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Originally published In Press as doi:10.1074/jbc.M409647200 on December 3, 2004

J. Biol. Chem., Vol. 280, Issue 9, 8079-8085, March 4, 2005
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Ultraviolet Irradiation Induces Smad7 via Induction of Transcription Factor AP-1 in Human Skin Fibroblasts*

Taihao Quan, Tianyuan He, John J. Voorhees, and Gary J. Fisher{ddagger}

From the Department of Dermatology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0609

Received for publication, August 23, 2004 , and in revised form, November 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Smad7 functions as an endogenous negative regulator of transforming growth factor-{beta} (TGF-{beta})/SMAD signaling. The TGF-{beta}/SMAD pathway is a major regulator of collagen production in connective tissue. Reduced expression of SMAD7 has been reported in TGF-{beta}-mediated fibrotic diseases, characterized by overproduction of collagen. Solar ultraviolet (UV) irradiation reduces collagen production by fibroblasts in human skin in vivo. We have investigated regulation of Smad7 gene expression by UV irradiation in human skin fibroblasts. UV irradiation transiently increased SMAD7 mRNA and protein levels. Induction of SMAD7 mRNA and protein was maximal within 5 h and returned to initial basal levels 24 h post-UV irradiation. UV irradiation induced Smad7 promoter-reporter activity 3-fold. The Smad7 promoter contains functional enhancer sequences that bind transcription factors SMAD3 and activator protein-1 (AP-1). UV irradiation reduced protein binding to the Smad3 enhancer and increased binding to the AP-1 enhancer. Deletion of the AP-1 binding site in the Smad7 promoter completely abolished UV stimulation of SMAD7 transcription. Deletion of the Smad3 element had no effect on UV irradiation-induced promoter activity. UV irradiation increased mRNA and protein expression of the AP-1 family members, c-Jun and c-Fos, which bound to the AP-1 element in the Smad7 promoter. Furthermore, overexpression of dominant negative c-Jun substantially reduced UV irradiation induction of SMAD7 transcription. These data demonstrate that induction of Smad7 gene expression by UV irradiation is mediated via induction of the transcription factor AP-1 in human skin fibroblasts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-{beta} (TGF-{beta})1 regulates a wide variety of cellular functions, including proliferation, differentiation, adhesion, migration, extracellular matrix production, and extracellular matrix degradation (1–3). TGF-{beta} initiates its effects on cellular functions by binding to the cell surface TGF-{beta} receptor complex. Receptor binding triggers phosphorylation and activation of SMAD2 and SMAD3, which regulate expression of TGF-{beta} target genes (4–6). Activation of SMAD2/3 is antagonized by the endogenous negative regulator SMAD7 (7). Expression of SMAD7 is inducible by TGF-{beta} itself, and this induction plays a critical role in negative feedback mechanisms that regulate TGF-{beta}/Smad-mediated biological responses (7–10).

Aberrant expression of SMAD7 disrupts the delicate balance inherent in TGF-{beta}/SMAD signaling and thereby alters TGF-{beta}-dependent cellular responses (2, 11–13). For example, the level of SMAD7 expression is reportedly reduced in the fibrotic disease scleroderma (14). This reduction could permit unrestrained activation of the TGF-{beta}/Smad pathway, resulting in excessive production and deposition of collagen, which is observed in this disease (14). Conversely, SMAD7 is reportedly overexpressed in inflammatory bowel disease (15). This overexpression could down-regulate TGF-{beta}-mediated immunosuppression, resulting in hypersecretion of proinflammatory cytokines, which is observed in this disease.

