Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.

Smad proteins transduce signals from TGF-beta receptors and regulate transcription of target genes either directly or in combination with other sequence-specific transcription factors. AP-1 sites and their cognate transcription factors also play important roles in the gene regulatory activities of TGF-beta. In this report, we have investigated the functional interactions of the Smad and AP-1 transcription factors. We demonstrate that Smad and AP-1 complexes specifically bind to their cognate cis-elements and do not interact with each other on-DNA, whereas off-DNA interactions occur between Smad3 and both c-Jun and JunB. Using both artificial constructs specific for either the Smad or AP-1 signaling pathways or natural promoters known to be TGF-beta-responsive, we have determined that Jun family members downregulate Smad3-mediated gene transactivation whereas AP-1-dependent promoters are synergistically activated by Smad3 and Jun proteins. We propose a model where the presence of Smad- and/or AP-1-specific cis-elements within TGF-beta-responsive genes allows dynamic modulation of gene expression, in contrast to the existing model where interactions between Smad and AP-1 proteins are merely an on/off mechanism to regulate TGF-beta/Smad targets.

Fibroblast growth factor (FGF) receptor 1-IIIb is a naturally occurring functional receptor for FGFs that is preferentially expressed in the skin and the brain.

Fibroblast growth factors (FGFs) transmit their signals through four transmembrane receptors that are designated FGFR1-4. Alternative splicing in the extracellular region of FGFR1-3 generates receptor variants with different ligand binding affinities. Thus two types of transmembrane receptors (IIIb and IIIc isoforms) have been identified for FGFR2 and FGFR3, and the existence of analogous variants has been postulated for FGFR1 based on its genomic structure. However, only a single full-length transmembrane FGFR1 variant (FGFR1-IIIc) has been identified so far. Here we describe the cloning of a full-length cDNA encoding FGFR1-IIIb from a mouse skin wound cDNA library. This receptor isoform was expressed at the highest levels in a subset of sebaceous glands of the skin and in neurons of the hippocampus and the cerebellum. FGFR1-IIIb was expressed in L6 rat skeletal muscle myoblasts and used in cross-linking and receptor binding studies. FGF-1 was found to bind the receptor with high affinity, whereas FGF-2, -10, and -7 bound with significantly lower affinities. Despite their apparently similar but low affinities, FGF-10 but not FGF-7 induced the activation of p44/42 mitogen-activated protein kinase in FGFR1-IIIb-expressing L6 myoblasts and stimulated mitogenesis in these cells, demonstrating that this new receptor variant is a functional transmembrane receptor for FGF-10.

Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(I) collagen (COL1A2) gene transcription in human dermal fibroblasts.

Among its plethora of activities as an inflammatory mediator, TNF-alpha has potent regulatory control on extracellular matrix production and degradation. Earlier studies have documented that TNF-alpha inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5' end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-alpha response element of the COL1A2 promoter. The TNF-alpha response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-kappa B (AGAGTTTCCC) binding sites. TNF-alpha effect was eliminated by a 2-bp substitution mutation in the NF-kappa B1 binding half site of the NF-kappa B cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-kappa B heterodimers as well as NF-kappa B1 and RelA homodimers, but not AP-1, were capable of binding this element. Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-alpha stimulation, which reached a plateau by 1 h and was not affected by preincubation of cells with cycloheximide. Gel supershift assays identified the complex as the NF-kappa B (p50/p65) heterodimer, whereas Abs to nuclear factor of activated T cells (NF-AT) and Jun family members failed to recognize the complex. These data suggest that in fibroblasts TNF-alpha activates and initiates the nuclear translocation of NF-kappa B that binds a divergent NF-kappa B element and plays a critical role in the observed inhibition of alpha 2(I) collagen gene transcription.

Cooperation between SMAD and NF-kappaB in growth factor regulated type VII collagen gene expression.

We have previously demonstrated that transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, synergistically enhance the expression of type VII collagen gene (COL7A1) in human dermal fibroblasts in culture (Mauviel et al., 1994). Recently, we identified a SMAD-containing complex, rapidly induced by TGF-beta and binding the region [-496/-444] of the COL7A1 promoter, responsible for COL7A1 gene transactivation (Vindevoghel et al., 1998a). In this report, we demonstrate that TGF-beta and TNF-alpha response elements are distinct entities within the COL7A1 promoter. In particular, we demonstrate that the TNF-alpha effect is mediated by NF-kappaB1/RelA (p50/p65) and RelA/RelA (p65/p65) NF-kappaB complexes binding the TNF-alpha response element (TaRE) located in the region [-252/-230], with RelA acting as the transcriptional activator. Finally, we provide definitive evidence for the role of both TGF-beta and TNF-alpha response elements as enhancer sequences, functioning in the context of a heterologous promoter in an additive manner in response to TGF-beta and TNF-alpha. This study provides the first identification of a functional interaction between the two immediate-early transcription factors, SMAD and NF-kappaB, to activate the expression of an extracellular matrix-related gene, COL7A1.

