Oncogene (1999) 18, 1837 ± 1844 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Cooperation between SMAD and NF-kB in growth factor regulated type VII collagen gene expression Atsushi Kon1,3, Laurence Vindevoghel1,3, David J Kouba1,3, Yasuo Fujimura5, Jouni Uitto1,2,3,4 and Alain Mauviel*,1,3 1Department of Dermatology and Cutaneous Biology, Jeerson Medical College, Philadelphia, Pennsylvania, USA; 2Department of Biochemistry and Molecular Pharmacology, Jeerson Medical College, Philadelphia, Pennsylvania, USA; 3Jeerson Institute of Molecular Medicine, Jeerson Medical College, Philadelphia, Pennsylvania, USA; 4Kimmel Cancer Centre, Jeerson Medical College, Philadelphia, Pennsylvania, USA; 5Department of Human Genetics, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania, USA We have previously demonstrated that transforming mechano-bullous skin disease characterized by ex- growth factor-b (TGF-b) and pro-in¯ammatory cyto- treme fragility of the skin and leading to development kines, such as tumor necrosis factor-a (TNF-a)or of sub-lamina densa blisters (reviewed in Christiano interleukin-1b, synergistically enhance the expression of and Uitto, 1996). type VII collagen gene (COL7A1) in human dermal We have previously shown that pro-in¯ammatory ®broblasts in culture (Mauviel et al., 1994). Recently, we cytokines, such as TNF-a and IL-1, as well as TGF-b, identi®ed a SMAD-containing complex, rapidly induced are potent inducers of COL7A1 expression (Mauviel et by TGF-b and binding the region [7496/7444] of the al., 1994). The regulation of COL7A1 by cytokines and COL7A1 promoter, responsible for COL7A1 gene growth factors diers from that of other collagens, as transactivation (Vindevoghel et al., 1998a). In this exempli®ed by type I collagen. Speci®cally, we report, we demonstrate that TGF-b and TNF-a response previously demonstrated that TGF-b has an additive, elements are distinct entities within the COL7A1 if not synergistic, eect with pro-in¯ammatory promoter. In particular, we demonstrate that the TNF- cytokines, such as IL-1 or TNF-a, on type VII a eect is mediated by NF-kB1/RelA (p50/p65) and collagen gene expression, as measured at both protein RelA/RelA (p65/p65) NF-kB complexes binding the and mRNA levels (Mauviel et al., 1994). In contrast, TNF-a response element (TaRE) located in the region the eects of TGF-b and TNF-a on type I collagen [7252/7230], with RelA acting as the transcriptional gene expression are antagonistic, TGF-b being a activator. Finally, we provide de®nitive evidence for the powerful activator of type I collagen gene promoters role of both TGF-b and TNF-a response elements as (both COL1A1 and COL1A2) while TNF-a exerts an enhancer sequences, functioning in the context of a inhibitory eect (Varga et al., 1987; Inagaki et al., heterologous promoter in an additive manner in response 1995; Chung et al., 1996). to TGF-b and TNF-a. This study provides the ®rst Recently, we have demonstrated that TGF-b identi®cation of a functional interaction between the two upregulation of COL7A1 gene expression occurs at immediate-early transcription factors, SMAD and NF- the transcriptional level and we identi®ed the TGF-b kB, to activate the expression of an extracellular matrix- responsive region of the COL7A1 promoter as a target related gene, COL7A1. of the SMAD signaling pathway (Vindevoghel et al., 1998a). Speci®cally, we have shown that a SMAD3- Keywords: SMAD; NF-kB; TGF-b; TNF-a; transcrip- containing complex rapidly forms with the COL7A1 tion; promoter promoter in response to TGF-b. The resulting gene transactivation occurs in a SMAD4-dependent manner (Vindevoghel et al., 1998b). In this study, we have examined whether the Introduction additive eect of TGF-b with TNF-a on type VII collagen gene expression is exerted at the transcrip- Type VII collagen is the predominant, if not the tional level by activation of the corresponding exclusive, component of the anchoring ®brils, attach- promoter. We demonstrate that the two cytokines ment structures stabilizing the association of the independently activate the COL7A1 promoter through cutaneous basement membrane to the underlying distinct response elements, TGF-b acting via direct dermis (reviewed in Burgeson, 1993; Christiano and SMAD binding to the 7496/7444 region of the Uitto, 1996). In the skin, type VII collagen is promoter, whereas TNF-a eect is mediated by synthesized by both dermal ®broblasts and epidermal binding of NF-kB1/RelA NF-kBtoacis-element keratinocytes. Alterations in the type VII collagen located between nucleotides 7252 and 7230, i.e. protein structure or lack of its expression due to approximately 200 bp downstream from the TGF-b mutations in the corresponding gene COL7A1 are the response element. These data represent the ®rst hallmark of dystrophic epidermolysis bullosa, a demonstration of additive transcriptional activity of two immediate-early transcription factors, SMAD and NF-kB, in the context of the activation of an extracellular matrix gene, COL7A1, by cytokines *Correspondence: A Mauviel