We have reported previously that ultraviolet (UV) irradiation induces SMAD7 in human skin in vivo and that this induction contributes to impairment of TGF-{beta}/SMAD signaling (16, 17). We observed that UV irradiation induces SMAD7 in both the outer compartment (epidermis) and inner compartment (dermis) of human skin (16). In the outer compartment of skin, TGF-{beta} is a powerful negative regulator of cell (keratinocyte) proliferation (13, 18–21). Therefore, induction of SMAD7 contributes to keratinocyte hyperplasia, which occurs in response to UV irradiation (16, 22, 23). Increased keratinocyte growth is a characteristic of UV irradiation-induced skin cancer. In the inner compartment of skin, TGF-{beta} is a major regulator of extracellular matrix production and deposition by fibroblasts (2, 3, 24, 25). In dermal fibroblasts, impairment of the TGF-{beta}/SMAD pathway causes reduced production of type I procollagen, thereby leading to loss of collagen (26). Reduced collagen is a key factor in premature skin aging (photoaging), which is caused by UV irradiation (27). Therefore, induction of SMAD7, with concomitant reduction of TGF-{beta} responsiveness, may be a key mediator of UV irradiation-induced skin cancer and photoaging. Accordingly, understanding mechanisms by which UV irradiation induces Smad7 gene expression is vital to our knowledge of the pathophysiology of these common diseases.

The 5'-flanking region of the Smad7 promoter contains binding sequences for the transcription factors SMAD3, AP-1, and Sp-1 (28). These regulatory elements and flanking sequences of the Smad7 gene promoter are highly conserved in human, rat, and murine genes (29). SMAD3, AP-1, and Sp-1 binding sequences are necessary for optimal induction of SMAD7 transcription by TGF-{beta} (28, 30, 31). Disruption of the Smad3 element abolishes TGF-{beta} inducibility but does not greatly impair basal expression. Disruption of the AP-1 and Sp-1 binding elements substantially impairs basal and TGF-{beta}-stimulated Smad7 expression.

Exposure of human skin in vivo to UV irradiation activates multiple cell surface cytokine and growth factor receptors, mitogen-activated protein kinase pathways, and rapidly induces transcription factor AP-1 activity (23, 27, 32, 33). Based on these findings, we hypothesized that AP-1 plays a major role in induction of Smad7 expression by UV irradiation. To test this hypothesis, we have investigated the mechanism of Smad7 induction by UV irradiation. Our data demonstrate that the AP-1 family members c-Jun and c-Fos mediate activation of Smad7 transcription by UV irradiation in human skin dermal fibroblasts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's medium, fetal bovine serum, trypsin solution, penicillin/streptomycin, and L-glutamine were purchased from Invitrogen. [{gamma}-32P]ATP and [{alpha}-32P]dCTP were obtained from PerkinElmer Life Sciences. The c-Jun and c-Fos antibodies were purchased from Transduction Laboratories (San Diego, CA), and SMAD3 was obtained from Zymed Laboratories (South San Francisco, CA). The SMAD7 primary antibody was generously provided by Dr. Peter ten Dijke. The Smad7 promoter-reporter constructs were kindly provided by Rainer Heuchel. All other reagents were purchased from Sigma.

Cell Culture and UV Irradiation—Human skin primary fibroblasts were prepared from a keratome biopsy of healthy adult normal human skin as described previously (34). The cells used for this study were between passages 3 and 6. UV irradiation was performed as described previously (17, 35). Briefly, subconfluent cells were exposed to UV irradiation (50 mJ/cm2) using an Ultralite Panelite lamp containing six FS24T12 UVB-HO bulbs (Daavlin, Byran, OH). A Kodacel filter was used to eliminate wavelengths below 290 nm (UVC). The irradiation intensity was monitored with an IL400A phototherapy radiometer and a SED240/UVB/W photodetector (International Light, Newbury, MA).

RNA Isolation and Northern Blot Analysis—Total RNA from cultured human skin fibroblasts was prepared using a commercial kit (RNeasy Midi kit, Qiagen, Chatsworth, CA) according to the manufacturer's protocol. Northern blot analysis was performed as described previously (17). Briefly, total RNA (20 µg) was resolved by 1.2% agarose electrophoresis, transferred to nylon membranes, and hybridized with Smad7 cDNA probes (17) labeled with [32P]dCTP by random priming. Each blot was stripped and rehybridized with the 36B4 internal control gene transcript to monitor the sample load in each lane. The intensities of each band were quantified by a Storm PhosphorImager (Amersham Biosciences).