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

Smad-dependent transcriptional activation of human type VII collagen gene (COL7A1) promoter by transforming growth factor-beta.

We have previously shown that transforming growth factor-beta (TGF-beta) increases type VII collagen gene (COL7A1) expression in human dermal fibroblasts in culture (Mauviel, A., Lapière, J.-C., Halcin, C., Evans, C. H., and Uitto, J. (1994) J. Biol. Chem. 269, 25-28). To gain insight into the molecular mechanisms underlying the up-regulation of COL7A1 by this growth factor, we performed transient cell transfections with a series of 5'-deletion promoter/chloramphenicol acetyltransferase reporter gene constructs. We identified a 68-base pair region between nucleotides -524 and -456, relative to the transcription start site, as critical for TGF-beta response. Using electrophoresis mobility shift assays (EMSAs) with an oligonucleotide spanning the region from -524 to -444, we discovered that a TGF-beta-specific protein-DNA complex was formed as early as 11 min after TGF-beta stimulation and persisted for 1 h after addition of the growth factor. Deletion analysis of the TGF-betaresponsive region of the COL7A1 promoter by EMSA identified segment -496/-444 as the minimal fragment capable of binding the TGF-beta-induced complex. Furthermore, two distinct segments, -496/-490 and -453/-444, appeared to be necessary for TGF-beta-induced DNA binding activity, suggesting a bipartite element. Supershift experiments with a pan-Smad antibody unambiguously identified the TGF-beta-induced complex as containing a Smad member. This is the first direct identification of binding of endogenous Smad proteins to regulatory sequences of a human gene.

A GT-rich sequence binding the transcription factor Sp1 is crucial for high expression of the human type VII collagen gene (COL7A1) in fibroblasts and keratinocytes.

Type VII collagen is the major component of anchoring fibrils, structural elements that stabilize the attachment of the basement membrane to the underlying dermis. In this study, we have dissected the human type VII collagen gene (COL7A1) promoter to characterize the cis-elements responsible for the expression of the gene in cultured fibroblasts and keratinocytes. Using transient cell transfections with various 5' end deletion COL7A1 promoter/chloramphenicol acetyltransferase reporter gene plasmid constructs, we determined that the region between nucleotides -524 and -456, relative to the transcription start site, is critical for high promoter activity in both cell types studied. Gel mobility shift assays using several DNA fragments spanning this region identified a GT-rich sequence between residues -512 and -505, necessary for the binding of nuclear proteins to this region of the promoter. Point mutations abolished the binding of nuclear proteins in gel shift assays and drastically diminished the activity of the promoter in transient cell transfections. Supershift assays with antibodies against various transcription factors including Sp1, Sp3, c-Jun/AP-1, and AP-2, and competition experiments with oligonucleotides containing consensus sequences for Sp1 and AP-1 binding identified Sp1 as the transcription factor binding to this region of the COL7A1 promoter. Indeed, recombinant human Sp1 was shown to bind the COL7A1 promoter GT-rich element but not its mutated form in gel mobility shift assays. In addition, co-transfection of pPacSp1, an expression vector for Sp1, together with the COL7A1 promoter/chloramphenicol acetyltransferase construct into Sp1-deficient Drosophila Schneider SL2 cells unequivocally demonstrated that Sp1 is essential for high expression of the COL7A1 gene. These data represent the first in-depth analysis of the human COL7A1 promoter transcriptional control.

Cytotoxicity of sulphur mustard on a human keratinocyte cell line: direct effects compared to conditioned-medium effects.

Direct toxic effects of sulphur mustard (SM) were compared to SM-conditioned medium effects in a human keratinocyte cell line. Cytotoxicity was evaluated by measurement of cell viability with the Neutral Red uptake assay. Culture directly exposed to SM exhibited cytotoxic dose-response curves after 24, 48 or 72 h. Inhibitory concentration 50 (IC50) appeared to be more than 10 times lower after 72 h than the corresponding value after 24 h. In contrast, no cytotoxic activity was observed in media which were collected 4 h or 24 h after SM exposure. Cell viability was unchanged even if observation was extended to 48 h. Results suggested that SM cytotoxicity was not due to potential mediators secreted by human keratinocytes.