Spl Phosphorylation Induced by Serum Stimulates the Human α2(I) Collagen Gene Expression

Ihn, Hironobu; Ihn, Yoshie; Trojanowska, Maria

doi:10.1046/j.0022-202x.2001.01371.x

Cited by 23 publications

(23 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Phosphorylated levels of Smad3 or p85 were examined by immunoprecipitation using anti-Smad3 and antiphosphoserine-specific antibodies or anti-p85 and anti-phosphotyrosine antibodies, respectively (Ihn et al, 2001a;Yamane et al, 2003b). The same membrane was then stripped and reprobed with anti-Smad2/3 (N-19) or p85 antibody to show the total amount of Smad3 or p85, respectively.…”

Section: Methodsmentioning

confidence: 99%

Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on Transforming Growth Factor-β1-Induced Extracellular Matrix Expression

Ihn²,

2005

Self Cite

View full text Add to dashboard Cite

This is the first report that characterizes specific inhibitor of Smad3 (SIS3) as a potent and selective inhibitor of Smad3 function. In the reporter assay, the increased luciferase activity of p3TP-lux by the overexpression of constitutively active form of ALK-5 was abrogated by the treatment with SIS3 in a dosedependent manner. Immunoprecipitation revealed that SIS3 attenuated the transforming growth factor (TGF)-␤1-induced phosphorylation of Smad3 and interaction of Smad3 with Smad4. On the other hand, this reagent did not affect the phosphorylation of Smad2. Thereafter, we evaluated the ability of SIS3 in the suppression of the TGF-␤1-induced type I procollagen up-regulation in human dermal fibroblasts. We found that the addition of SIS3 attenuated the effects of TGF-␤1 by reducing the transcriptional activity. SIS3 also inhibited the myofibroblast differentiation of fibroblasts by TGF-␤1. Moreover, we demonstrated that SIS3 completely diminished the constitutive phosphorylation of Smad3 as well as the up-regulated type I collagen expression in scleroderma fibroblasts. Together, our study suggested that SIS3 is a useful tool to evaluate the TGF-␤-regulated cellular mechanisms via selective inhibition of Smad3.Transforming growth factor (TGF)-␤1 plays a critical role in a variety of biological processes, including proliferation, differentiation, extracellular matrix production, and apoptosis. The diverse cellular responses elicited by TGF-␤1 are triggered by the activation of serine/threonine kinase TGF-␤ receptors. On activation by TGF-␤1 or related ligands, signaling from the receptors to the nucleus is mediated by phosphorylation of cytoplasmic mediators called Smads. The receptor-associated Smads, such as Smad2 and Smad3, interact directly with, and are phosphorylated by, activated TGF-␤ receptor type I (Nakao et al., 1997). They are ligandspecific and form, on phosphorylation, heteromeric complexes with Smad4. The latter functions as a common mediator for all Smad pathways. These complexes then are translocated into the nucleus, where they function as transcription factors, possibly in association with other proteins, such as Sp1. The third group of Smad proteins, the inhibitory Smads such as Smad6 or Smad7, prevents phosphorylation and/or nuclear translocation of receptor-associated Smads.TGF-␤1 has been implicated in the development of fibrotic condition, including skin, lung, or liver. Systemic sclerosis or scleroderma is an acquired disorder that typically results in fibrosis of the skin and internal organs. Fibroblasts from affected scleroderma skin cultured in vitro produce excessive amounts of extracellular matrix (ECM), various collagens, mainly type I and III collagens, and display increased transcription of corresponding genes (Hitraya and Jimenez, 1996). Many of the characteristics of scleroderma fibroblasts resemble those of normal fibroblasts stimulated by TGF-␤1 (LeRoy et al., 1989), suggesting that activation of dermal fibroblast in scleroderma may be a result of stimulation by aut...

show abstract

Section: Methodsmentioning

confidence: 99%

Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on Transforming Growth Factor-β1-Induced Extracellular Matrix Expression

Ihn²,

2005

Self Cite

View full text Add to dashboard Cite

show abstract

“…Monolayers were washed, and the cells were transfected by the Lipofectin technique (FuGENE 6 Transfectin Reagent; Roche Applied Science) (12)(13)(14)(15) with Ϫ3500 COL1A2/chloramphenicol acetyltransferase constructs. pSV-␤-galactosidase control vector (Promega) was cotransfected to normalize for transfection efficiency (12)(13)(14)(15)(17)(18)(19)(20)(21)(22)(23). After incubation overnight, the medium was replaced with MEM containing 0.1% bovine serum albumin or with MEM containing various amounts of cytokines, and incubation was continued for 48 h. The cells were harvested in 0.25 M Tris-HCl (pH 8) and fractured by freeze thawing.…”

Section: Methodsmentioning

confidence: 99%

“…The remaining cells were washed with cold phosphate-buffered saline twice and lysed in lysis buffer (5 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1 g/ml aprotinin, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, and 100 g/ml phenylmethylsulfonyl fluoride). Aliquots of conditioned media (normalized for cell numbers) or cell lysates (normalized for protein concentrations as measured by the Bio-Rad reagent) were subjected to electrophoresis on 7.5% gradient sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose filters (17)(18)(19)). The nitrocellulose filters were then incubated with antibody against human type I collagen, ␤-actin, or STAT6.…”

Section: Methodsmentioning

confidence: 99%

Interleukin-13 Stimulates the Transcription of the Human α2(I) Collagen Gene in Human Dermal Fibroblasts

