transcriptional activation of Smad3. Luciferase assays using natural TGF-beta-responsive reporters also revealed the functional importance of H2 in the Smad3 MH1 domain in direct DNA binding. Smad3 thus binds to DNA directly through the beta-hairpin loop, and H2 supports specific DNA binding of Smad3.
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PMID:Alpha-helix 2 in the amino-terminal mad homology 1 domain is responsible for specific DNA binding of Smad3. 1138 74

We have studied the effects of two new synthetic triterpenoids, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative, 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), on transforming growth factor (TGF)-beta/Smad signaling. These agents, at nanomolar concentrations, increase the expression of TGF-beta-dependent genes, such as those for plasminogen activator inhibitor 1 and the type II TGF-beta receptor, and they synergize with TGF-beta in this regard. They prolong the activation of Smad2 induced by TGF-beta and markedly enhance the ability of Smad3 to activate a Smad binding element, CAGA-luciferase. In transfection assays, they reverse the inhibitory effects of Smad7. CDDO and CDDO-Im also enhance Smad signaling in the pathways of two other members of the TGF-beta superfamily, namely, activin and bone morphogenetic protein. Finally, these triterpenoids induce expression of the transcriptional coactivator p300-CBP-associated factor and synergize with TGF-beta in this regard. These are the first studies to report enhancement of Smad signaling by synthetic triterpenoids and should further their optimal use for applications in prevention or treatment of diseases in which there is aberrant function of TGF-beta.
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PMID:Synthetic triterpenoids enhance transforming growth factor beta/Smad signaling. 1264 1

Transforming growth factor-beta (TGF-beta)-induced alpha-smooth muscle actin (ASMA) expression is a key indicator of myofibroblast differentiation from fibroblasts. Recent studies suggest that a TGF-beta control element is important in the regulation of the ASMA gene promoter by TGF-beta. In this study, the role of Smad3, a key component of the Smad pathway that mediates TGF-beta signaling in regulation of ASMA gene expression, is investigated. All members of the Smad family were expressed in rat lung fibroblasts, and Smad3 expression was elevated upon TGF-beta 1 treatment. Transfection with a Smad3-expressing plasmid markedly increased Smad3 and ASMA protein expression, whereas transfection with an antisense Smad3 plasmid suppressed Smad3 and ASMA expression. Similar effects were noted when the cloned rat ASMA promoter-luciferase reporter gene construct was used to monitor transcriptional activation of the ASMA gene. Electrophoretic mobility shift assays and DNA affinity precipitation indicated Smad3 binding to at least two regions of the promoter containing CAGA motifs, termed Smad3-binding elements (SBEs). Mutation of one of the SBEs decreased promoter activity significantly, indicative of a functional role for this SBE. Taken together, these findings suggest a role for Smad3 in TGF-beta regulation of ASMA gene expression in myofibroblast differentiation.
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PMID:Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression. 1270 45