Received 10 August 1998; revised 7 October 1998; accepted 15 often characterized as antagonistic to each other in a October 1998 number of experimental systems. SMAD and RelA/NF-kB coordinated transcriptional activities AKonet al 1838 Cells were subsequently treated with TNF-a for 40 h, Results at which point CAT activity was determined. As shown in Figure 2, a stimulatory eect of TNF-a, varying TNF-a and TGF-b exert an additive eect on the from 2.5 ± 4.7-fold, was observed with constructs COL7A1 promoter containing at least 7396 bp of COL7A1 5' regulatory To determine whether the additive eect of TGF-b and sequences. TNF-a responsiveness was totally abolished TNF-a on COL7A1 gene expression, as determined at by further 5' deletion to position 7230 of the both the mRNA and protein levels (Mauviel et al., promoter, indicating that the TNF-a responsive 1994), was exerted, at least in part, at the transcrip- sequences are located between nucleotides 7396 and tional level through activation of the corresponding 7230. promoter, transient cell transfection experiments were performed in human dermal ®broblast cultures. These The 7271/7201 COL7A1 promoter fragment binds a studies utilized a COL7A1 promoter/CAT reporter rapidly-induced, TNF-a-responsive nuclear factor gene construct containing 722 bp of 5' end regulatory sequences of the human COL7A1 gene (Vindevoghel et The transfection experiments described above undoubt- al., 1997). As shown in Figure 1, TGF-b and TNF-a edly identi®ed the TNF-a responsive region of the both enhanced COL7A1 promoter activity (6.8- and COL7A1 promoter as being distinct from the recently 3.6-fold, respectively). An additive eect was observed characterized TGF-b response element (Vindevoghel et (13.3-fold) when the two cytokines were added al., 1998a). A schematic representation is provided in simultaneously to the ®broblast culture medium. Figure 3a, indicating the distinct growth factor/ These data establish that both cytokines exert their cytokine responsive regions. To determine whether enhancing activity on COL7A1 gene expression, at the region of COL7A1 promoter conferring TNF-a least in part, at the transcriptional level. responsiveness contained TNF-a-speci®c cis-elements, electrophoretic mobility shift assays (EMSAs) were performed with two overlapping DNA probes covering Delineation of the TNF-a responsive region within the the entire region between nucleotides 7410 and 7201 COL7A1 promoter (see Figure 3a). As shown in Figure 3b, no dierence in We have recently demonstrated that the TGF-b the binding pattern of nuclear extracts from control or responsive sequences reside between nucleotides 7496 TNF-a-treated ®broblasts could be observed with the and 7444, relative to the transcription start site of the probe spanning the region 7410/7300. On the other COL7A1 gene (Vindevoghel et al., 1998a). To hand, a speci®c TNF-a-induced band with retarded characterize the TNF-a responsive region of the mobility was observed with the 7319/7201 probe at COL7A1 promoter, ®broblast cultures were trans- 1 h following TNF-a addition to the ®broblast fected with a series of 5' end deletion COL7A1 medium. Time-course experiments indicated a progres- promoter/CAT constructs (Vindevoghel et al., 1997). sive enhancement of DNA binding activity, detectable as early as 5 min after TNF-a addition, with a maximal intensity between 15 and 60 min (Figure 3c). The retarded DNA-protein complex appeared to consist of two closely migrating bands. Figure 2 Delineation of the TNF-a responsive region within the Figure 1 TNF-a and TGF-b exert an additive eect on the COL7A1 promoter. Con¯uent ®broblast cultures were transfected COL7A1 promoter. Con¯uent ®broblast cultures were transfected with various 5' deletion/CAT constructs of the human COL7A1 with the 7722COL7A1/CAT construct of the human COL7A1 promoter by the calcium phosphate/DNA co-precipitation promoter by the calcium phosphate/DNA co-precipitation procedure, as described in Materials and methods. After glycerol procedure, as described in Materials and methods. After glycerol shock, the cultures were incubated in fresh medium containing shock, the cultures were incubated in fresh medium containing 1% fetal calf serum, without (7) or with (+) TNF-a (10 ng/ml) 1% fetal calf serum. TNF-a and/or TGF-b (10 ng/ml) were added added to the medium 4 h later. After 40 h of incubation, cell to the medium 4 h later. After 40 h of incubation, cell extracts extracts were assayed for CAT activity with [14C]chloramphenicol 14 were assayed for CAT activity with [ C]chloramphenicol as a as a substrate, using identical amounts of protein. A substrate, using identical amounts of protein. The ®gure shows a representative autoradiogram of the CAT assay, depicting the representative autoradiogram of the CAT assay, depicting the separation of acetylated (AC) and unacetylated (C) forms of separation of acetylated (AC) and unacetylated (C) forms of [14C]chloramphenicol by a thin-layer chromatography is shown, 14 [ C]chloramphenicol by a thin-layer chromatography.