Transforming Growth Factor-β Regulates DNA Binding Activity of Transcription Factor Fli1 by p300/CREB-binding Protein-associated Factor-dependent Acetylation

ABSTRACT Previous studies have shown that transforming growth factor β (TGF-β)-induced collagen gene expression involves acetylation-dependent dissociation from the human α2(I) collagen (COL1A2) promoter of the transcriptional repressor Fli1. The goal of this study was to elucidate the regulatory steps preceding the acetylation of Fli1. We first showed that TGF-β induces Fli1 phosphorylation on a threonine residue(s). The major phosphorylation site was localized to threonine 312 located in the DNA binding domain of Fli1. Using several independent approaches, we demonstrated that Fli1 is directly phosphorylated by protein kinase C δ (PKC δ). Additional experiments showed that in response to TGF-β, PKC δ is recruited to the collagen promoter to phosphorylate Fli1 and that this step is a prerequisite for the subsequent interaction of Fli1 with p300/CREB-binding protein-associated factor (PCAF) and an acetylation event. The phosphorylation of endogenous Fli1 preceded its acetylation in response to TGF-β stimulation, and the blockade of PKC δ abrogated both the phosphorylation and acetylation of Fli1 in dermal fibroblasts. Promoter studies showed that a phosphorylation-deficient mutant of Fli1 exhibited an increased inhibitory effect on the COL1A2 gene, which could not be reversed by the forced expression of PCAF or PKC δ. These data strongly suggest that the phosphorylation-acetylation cascade triggered by PKC δ represents the primary mechanism whereby TGF-β regulates the transcriptional activity of Fli1 in the context of the collagen promoter.

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Transforming growth factor-β1 differentially mediates fibronectin and inflammatory cytokine expression in kidney tubular cells

AJP Renal Physiology ◽

10.1152/ajprenal.00013.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. F1070-F1077 ◽

Cited By ~ 34

Author(s):

Weier Qi ◽

Xinming Chen ◽

John Holian ◽

Ellein Mreich ◽

Stephen Twigg ◽

...

Keyword(s):

Growth Factor ◽

Dna Binding ◽

Transforming Growth Factor ◽

Human Kidney ◽

Transforming Growth Factor Β ◽

Binding Activity ◽

Chemokine Production ◽

Dna Binding Activity ◽

Fibronectin Expression ◽

Proximal Tubular

Transforming growth factor-β1 (TGF-β1) is not only an important fibrogenic but also immunomodulatory cytokine in the human kidney. We have recently demonstrated that TGF-β1 induces interleukin-8 (IL-8), macrophage chemoattractant protein-1 (MCP-1), and fibronectin production in renal proximal tubular (HK-2) cells. However, the unique dependence of IL-8, MCP-1, and fibronectin on TGF-β1 expression is unknown. The TGF-β1 gene was effectively silenced in HK-2 cells using small-interference (si) RNA. Basal secretion of IL-8 and MCP-1 decreased (both P < 0.05) but, paradoxically, fibronectin increased ( P < 0.05) in TGF-β1-silenced cells compared with cells transfected with nonspecific siRNA. Significant increases were observed in mRNA for the TGF-β2 ( P < 0.05), TGF-β3 ( P < 0.05) isoforms and pSmad2 ( P < 0.05), which were reflected in protein expression. Concurrent exposure to pan-specific TGF-β antibody reversed the observed increase in fibronectin expression, suggesting that TGF-β2 and TGF-β3 isoforms mediate the increased fibronectin expression in TGF-β1-silenced cells. An increase in the DNA binding activity of activator protein-1 (AP-1; P < 0.05) was also observed in TGF-β1-silenced cells. In contrast, nuclear factor-κB (NF-κB) DNA binding activity was significantly decreased ( P < 0.0005). These studies demonstrate that TGF-β1 is a key regulator of IL-8 and MCP-1, whereas fibronectin expression is regulated by a complex interaction between the TGF-β isoforms in the HK-2 proximal tubular cell line. Decreased expression of TGF-β1 reduces chemokine production in association with reduced NF-κB DNA binding activity, suggesting that immunomodulatory pathways in the kidney are specifically dependent on TGF-β1. Conversely, decreased expression of TGF-β1 results in increased TGF-β2, TGF-β3, AP-1, and pSmad2 that potentially mediates the observed increase in fibronectin.