Bleomycin produces its fibrogenic effect, at least in part, by TGF-beta1 secretion. Treatment of IMR-90 human embryonic lung fibroblasts with bleomycin at 0.5 microg/ml results in a 1.6-fold increase of TGF-beta1 as determined by a specific ELISA assay for TGF-beta1 after acidification of the conditioned media. This elevation of TGF-beta1 secretion is furthermore enhanced in vivo by TGF-beta1 autoinduction of the TGF-beta1 gene. To demonstrate TGF-beta1 autoinduction, the fibroblasts were pretreated with 12.5 ng/ml TGF-beta1, washed extensively to remove any residual TGF-beta1, and then allowed to incubate for 24 h in AIM V synthetic serum-free media. The media when assayed using the ELISA assay contained a 1.6-fold increase of TGF-beta1. The distal promoter of the human TGF-beta1 gene contains a Smad 3 element (CAGGACA), which is homologous to the Smad 3 binding element motif (CAGA). The nuclear extracts of human embryonic lung fibroblasts treated for either 15 min or 24 h with TGF-beta1 did not demonstrate specificity of binding of a protein(s) to the homologous Smad 3 element as determined by cold wild-type oligodeoxynucleotide competition experiments. However, specific Smad 3 binding to the Smad 3 element (GTCTAGAC) found in proximal promoter of the Smad 7 gene was observed by cold oligo competition and supershift assays using a goat polyclonal Smad 3 antibody in the presence and absence of an N-terminal Smad 3 peptide. To determine the functionality of this Smad 3 binding to the Smad 3 element in the proximal promoter of the Smad 7 inhibitory gene to TGF-beta1 secretion, fibroblasts were transiently pretransfected with double-stranded phosphorothioate oligo "decoys" containing the Smad 7/Smad 3 element in the presence of plasmin to convert latent TGF-beta1 to active TGF-beta1. Under these conditions, which simulate the in vivo situation of 2.2-fold increase of total active TGF-beta1 was observed. Fibroblasts were also pretransfected with these double-stranded oligo "decoys," washed, then treated with TGF-beta1, washed and incubated in AIM V for an additional 24 h. In this latter experiment, a superinduction of TGF-beta1 secretion was observed. We propose that these oligo "decoys" bind Smad 3 preventing this initiation factor from binding to the Smad 7/Smad 3 element thereby decreasing the transcription of the Smad 7 gene. The decrease of the inhibitory Smad 7 would result in less binding of this Smad inhibitor to the Type I TGF-beta receptor and less antagonism of active TGF-beta1, more autoinduction of the TGF-beta1 gene, and more of the fibrogenic effects of TGF-beta1.
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PMID:TGF-beta1-induced Smad 3 binding to the Smad 7 gene: knockout of Smad 7 gene transcription by sense phosphorothioate oligos, autoregulation, and effect on TGF-beta1 secretion: bleomycin acts through TGF-beta1. 1276 81

Growth differentiation factor-9 (GDF-9) is an oocyte-derived growth factor and a member of the TGF-beta superfamily that includes TGF-beta, activin, and bone morphogenetic proteins (BMPs). GDF-9 is indispensable for the development of ovarian follicles from the primary stage, and treatment with GDF-9 enhances the progression of early follicles into small preantral follicles. Similar to other TGF-beta family ligands, GDF-9 likely initiates signaling mediated by type I and type II receptors with serine/threonine kinase activity, followed by the phosphorylation of intracellular transcription factors named Smads. We have shown previously that GDF-9 interacts with the BMP type II receptor (BMPRII) in granulosa cells, but the type I receptor involved is unknown. Using P19 cells, we now report that GDF-9 treatment stimulated the CAGA-luciferase reporter known to be responsive to TGF-beta mediated by the type I receptor, activin receptor-like kinase (ALK)5. In contrast, GDF-9 did not stimulate BMP-responsive reporters. In addition, treatment with GDF-9 induced the phosphorylation of Smad2 and Smad3 in P19 cells, and the stimulatory effect of GDF-9 on the CAGA-luciferase reporter was blocked by the inhibitory Smad7, but not Smad6. We further reconstructed the GDF-9 signaling pathway using Cos7 cells that are not responsive to GDF-9. After overexpression of ALK5, with or without exogenous Smad3, the Cos7 cells gained GDF-9 responsiveness based on the CAGA-luciferase reporter assay. The roles of ALK5 and downstream pathway genes in mediating GDF-9 actions were further tested in ovarian cells. In cultured rat granulosa cells from early antral follicles, treatment with GDF-9 stimulated the CAGA-luciferase reporter activity and induced the phosphorylation of Smad3. Furthermore, transfection with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 actions. In conclusion, although GDF-9 binds to the BMP-activated type II receptor, its downstream actions are mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins. Because ALK5 is a known receptor for TGF-beta, diverse members of the TGF-beta family of ligands appear to interact with a limited number of receptors in a combinatorial manner to activate two downstream Smad pathways.
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PMID:Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5. 1468 52

Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.
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PMID:Identification and characterization of a human smad3 splicing variant lacking part of the linker region. 1498 Jul 11