Quantitative Real-time RT-PCR—Quantitative real-time RT-PCR was performed as described previously (35). PCR primers and probes were purchased from the Applied Biosystems custom oligonucleotide synthesis service. The c-Jun and c-Fos primers and FAM-labeled probes for real-time RT-PCR were as follows: c-Jun sense primer, 5'-CCTCAACGCCTCGTTCCT-3'; antisense primer, 5'-TCAGGGTCATGCTCTGTTTCAG-3'; probe, 5'-CCGAGAGCGGACCTTATGGCTACAGTAACCCC-3'; c-Fos sense primer, 5'-GCCGAGCGCAGAGCATT-3'; antisense primer, 5'-CCCTTCGGATTCTCCTTTTCTC-3'; and probe, 5'-TCTTCTGGAGATAACTGTTCCACCTTGCC-3'. Primers and VIC-labeled probe for 36B4 were described previously (35). Multiplex PCR reactions contained primers and probes for the target gene and 36B4. Target gene and 36B4 mRNA levels were quantified based on standard curves, and gene levels were normalized to the housekeeping gene 36B4 (a ribosomal protein used as an internal control for quantitation).

Western Analysis—Western blot analysis was performed as described previously (17). Briefly, proteins from whole cell extract were resolved on 12% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and blocked with PBST (0.1% Tween 20 in phosphate-buffered saline) containing 5% milk. Primary antibodies were diluted in the PBST solution (1:1000) and incubated with a polyvinylidene difluoride membrane for 2 h at room temperature. Blots were washed three times with the PBST solution and incubated with the appropriate secondary antibody for 1 h at room temperature. After washing three times with PBST, the blots were developed with ECF (Vistra ECF Western blotting system, GE Healthcare, Piscataway, NJ) following the manufacturer's protocol. The intensities of each band were quantified by a Storm PhosphorImager (Amersham Biosciences).

Immunohistology—Immunohistology was performed as described previously (17). Cells (1 x 104) were plated on 8-well chamber slides and exposed to UV irradiation as described under "Cell Culture and UV Irradiation." Cells were washed once with phosphate-buffered saline and fixed in 2% paraformaldehyde for 2 h at room temperature. The slides were preincubated with normal rabbit serum for 1 h. Subsequently the slides were incubated for 1 h at room temperature with rabbit polyclonal antibody against Smad7. The slides were then incubated with a biotinylated anti-rabbit antibody for 30 min. The slides were then incubated with avidin-biotin peroxidase complex for 30 min. 3-Amino-9-ethyl carbazole (Sigma) was used as a chromogen. Between steps, the slides were rinsed for 10 min in Tris-buffered saline with 0.1% Triton X-100. All sections were lightly counterstained with hematoxylin.

Electrophoretic Mobility Shift Assay (EMSA)—EMSA was performed as described previously (17) using nuclear extracts from the human skin fibroblasts. Oligonucleotides for EMSA probes, designed from the Smad7 promoter, were as follows: Smad binding element (SBE) probe, 5'-TTTTAAAGCGACAGGGTGTCTAGACGGCCACG-3'; AP-1 binding element (AP-1E) probe, 5'-GGCCACGTGACGAGGCCGGAGCCGG-3'; SBE/AP-1E probe, 5'-TTTTAAAGCGACAGGGTGTCTAGACGGCCACGTGACGAGGCCG-GAGCCGG-3'. The SBE, AP-1E, and SBE/AP-1E probes were synthesized by Operon Technologies, Inc. (Alameda, CA). AP-1 consensus (5'-CGCTTGATGACTCAGCCGGAA-3') and mutant (5'-CGCTTGATGACTTGGCCGGAA-3') probes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For competition experiments, a 100-fold molar excess of cold competitors was preincubated with nuclear extract for 30 min on ice before the labeled probes were added. The gel was transferred to Whatman No. 3MM paper, vacuum-dried, and quantified by a Storm PhosphorImager (Amersham Biosciences).