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CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor β transcriptional responses in endothelial cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.16.9506 ◽

1998 ◽

Vol 95 (16) ◽

pp. 9506-9511 ◽

Cited By ~ 127

Author(s):

James N. Topper ◽

Maria R. DiChiara ◽

Jonathan D. Brown ◽

Amy J. Williams ◽

Dean Falb ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Transforming Growth Factor ◽

Binding Protein ◽

Transforming Growth Factor Β ◽

Cell Types ◽

Creb Binding Protein ◽

Transcriptional Responses ◽

Smad Proteins ◽

Smad Protein

The transforming growth factor-β (TGF-β) superfamily of growth factors and cytokines has been implicated in a variety of physiological and developmental processes within the cardiovascular system. Smad proteins are a recently described family of intracellular signaling proteins that transduce signals in response to TGF-β superfamily ligands. We demonstrate by both a mammalian two-hybrid and a biochemical approach that human Smad2 and Smad4, two essential Smad proteins involved in mediating TGF-β transcriptional responses in endothelial and other cell types, can functionally interact with the transcriptional coactivator CREB binding protein (CBP). This interaction is specific in that it requires ligand (TGF-β) activation and is mediated by the transcriptional activation domains of the Smad proteins. A closely related, but distinct endothelial-expressed Smad protein, Smad7, which does not activate transcription in endothelial cells, does not interact with CBP. Furthermore, Smad2,4–CBP interactions involve the COOH terminus of CBP, a region that interacts with other regulated transcription factors such as certain signal transduction and transcription proteins and nuclear receptors. Smad–CBP interactions are required for Smad-dependent TGF-β-induced transcriptional responses in endothelial cells, as evidenced by inhibition with overexpressed 12S E1A protein and reversal of this inhibition with exogenous CBP. This report demonstrates a functional interaction between Smad proteins and an essential component of the mammalian transcriptional apparatus (CBP) and extends our insight into how Smad proteins may regulate transcriptional responses in many cell types. Thus, functional Smad–coactivator interactions may be an important locus of signal integration in endothelial cells.

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Regulatory domains of the A-Myb transcription factor and its interaction with the CBP/p300 adaptor molecules

Biochemical Journal ◽

10.1042/bj3240729 ◽

1997 ◽

Vol 324 (3) ◽

pp. 729-736 ◽

Cited By ~ 25

Author(s):

Valeria FACCHINETTI ◽

Livio LOFFARELLI ◽

Sabine SCHREEK ◽

Michael OELGESCHLÄGER ◽

Bernhard LÜSCHER ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Binding Protein ◽

Camp Response Element Binding ◽

Binding Activity ◽

Myb Transcription Factor ◽

Physical Interaction ◽

Creb Binding Protein ◽

Activation Domain ◽

Activation Potential

The A-Myb transcription factor belongs to the Myb family of oncoproteins and is likely to be involved in the regulation of proliferation and/or differentiation of normal B cells and Burkitt's lymphoma cells. To characterize in detail the domains of A-Myb that regulate its function, we have generated a series of deletion mutants and have investigated their trans-activation potential as well as their DNA-binding activity. Our results have allowed us to delineate the trans-activation domain as well as two separate regulatory regions. The boundaries of the trans-activation domain (amino acid residues 218–319) are centred on a sequence rich in charged amino acids (residues 259–281). A region (residues 320–482) localized immediately downstream of the trans-activation domain and containing a newly identified conserved stretch of 48 residues markedly inhibits specific DNA binding. Finally the last 110 residues of A-Myb (residues 643–752), which include a sequence conserved in all mammalian myb genes (region III), negatively regulate the maximal trans-activation potential of A-Myb. We have also investigated the functional interaction between A-Myb and the nuclear adaptor molecule CBP [cAMP response element-binding protein (CREB)-binding protein]. We demonstrate that CBP synergizes with A-Myb in a dose-dependent fashion, and that this co-operative effect can be inhibited by E1A and can also be observed with the CBP homologue p300. We show that this functional synergism requires the presence of the A-Myb charged sequence and that it involves physical interaction between A-Myb and the CREB-binding domain of CBP.

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