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J. Biol. Chem., Vol. 276, Issue 28, 26349-26356, July 13, 2001

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Ultraviolet Irradiation Blocks Cellular Responses to Transforming Growth Factor- by Down-regulating Its Type-II Receptor and Inducing Smad7^*

Taihao Quan, Tianyuan He, John J. Voorhees, and Gary J. Fisher^§

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

Received for publication, November 30, 2000, and in revised form, April 29, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transforming growth factor- (TGF-) is a multi-functional cytokine that regulates cell growth and differentiation. Cellular responses to TGF- are mediated through its cell surface receptor complex, which activates transcription factors Smad2 and Smad3. Here we report that UV irradiation of mink lung epithelial cells causes near complete inhibition of TFG--induced Smad2/3-mediated gene expression. UV irradiation inhibited TGF--induced phosphorylation of Smad2 and subsequent nuclear translocation and DNA binding of Smad2/3. Specific cell surface binding of TGF- was substantially reduced after UV irradiation. This loss of TGF- binding resulted from UV-induced down-regulation of TGF- type II receptor (TRII) mRNA and protein. UV irradiation significantly inhibited TRII promoter reporter constructs, indicating that UV reduction of TRII expression involved transcriptional repression. In contrast to its effects on TRII, UV irradiation rapidly induced Smad7 mRNA and protein. Smad7 is known to antagonize activation of Smad2/3 and thereby block TGF--dependent gene expression. UV irradiation stimulated Smad7 promoter reporter constructs, indicating that increased Smad7 expression resulted, at least in part, from increased transcription. Overexpression of Smad7 protein to the level induced by UV irradiation inhibited TGF--induced gene expression 30%. Maintaining TRII levels by overexpression of TRII prevented UV inhibition of TGF- responsiveness. Taken together, these data indicate that UV irradiation blocks cellular responsiveness to TGF- through two mechanisms that impair TGF- receptor function. The primary mechanism is down-regulation of TRII, and the secondary mechanism is induction of Smad7.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transforming growth factor-beta (TGF-)¹ family members are multifunctional cytokines whose cellular effects are dependent on cell type and cellular context. For example, TGF- stimulates proliferation of fibroblasts in connective tissue and inhibits growth of epithelial cells (1). The TGF-s play important roles in cellular differentiation and biosynthesis of extracellular matrix (2, 3). Impairment of TGF- responsiveness occurs in a variety of cancer cells and contributes to loss of growth control (4-7).

TGF- signal transduction is mediated by a complex of three transmembrane receptors, Type I (TRI), Type II (TRII), and Type III (TRIII) TGF- receptors. TRI and TRII possess intrinsic serine/threonine kinase activity. TRIII is a membrane proteoglycan that is thought to facilitate ligand binding to TRII (8-11). Binding of ligand to TGF- receptors induces formation of a heteromeric complex of TRI and TRII receptors (2, 12-15). Formation of this heteromeric complex enables the TRII to phosphorylate TRI, resulting in activation of TRI kinase (2, 13, 16-18). TRI phosphorylates and thereby activates transcription factors Smad2 and Smad3 (1, 2, 19, 20). Phosphorylated Smad2 and/or Smad3 then bind their common partner, Smad4, to form a heteromeric complex, which then translocates to and accumulates in the nucleus (19), where it acts as a transcription factor (2, 15). The actions of TGF- are antagonized by Smad7, which interacts stably with TRI to prevent phosphorylation and activation of receptor-regulated Smad2/3, thereby blocking TGF- signaling (21, 22).

Solar ultraviolet (UV) irradiation is a potent environmental hazard capable of damaging cellular DNA and causing mutations (23, 24). Recent studies demonstrate that UV irradiation of cells causes ligand-independent rapid activation and clustering of many different growth factor and cytokine cell surface receptors (24-26). The resulting stimulation of multiple signal transduction pathways leads to induction and activation of transcription factors, notably activator protein-1 and NF-B, which results in transcription of their target genes (25, 27-29).