Electrophoretic Mobility Shift Assay Coupled with Western Blot—The major shifted bands were excised from the EMSA gel, and then the proteins were extracted from the gel by incubation overnight at 37 °C in buffer (1x phosphate-buffered saline, 10 mM Tris-HCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.5% SDS, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride). Gel debris was removed by centrifugation, and the supernatant was concentrated by Centricon-10 (Amicon, Inc., Beverly, MA). The samples were then heated for 5 min at 95 °C and analyzed by Western blot.

Transient Transfection of Smad7 Promoter-Reporter Construct and Luciferase Assays—Cells were transfected with wild-type Smad7 promoter/luciferase construct (-613 to +112) or deletion constructs in the binding sites of Smad3 and AP-1 (28). Cells were co-transfected with the {beta}-galactosidase expression vector to provide an internal standard for transfection efficiency. In some studies, the dominant negative mutant c-Jun (TAM-67) (36) expression vector was transfected. All plasmids were transfected into dermal fibroblasts using FuGENE 6 (Roche Applied Science) or the human dermal fibroblasts nucleofector kit (Amaxa Biosystems, Cologne, Germany) according to the manufacturer's protocols. After 24 h of transfection, cells were exposed to UV irradiation and harvested 24 h post-UV irradiation. Aliquots containing identical {beta}-galactosidase activity were used for each luciferase assay. Luciferase activity was measured using an enhanced luciferase assay kit (Pharmingen) according to the manufacturer's protocol.

Statistical Analysis—Comparisons among treatment groups were made with the paired t test (two groups) or the repeated measures of analysis of variance (more than two groups). Multiple pairwise comparisons were made with the Tukey's Studentized range test. All p values are two-tailed and considered significant when <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
UV Irradiation Increases SMAD7 mRNA and Protein in Human Skin Fibroblasts—We first determined the kinetics of induction of SMAD7 mRNA and protein in human skin fibroblasts by UV irradiation. UV irradiation transiently increased the levels of SMAD7 mRNA (Fig. 1A) and protein (Fig. 1B). Induction of Smad7 mRNA was maximal 5 h post-UV irradiation and returned to the initial basal level by 24 h post-UV irradiation. Induction of the SMAD7 protein was maximal between 5 and 8 h following UV irradiation and returned to the basal level 24 h post-irradiation. Immunohistology revealed that the SMAD7 protein was predominantly located in the cytoplasm of untreated cells, and the SMAD7 protein increased in the cytoplasm 5–8 h post-UV irradiation (Fig. 1C). These data indicate that UV irradiation increases the levels of SMAD7 mRNA and protein in human skin fibroblasts.



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  		FIG. 1.
UV irradiation increases Smad7 mRNA and protein levels in human skin fibroblasts. A, UV irradiation induces Smad7 mRNA. Total RNA was prepared from cells at the indicated times post-UV irradiation (50 mJ/cm2). Smad7 and 36B4 (a ribosomal RNA used as an internal control for quantitation) mRNA were analyzed by Northern analysis. Inset shows representative Northern blot. Bars indicate means ± S.E. for -fold change in Smad7 mRNA levels following UV irradiation relative to levels in non-irradiated control cells (CTRL). *, p < 0.05 versus non-irradiated control cells. n = 3. B, UV irradiation induces SMAD7 protein. Whole cell extracts were prepared from cells at the indicated times post-UV irradiation (50 mJ/cm2). SMAD7 protein was quantified by Western analysis. Inset shows representative Western blot. Bars indicate means ± S.E. for -fold change in SMAD7 protein following UV irradiation relative to levels in non-irradiated control cells. *, p < 0.05 versus non-irradiated control cells. n = 3. C, cellular localization of the SMAD7 protein following UV irradiation. Cellular localization of the SMAD7 protein was determined by immunohistochemical staining at the indicated times post-UV irradiation (50 mJ/cm2) as described under "Experimental Procedures." Results are representative of three experiments.