Jinnin¹,

Ihn²,

Yamane

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Interleukin (IL)-13 is a novel lymphokine produced by activated Type 2 helper cells. In this study, we examined the target genes of IL-13 by the cDNA microarray analysis in human dermal fibroblasts. We focused on the human ␣2(I) collagen gene, which was one of the IL-13-induced genes by the microarray analysis. IL-13 induced type I collagen protein as well as mRNA in a dose-dependent manner. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the IL-13-mediated up-regulation of ␣2(I) collagen mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, IL-13 treatment induced the promoter activity of ␣2(I) collagen by nuclear run-on transcription assay and chloramphenicol acetyltransferase assay. IL-13-mediated transcriptional activation of ␣2(I) collagen gene or type I collagen protein up-regulation was inhibited by the treatment of fibroblasts with a selective phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or STAT6 antisense oligonucleotide, but not by PD98059, a specific inhibitor of MEK/ERK, or SB202190 or SB203580, specific inhibitors of p38 MAPK; IL-13 induced the phosphorylation of PI3K p85 regulatory subunit and STAT6. These results suggest that IL-13 may play a role in the regulation of extracellular matrix and indicate the possible therapeutic value of the blockade of IL-13 signaling pathways via PI3K and STAT6 in fibrosis.

show abstract

“…Interestingly, phosphorylation has been implicated in changes in Sp1 binding and transcriptional activity (26,27). Although tyrosine residues are present, the vast majority of Sp1 phosphorylation in rat EC is on serine residues (25,28,29).…”

Section: Cyclic Strain Decreases the Affinity Of Sp1 To Mt1-mmp Promomentioning

confidence: 99%

Transcription Factor Sp1 Phosphorylation Induced by Shear Stress Inhibits Membrane Type 1-Matrix Metalloproteinase Expression in Endothelium

Yun

Dardik²,

Haga³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Membrane type 1-matrix metalloproteinase (MT1-MMP) plays a key role in endothelial cell migration, matrix remodeling, and angiogenesis. Previous studies demonstrated that a mechanical force, cyclic strain, increases MT1-MMP expression by displacing Sp1 with increased Egr-1 expression and binding to the promoter site. However, the effect of shear stress (SS) on MT1-MMP expression is poorly understood. Although Egr-1 mRNA transcription and protein was induced (7.6-fold) in response to SS (n ‫؍‬ 5, 0 -8 h, p < 0.05), SS decreased MT1-MMP mRNA transcription and protein levels in a time-dependent fashion (10, 50, and 90% reduction at 1, 4, and 8 h, respectively; n ‫؍‬ 5, p < 0.05). Egr-1 protein was increased after SS and cyclic strain, but Sp1 was serine-phosphorylated only after SS. SS increased Sp1 DNA binding (3.8-, 5.8-, and 2.4-fold increase at 1, 4, and 8 h, respectively; n ‫؍‬ 5, p < 0.05) that was inhibitable by calf intestinal phosphatase. Thus, SS inhibits MT1-MMP expression despite Egr-1 up-regulation by inducing the serine phosphorylation of Sp1, which in turn increases its binding affinity for its site on the MT1-MMP promoter, reducing the ability of Egr-1 to displace it. These data illustrate the complex control of microvascular endothelial cell MT1-MMP expression in response to distinct environmental stimuli (cyclic strain versus shear stress), consisting of both the modulation of specific transcription factor expression (Egr-1) as well as transcription factor post-translational modification (serine phosphorylation of Sp1).Angiogenesis is critical for different physiologic and pathologic processes including wound healing, tissue remodeling, chronic inflammatory disease, and tumorigenesis (1-3). Angiogenesis requires endothelial cells (EC) 1 to coordinate multiple functions including reduction of intercellular adhesion, degradation of subendothelial matrix, migration, proliferation, and formation of new capillary tubes (4). The matrix metalloproteinases (MMPs) are a family of structurally related zinc endopeptidases capable of proteolysis of numerous components of the extracellular matrix (5). Production and activation of MMPs correlate strongly with migration and invasion in endothelium (6, 7). Membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), one of the members of the family of matrix-bound MMPs by a C-terminal transmembrane domain, is identified as a physiological activator of pro-MMP-2 on the cell surface as well as a functional enzyme that degrades a number of extracellular matrix components (8, 9). It is not surprising that MT1-MMP has been shown to be a key regulator of angiogenesis (10, 11).MT1-MMP mRNA and protein levels are expressed at low levels in most noninvasive cells but are up-regulated during EC migration and matrix remodeling, and this up-regulation correlates with MMP-2 activation (11). The up-regulation of MT1-MMP mRNA and protein occurs in microvascular EC cultured in a three-dimensional collagen type-I matrix as a consequence of increased production and promoter bindi...

show abstract

Spl Phosphorylation Induced by Serum Stimulates the Human α2(I) Collagen Gene Expression

Cited by 23 publications

References 42 publications

Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on Transforming Growth Factor-β1-Induced Extracellular Matrix Expression

Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on Transforming Growth Factor-β1-Induced Extracellular Matrix Expression

Interleukin-13 Stimulates the Transcription of the Human α2(I) Collagen Gene in Human Dermal Fibroblasts

Transcription Factor Sp1 Phosphorylation Induced by Shear Stress Inhibits Membrane Type 1-Matrix Metalloproteinase Expression in Endothelium

Contact Info

Product

Resources

About