Despite widespread interest in mechanisms of action of UV irradiation, relatively little is known regarding the effect of UV irradiation on TGF- receptor activation or on TGF-/Smad signaling. We report here that in contrast to growth factor- and cytokine receptor-mediated signal transduction, UV irradiation impairs TGF- receptor-mediated signal transduction. This impairment of TGF- responsiveness results from down-regulation of TRII and induction of Smad7 by UV irradiation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Dulbecco's modified Eagle's medium, fetal bovine serum, trypsin solution, penicillin, streptomycin, L-glutamine, and G418 (geneticin) were purchased from Life Technologies, Inc. TRI, TRII, Smad2, Smad3, and Smad7 primary and secondary antibodies for Western analysis were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-Smad2 was obtained from Upstate Biotechnology (Lake Placid, NY). Fluorescein isothiocynate-conjugated anti-rabbit IgG and anti-goat IgG secondary antibodies for immunofluorescence confocal laser microscopy were purchased from Roche Molecular Biochemicals. Human recombinant TGF- 1 was purchased from R&D Systems (Minneapolis, MN). ¹²⁵I-TGF- 1, [-³²P]ATP, and [-³²P]dCTP were obtained from PerkinElmer Life Sciences. The cross-linking agent disuccinimidyl suberate was purchased from Pierce. All other reagents were purchased from Sigma.

Cell Culture and UV Irradiation-- Mink lung epithelial cells (MLECs) and MLECs stably transfected with a plasminogen activator inhibitor-1 (PAI-1) promoter/TGF-/luciferase reporter gene were generously provided by Dr. Daniel Rifkin of the New York University Medical Center. MLECs were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine, and G418 (geneticin, 200 µg/ml, to stably transfected cells only) in a humidified incubator with 5% CO₂ at 37 °C.

Before UV irradiation, sub-confluent cells were incubated overnight in serum-free media. The next morning, the media were collected, and cells were covered with a thin layer of PBS and irradiated with UV (20 mJ/cm²) using a Daavlin Spectra panel lamp (Bryan, OH) containing six FS24T12UVB-HO bulbs. A Kodacel filter was used to eliminate wavelengths below 290 nm (UVC). The irradiation intensity was monitored with an IL400A radiometer and a SED240/UVB/W photodetector (International Light, Newbury, MA). After irradiation, the PBS was aspirated, and the original media were put back into the plates. Cellular viability 24 h after UV irradiation was near 100%, based on cell morphology and number. For TGF- 1 treatment, TGF- 1 (1 ng/ml) was added at the indicated times post-UV for 1 h.

Immunofluorescence Confocal Laser-scanning Microscopy-- MLECs (1 × 10⁴) were plated on 8-well chamber slides and exposed to UV (20 mJ/cm²). TGF-1 (1 µg/ml) was added at the indicated times post-UV for 1 h. Cells were rinsed once with PBS and fixed in 5% paraformaldehyde for 2 h at room temperature. The cells were incubated with 0.5% Nonidet P-40 and then blocked with 2% bovine serum albumin (BSA). Cells were then incubated overnight with primary antibody (anti-Smad2 or anti-Smad3, 1:500 dilution) at 4 °C. Slides were subsequently washed with PBS five times and incubated with primary antibody for 1 h. Fluorescein isothiocynate-labeled secondary antibodies were then added for 1 h. The fluorescence-stained cells were observed and photographed with a Bio-Rad MRC 600 confocal microscope through a 60× objective.

RNA Isolation and Northern Blot Analysis-- Total RNA from MLECs was extracted with a commercial kit (RNeasy Midi Kit, Qiagen, Chatsworth, CA) according to the manufacturer's protocol. Samples of total RNA (30-50 µg) were resolved by 1.2% agarose electrophoresis, transferred to nylon membranes, and hybridized with TRI, TRII, and Smad7 cDNA probes labeled with [³²P]dCTP by random priming. Each blot was stripped and re-hybridized with 36B4 internal control gene transcript to monitor the sample load in each lane. The intensities of each band were quantified by STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and normalized to the 36B4 gene transcript.

cDNA probe was generated by digestion of Smad7 cDNA expression vector (30) (obtained from Dr. Peter ten Dijke, Ludwig Institute for Cancer Research, Uppsala, Sweden) with HindIIII and XbaI. cDNA probes for TRI and TRII were generated by polymerase chain reaction from human skin total RNA using the following primers: for TRI, 5'-CCTGGCCTTGGTCCTGTG-3' and 5'-TCTGTGGCTGAATCATGTCTTACT-3'; for TRII, 5'-AACTGTGTAAATTTTGTGATGTGA-3' and 5'-CGGGCCTCTGGGTCGTG-3'. The polymerase chain reaction products were sub-cloned into the pCRII vector (Invitrogen, Carlsbad, CA) and verified by restriction digestion and sequencing.