 
UV Irradiation Induces AP-1-dependent Smad7 Promoter Activity in Human Skin Fibroblasts—We next examined UV stimulation of Smad7 promoter activity. The Smad7 promoter/luciferase construct (-613 to +112) containing the Smad3, AP-1, and Sp-1 binding elements was transfected into skin fibroblasts, and reporter activity was determined following sham or UV irradiation. UV irradiation stimulated promoter activity 3-fold (Fig. 2). Deletion of the Smad3 element reduced basal and UV irradiation-stimulated reporter activity proportionally such that net induction by UV irradiation was not altered. Deletion of the AP-1 binding element also reduced basal reporter activity. However, in contrast to deletion of the Smad3 element, deletion of the AP-1 element abolished induction of reporter activity by UV irradiation. These data indicate that UV irradiation stimulates Smad7 promoter activity and that this stimulation is dependent on the presence of the AP-1 binding site in human skin fibroblasts.



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  		FIG. 2.
Deletion of the AP-1 binding site abolishes UV irradiation-stimulated Smad7 promoter activity. Human skin fibroblasts were transiently transfected with the wild-type Smad7 promoter/luciferase construct (-613 to +112) containing the SMAD3 element (SBE) and AP-1 element or constructs with SBE or AP-1E deleted (as shown in the figure). Cells were co-transfected with the {beta}-galactosidase expression vector. Cells were exposed to UV irradiation (50 mJ/cm2) 24 h after transfection, and cell lysates were prepared 24 h post-UV irradiation. Smad7 promoter/luciferase activities were normalized to {beta}-galactosidase activity. Bars indicate means ± S.E. -fold change in Smad7 promoter activity relative to non-irradiated wild-type SMAD7 control cells. *, p < 0.05 versus non-irradiated wild-type SMAD7 control cells. n = 3.

 
UV Irradiation Increases Protein Binding to the AP-1 but Not Smad3 Element in the Smad7 Promoter—We next examined protein binding to the Smad3 and AP-1 elements in non-irradiated and skin fibroblasts exposed to UV irradiation. EMSAs were performed using three probes with sequences identical to the Smad7 promoter. The SBE/AP-1E probe contained both the Smad3 and AP-1 elements; the SBE probe contained only the Smad3 element; and the AP-1E probe contained only the AP-1 binding element (Fig. 3A). Nuclear proteins from non-irradiated fibroblasts formed two major specific retarded complexes with the SBE/AP-1E probe (Fig. 3B). UV irradiation substantially increased the intensity of the upper retarded DNA-protein complex and reduced the intensity of the lower complex (Fig. 3B). The upper retarded DNA-protein complex was abolished by the excess unlabeled consensus AP-1 probe but not by the consensus SBE probe. In contrast, the lower complex was completely abolished by excess unlabeled consensus SBE but not consensus AP-1 probe (data not shown). These data indicate that the upper complex contains AP-1 family protein but not SMAD3 protein, and the lower complex contains SMAD3 protein but not AP-1 family protein. Nuclear proteins from non-irradiated cells formed a single retarded complex with the SBE probe (Fig. 3C). This single retarded DNA-protein complex migrated to the position of the lower band observed with the SBE/AP-1E probe (Fig. 3, B and C). UV irradiation reduced the intensity of the SBE complex similar to what was observed with the lower complex formed with the SBE/AP-1E probe (Fig. 3, B and C). The AP-1E probe formed a single specific retarded complex with nuclear proteins from non-irradiated fibroblasts (Fig. 3D). The migration of this complex was similar to the upper retarded band formed with the SBE/AP-1E probe (Fig. 3, B and D). UV irradiation increased the intensity of the AP-1 complex as was observed with the upper retarded band formed with the SBE/AP-1E probe (Fig. 3B).