Bone morphogenetic protein-7 (BMP7) is expressed in adult kidney and reduces renal fibrogenesis when given exogenously to rodents with experimental chronic nephropathies. In mesangial cells that regulate glomerular fibrosis in vivo, BMP7 inhibits transforming growth factor beta (TGF-beta)-driven fibrogenesis, primarily by preventing the TGF-beta-dependent down-regulation of matrix degradation and up-regulation of PAI-1. The signals and mechanisms of the BMP7 opposition to actions of TGF-beta are unknown. Here we show in mesangial cells that BMP7 reduces nuclear accumulation of Smad3 and blocks the transcriptional up-regulation of the TGF-beta/Smad3 target, CAGA-lux. Smad5 knock-down impairs the ability of BMP7 to interfere with the activation of CAGA-lux and the accumulation of PAI-1 by TGF-beta indicating that Smad5 is required. Smad5 knock-down also reduces the rise in Smad6 upon BMP7. Forced expression of smad5 (found to be the preferred BMP7-induced receptor-activated Smad signal in mesangial cells) or of smad6 mimics BMP7 in opposing the increase in transcriptional activation of PAI-1 and its secretion upon TGF-beta. This suggests a model for the BMP7-induced opposition to TGF-beta-dependent mesangial fibrogenesis requiring Smad5; the model involves the inhibitory Smad6 downstream of Smad5 as well as reduced availability of Smad3 in the nucleus. BMP7 does not require signaling through Erk1/2, p38, or JNK and does not utilize the TGF-beta transcriptional co-repressors Ski or SnoN in mesangial cells. These studies provide first insights into mechanisms through which BMP7 opposes TGF-beta-induced glomerular fibrogenesis.
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PMID:Bone morphogenetic protein-7 signals opposing transforming growth factor beta in mesangial cells. 1504 7

c-Ski inhibits transforming growth factor-beta (TGF-beta) signaling through interaction with Smad proteins. c-Ski represses Smad-mediated transcriptional activation, probably through its action as a transcriptional co-repressor. c-Ski also inhibits TGF-beta-induced downregulation of genes such as c-myc. However, mechanisms for transcriptional regulation of target genes by c-Ski have not been fully determined. In this study, we examined how c-Ski inhibits both TGF-beta-induced transcriptional activation and repression. DNA-affinity precipitation analysis revealed that c-Ski enhances the binding of Smad2 and 4, and to a lesser extent Smad3, to both CAGA and TGF-beta1 inhibitory element probes. A c-Ski mutant, which is unable to interact with Smad4, failed to enhance the binding of Smad complex on these probes and to inhibit the Smad-responsive promoter. These results suggest that stabilization of inactive Smad complexes on DNA is a critical event in c-Ski-mediated inhibition of TGF-beta signaling.
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PMID:c-Ski inhibits the TGF-beta signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements. 1510 21

Amadori-modified glycated albumin stimulates extracellular matrix and transforming growth factor-beta (TGF-beta) expression in cultured mesangial cells. Smad proteins transduce the TGF-beta-mediated signal, and Smad-binding CAGA sequences are present in the plasminogen activator inhibitor-1 (PAI-1) promoter. This study examined whether glycated albumin induces PAI-1 transcription in human mesangial cells (HMC) through Smad-binding sites in the PAI-1 promoter. Quiescent HMC were exposed to 200 microg/ml bovine serum albumin (BSA) or glycated BSA (Gly-BSA) for 12-72 h. At 24 h, Gly-BSA stimulated TGF-beta1 and PAI-1 mRNA expression in HMC to 1.8 and 3.2 times that in the BSA-treated control cells. Gly-BSA also activated the PAI-1 promoter luciferase activity 2.3-fold. Gly-BSA-treated cells enhanced Smad2 and Smad3 protein levels 2.5 times the control levels in the nuclei. An electrophoretic mobility shift assay performed using CAGA sequences as a probe showed that Gly-BSA increased DNA/protein complexes. When nuclear extracts were preincubated with 100-fold molar excess of unlabeled CAGA oligonucleotide, the formation of complex was prevented. The DNA-binding protein was shown to be Smad3 by antibody supershift. Transfection of phosphorothioate CAGA oligonucleotide, a CAGA antisense analog, inhibited Gly-BSA-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked Gly-BSA-induced PAI-1 promoter luciferase activity. These results indicate that Gly-BSA increases DNA binding activity of Smad3 and that it stimulates PAI-1 transcription through Smad-binding CAGA sequences in the PAI-1 promoter in HMC. Thus progression of diabetic nephropathy may be promoted by PAI-1 upregulation mediated by the glycated albumin-induced Smad/DNA interactions.
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PMID:Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells. 1519 28


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