Western Analysis-- Nuclear extracts were prepared as described previously (31). Membrane fractions were prepared according to a method previously described (32) with slight modification. Briefly, MLECs were washed twice with PBS and suspended in fresh hypotonic buffer (20 mM Tris, pH 7.5, 10 mM NaCl, 0.3 M phenylmethylsulfonyl fluoride). Cells were then harvested on ice by scraping and homogenized with a Dounce homogenizer. Crude nuclei were pelleted at 2000 × g for 10 min, and the supernatant was then spun at 25,000 × g for 30 min at 4 °C. To obtain a crude membrane fraction, the membrane pellet was re-suspended in whole cell extraction buffer (25 mM HEPES, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol, 20 mM -glycerol phosphate, 0.1 mM Na₃VO₄, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride) and stored at 80 °C. Concentrations of membrane or nuclear proteins were measured by Bio-Rad protein assay (Bio-Rad) using BSA as a standard. Nuclear or membrane proteins were resolved on 8% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and reacted with primary antibodies. Blots were visualized and quantified with enhanced chemifluorescence (ECF) (Vistra ECF Western blotting system, Amersham Pharmacia Biotech) following the manufacturer's protocol. The intensities of each band were quantified by STORM PhosphorImager.

Whole Cell Binding and Cross-linking of ¹²⁵I-TGF-1-- Binding of ¹²⁵I-TGF- to MLECs was performed according to the method described (33) with minor modifications. Sub-confluent cells were incubated overnight in complete medium. Cells were then irradiated with UV as described above, and incubation was continued for 1, 3, 5, 8, 16, or 24 h after exposure. Cells were washed once with Krebs-Ringer-Hepes/BSA binding buffer (50 mM Hepes, pH 7.5, 128 mM NaCl, 1.3 mM CaCl₂, 5 mM KCl, 0.5% BSA) and then incubated in Krebs-Ringer-Hepes/BSA for 30 min at 37 °C. Cells were washed with 0.1% glacial acetic acid for 5 min at room temperature, placed in Krebs-Ringer-Hepes/BSA, and then 0.2 nM ¹²⁵I-TGF- 1 was added for 3-4 h at 4 °C. Nonspecific binding was determined by addition of a 100-fold excess of unlabeled TGF- 1. Cells were harvested by scraping, and radioactivity was measured as described previously (34). Cross-linking of ¹²⁵I-TGF- 1 to its receptors was carried out as previously described (35), with minor modifications. Briefly, sub-confluent cells were treated with ¹²⁵I-TGF-1 as described above for the binding assay. Reactions were halted by adding glycine. ¹²⁵I-TGF- 1 was cross-linked to receptors by the addition of disuccinimidyl suberate (0.5 mg/ml). Cellular proteins were solubilized then resolved on 8% SDS-polyacrylamide electrophoresis gels.

Electrophoretic Mobility Shift Assay-- Electrophoretic mobility shift assays were performed with nuclear extracts from MLECs. Double-stranded oligodeoxynucleotides containing the TGF- response element in the PAI-1 promoter (5'-TCGAGAGCCAGACAAGGAGCCAGACAAGGAGCCAGACAC-3' and its complementary strand) and the consensus Smad binding element (5'-GGATAGCGTCTAGACATAGTCTAGACTGAGT-3' and its complementary strand) were used as probes (36, 37). All oligonucleotides were synthesized by Operon Technologies, Inc. (Alameda, CA). Electrophoretic mobility shift assays were performed as described previously (38) with minor modifications. Briefly, synthetic single strand oligonucleotides were incubated in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.3 M NaCl at 75 °C for 15 min and placed at room temperature overnight. The annealed double-stranded oligonucleotides were 5'-end-labeled with [-³²P]ATP using T4 polynucleotide kinase (Life Technologies Inc.). The end-labeled probe was purified with a G50 column (Roche Molecular Biochemicals). Approximately 2 × 10⁵ cpm of end-labeled DNA probe was incubated with 5-10 µg of MLEC nuclear extract in a volume of 20 µl. For competition experiments, a 10-50-fold molar excess of unlabeled DNA probe was preincubated with nuclear extract for 30 min on ice before labeled probe was added. After 30 min of incubation on ice, the protein-DNA complexes were electrophoresed on 4% polyacrylamide gel at 200 V in 0.5 × TBE (1.0 M Tris, 0.9 M boric acid, 0.01 M EDTA). The gel was transferred to Whatman No. 3MM paper, vacuum-dried, and scanned by STORM PhosphorImager.