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  		FIG. 3.
UV irradiation increases protein binding to the AP-1 but not the SMAD3 element of the Smad7 promoter. A, nucleotide sequence of the Smad7 proximal promoter. SMAD3 (SBE) and AP-1E binding sites are marked in capital letters. Heavy lines under the sequence denote EMSA probes containing SBE/AP-1E, SBE, and AP-1E. Arrow indicates transcription start site. B–E, nuclear extracts were prepared at the indicated times (Hrs) post-UV irradiation (50 mJ/cm2), and DNA-protein complex formation was analyzed by EMSA. Closed triangles indicate specific retarded complexes. Open triangles indicate nonspecific bands. Results are representative of three experiments. B, UV irradiation alters DNA-protein complex formation with the SBE/AP-1E probe. C, UV irradiation reduces DNA-protein complex formation with the SBE probe. D,UV irradiation increases DNA-protein complex formation with the AP-1E probe. E, competition of protein binding to the AP-1E probe by 100-fold excess unlabeled wild-type (AP-1) or mutant (AP-1 mut) AP-1 binding element. Nuclear extracts were prepared 4 h post-UV irradiation.

 
To confirm the specificity of the retarded complexes formed with the AP-1E probe induced by UV irradiation, we performed competition EMSA. DNA-protein complex formation with the labeled AP-1E probe was completely abolished by the excess unlabeled consensus AP-1 but not by the mutant AP-1 probe (Fig. 3E).

To identify proteins bound to the SBE/AP-1E, SBE, and AP-1E probes, we performed supershifts using antibodies to SMAD and AP-1 family members. However, these experiments did not yield reproducible results. Therefore, we performed EMSA coupled with Western blot analysis for the identification of proteins bound to the probes. The upper retarded complex formed by cells exposed to UV irradiation with the SBE/AP-1E probe, the lower retarded complex formed by non-irradiated cells with the SBE/AP-1E probe, the complex formed by non-irradiated cells with the SBE probe, the complex formed by cells exposed to UV irradiation with the AP-1E probe, and equivalent gel areas from lanes without probes (used for control) were excised and subjected to SDS-PAGE immunoblotting with antibodies to c-Jun, c-Fos, and SMAD3 (Fig. 4). c-Jun and c-Fos were readily detected in the upper retarded complex (Fig. 4, lanes 1 and 2), and SMAD3 was detected in the lower retarded complex (Fig. 4, lane 3) formed with the SBE/AP-1E probe. The AP-1E probe bound c-Jun and c-Fos (Fig. 4, lanes 4 and 5) but not SMAD3 (data not shown), whereas the SBE probe bound SMAD3 (Fig. 4, lane 6) but not c-Jun or c-Fos (data not shown). No immunoreactivities were detected in extracts from the gel control lanes (data not shown). These data demonstrate that UV irradiation increases binding of c-Jun and c-Fos and reduces binding of SMAD3 to the Smad7 promoter.



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  		FIG. 4.
Identification of Smad7 proximal promoter DNA-binding proteins by EMSA coupled with Western analysis. Specific upper and lower shifted complexes formed by exposure to UV irradiation (SBE/AP-1E, SBE, and AP-1E) or non-irradiated probes from the proximal Smad7 promoter (as described in the legend to Fig. 3) were excised and subjected to Western analysis with antibodies to c-Jun, c-Fos, and SMAD3. Lanes 1 and 2, c-Jun and c-Fos proteins were detected in the upper complex formed by cells exposed to UV irradiation (2 h) with the SBE/AP-1E probe. Lane 3, SMAD3 protein was detected in the lower complex formed by non-irradiated cells with the SBE/AP-1E probe. Lanes 4 and 5, c-Jun and c-Fos proteins were detected in the complex formed by cells exposed to UV irradiation (4 h) with the AP-1E probe. Lane 6, SMAD3 protein was detected in the complex formed by non-irradiated cells with the SBE probe. Results are representative of three independent experiments.

 
Overexpression of Dominant Negative c-Jun Abolished UV Irradiation-induced Activation of Smad7 Promoter—The data presented in Figs. 2, 3, and 4 indicate that AP-1 plays a major role in UV irradiation-induced Smad7 gene expression. To further substantiate this conclusion, UV irradiation-induced c-Jun and c-Fos mRNA and protein levels were determined by quantitative real-time RT-PCR and Western analysis, respectively. UV irradiation-induced c-Jun and c-Fos mRNA (Fig. 5, A and C) within 2 h, and maximum induction of the c-Jun and c-Fos proteins (Fig. 5, B and D) was observed at 8 h post-UV irradiation in human skin fibroblasts.



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  		FIG. 5.
UV irradiation induces c-Jun and c-Fos in human skin fibroblasts. Cells were exposed to UV irradiation (50 mJ/cm2), and total RNA or whole cell protein extracts were prepared at the indicated times post-irradiation. c-Jun (A) and c-Fos (C) mRNA levels were quantified by real-time RT-PCR, normalized to mRNA for 36B4 (which encodes a ribosomal protein) used as an internal control. c-Jun (B) and c-Fos (D) protein levels were determined by Western analysis. Insets in B and D show representative Western blots. Bars indicate means ± S.E. -fold change relative to levels in non-irradiated control cells (CTRL). *, p < 0.05 versus control. n = 3–5.

 
Finally, we utilized dominant negative mutant c-Jun to examine AP-1 function. As shown in Fig. 6, overexpression of dominant negative c-Jun reduced basal Smad7 promoter activity ~50%. Importantly, induction of Smad7 promoter activity by UV irradiation was essentially abolished by overexpression of dominant negative c-Jun. These data demonstrate that induction of AP-1 by UV irradiation functions to induce Smad7 gene expression in human skin fibroblasts.



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  		FIG. 6.
Overexpression of dominant negative c-Jun abolished UV irradiation-induced Smad7 promoter activity in human skin fibroblasts. Cells were co-transfected with the Smad7 promoter/luciferase construct (-613 to +112) and empty or dominant negative (dn) c-Jun expression vector. Cells were exposed to UV irradiation (50 mJ/cm2) 24 h after transfection. Cell lysates were prepared 24 h post-UV irradiation, and promoter activity was determined by luciferase assay and normalized to {beta}-galactosidase activity. Bars indicate means ± S.E. for -fold change in Smad7 promoter activity relative to activity in non-irradiated control cells (CTRL). *, p < 0.05 versus cells exposed to UV irradiation. n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
SMAD7 functions as an endogenous negative feedback regulator of TGF-{beta}/SMAD signaling to modulate the intensity and duration of TGF-{beta} responses (7, 8). The molecular mechanisms underlying the antagonistic effect of SMAD7 on the TGF-{beta}/SMAD pathway are relatively well characterized (1). SMAD7 stably interacts with the activated TGF-{beta} receptor type I to prevent association and phosphorylation of SMAD2 and SMAD3 and thus inhibits SMAD2 and SMAD3 nuclear translocation (7). Furthermore, SMAD7 inhibits TGF-{beta} responsiveness by promoting degradation of TGF-{beta} receptor type I through recruitment of an E3 ubiquitin-protein isopeptide ligase to the TGF-{beta} receptor (37–40). In addition to its inhibitory effects at the TGF-{beta} receptor, SMAD7 can function as a transcription repressor in the nucleus by recruiting histone deacetylases to TGF-{beta}/Smad target genes (41).

We have reported previously that exposure of human skin in vivo to solar simulated UV irradiation induces SMAD7 and impairs the TGF-{beta}/SMAD signaling pathway (16, 17). The data presented here demonstrate that UV irradiation stimulates Smad7 promoter activity and that this activation is mediated by transcription factor AP-1, which is induced by UV irradiation, in human skin fibroblasts. The proximal promoter of the Smad7 gene contains binding elements for Smad3, AP-1, and Sp-1 (28). The SMAD3 binding element is required for induction of Smad7 by TGF-{beta} (30, 31), whereas the AP-1 and Sp-1 binding elements cooperate to enhance SMAD3-dependent Smad7 gene expression (28).

Here we provide several lines of experimental evidence that support the conclusion that AP-1 but not SMAD3 plays a critical role in activation of Smad7 promoter activity by UV irradiation (Figs. 2, 3, and 6). Interestingly, we found that UV irradiation reduced SMAD3 binding to its element in the Smad7 promoter. Inhibition of SMAD3 activation by UV irradiation may be mediated by several mechanisms. First, we have reported that UV irradiation down-regulates TGF-{beta} receptor type II, which results in reduced phosphorylation and nuclear translocation of SMAD3 in response to TGF-{beta} (26). Second, as shown here, UV irradiation induces SMAD7, which in turn interacts with TGF-{beta} receptor type I to prevent association and activation of SMAD3. Third, it has been shown that c-Jun, which is induced by UV irradiation, is able to interact physically with SMAD3 and thereby suppress SMAD3-driven gene transactivation (42–44).

UV irradiation activates multiple cell surface growth factor and cytokine receptors, which initiates intracellular signal transduction cascades (22, 23, 27, 33). These activated signaling cascades activate target transcription factors, including AP-1. We show here that UV irradiation induces c-Jun and c-Fos in human skin fibroblasts. A primary function of human skin fibroblasts is to produce type I collagen (and other extracellular matrix molecules), which is the major structural protein in skin connective tissue. This production of collagen by skin fibroblasts is strongly stimulated by TGF-{beta} (26, 45). We have reported previously that UV irradiation reduces type I procollagen production in human skin in vivo and human skin fibroblasts (46). This inhibition is mediated in part by UV-irradiated induction of AP-1 (46), which has been shown to interfere with TGF-{beta}-activated SMAD3 stimulation of type I procollagen promoter activity (45, 47).

The data presented here strongly suggest that AP-1 represses type I procollagen production by a second distinct mechanism, which involves stimulation of Smad7 expression, which impairs TGF-{beta} responsiveness. In addition to driving Smad7 expression, AP-1 drives induction of matrix metalloproteinases by UV irradiation (22, 23, 27, 33). UV irradiation-induced matrix metalloproteinases degrade skin collagen. This degradation of skin collagen is a major cause of premature skin aging and promotes a tissue environment conducive to skin cancer (33, 48, 49). The data presented herein support a new role for AP-1 in mediating UV irradiation-induced skin aging and cancer. By inducing Smad7 expression, AP-1 can interfere with type I procollagen production and thereby exacerbate UV irradiation-induced degradation and loss of type I collagen.


   FOOTNOTES
 
* This work was supported in part by the Babcock Foundation and National Institutes of Health Grant AG19364-02 (to G. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Dermatology, University of Michigan Medical School, 1150 W. Medical Center Dr., Medical Science I, Rm. 6447, Ann Arbor, MI 48109-0609. Tel.: 734-763-1469; Fax: 734-647-0076; E-mail: dianemch{at}umich.edu.

1 The abbreviations used are: TGF-{beta}, transforming growth factor-{beta}; Smad, Sma- and Mad-related protein; SBE, Smad binding element; EMSA, electrophoretic mobility shift assay; AP-1, activator protein-1; AP-1E, AP-1 binding element; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Rainer Heuchel and Dr. Peter ten Dijke, who kindly provided us with reagents. We are indebted to Suzan Rehbine for the procurement of tissue specimens, Kathy Bucknell for technical assistance, Laura VanGoor for the preparation of graphic material, Ted Hamilton for statistical analysis, and Diane Fiolek for administrative assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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