Transient Transfection and Luciferase Assays-- -Galactosidase expression vector (1 µg, pCMV, CLONTECH Laboratories, Inc., Palo Alto, CA) was co-transfected to provide an internal standard for transfection efficiency. Three luciferase promoter reporter constructs were utilized: 1) Smad binding element X4 (39), containing four repetitions of the GTCTAGAC Smad3/4 binding motif (provided by Dr. Bert Vogelstein of the Johns Hopkins Oncology Center, Baltimore, MD), 2) TRII promoter (40) (provided by Dr. Seong-Jin Kim of the NCI, National Institutes of Health, Bethesda, Maryland), and 3) Smad7 promoter (41) (provided by Dr. Yan Chen of the Indiana University School of Medicine). All plasmids were introduced into cells using Fugene 6 (Roche Molecular Biochemicals) according to the manufacturer's protocol. Where indicated in the text, cells were UV-irradiated as described above 24 h after transfection, and 8 h later, TGF-1 (1 ng/ml) was added to the medium. Twenty-four h after treatment, cells were washed once with PBS, harvested in lysis buffer (Pharmingen International, San Diego, CA), and assayed for -galactosidase activity (42). Aliquots containing identical -galactosidase activity were used for each luciferase assay (43). Luciferase activity was measured using an enhanced luciferase assay kit (Pharmingen International) according to the manufacturer's protocol. Smad7 (30) or TRII (44) were overexpressed by transient transfection in MLECs stably expressing the TGF--inducible PAI promoter luciferase reporter. TRII-coding region was generated by polymerase chain reaction using the primers 5'-GAATTCGTCTGCCATGGGTCGGGGGC-3' and 5'-TTCAGGAATCTTCTCCTCC-3'. The polymerase chain reaction product was cloned into pcDNA 3.1 (Invitrogen) and verified by restriction digestion and sequencing.

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 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 Represses Transactivation by TGF--- We initially investigated the effect of UV irradiation on TGF--induced gene expression using two well characterized TGF--regulated luciferase reporter constructs: 1) an 800-base pair fragment (799/+71) of the human PAI-1 gene (45) and 2) four repetitions of the GTCTAGAC Smad3/4 binding element upstream of a SV40 minimal promoter (39). As shown in Fig. 1, TGF- treatment activated these reporter constructs more than 10-fold. Pretreatment of cells with UV irradiation 8 h before the addition of TGF-1 repressed activation of both reporter genes almost completely (Fig. 1). UV treatment alone had no significant effect either on the PAI-1 reporter gene or on the Smad binding element reporter construct (Fig. 1). Taken together, these data indicate that UV irradiation inhibits TGF- responsiveness.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   UV irradiation impairs TGF--induced gene transcription. MLECs stably expressing a PAI promoter-luciferase reporter gene (open bars) or MLECs transiently co-transfected with a luciferase reporter construct containing four repetitions of the GTCTAGAC Smad3/4 binding element and an expression vector for -galactosidase (hatched bars) were exposed to UV irradiation (20 mJ/cm2) 8 h before addition of TGF- 1 (1 ng/ml) for 16-24 h. Cell lysates were normalized to cell number for stable transfectants or -galactosidase activity for transient transfectants and assayed for luciferase activity. Bar heights are the means ± S.E. for fold change in luciferase activity relative to activity in control cells. n = 3-4. *, p < 0.05 versus untreated cells; , p < 0.05 versus TGF--treated cells.

UV Irradiation Inhibits Smad2 Phosphorylation and Nuclear Translocation-- Binding of TGF- to its receptors results in phosphorylation of Smad2 and Smad3, which then associate with Smad4 and translocate into the nucleus. The reduced responsiveness of TGF--regulated reporter constructs to TGF- after exposure of cells to UV irradiation could result from decreased Smad2/3 activation. To examine this possibility, we next examined whether UV irradiation inhibits phosphorylation of Smad2 and nuclear translocation of Smad2 and Smad3. In untreated cells, the basal level of phosphorylated Smad2 was minimally detectable by Western analysis. Treatment of cells with TGF- 1 increased Smad2 phosphorylation ~4.5-fold (Fig. 2). Treatment of cells with UV irradiation alone did not alter the level of phosphorylated Smad2 (data not shown). However, pretreatment of cells with UV (20 mJ/cm²) before the addition of TGF- 1 resulted in a time-dependent inhibition of Smad2 phosphorylation. Although TGF-1-induced Smad2 phosphorylation was not sufficiently changed at 1 and 3 h post-UV, it was almost completely blocked at 8 and 16 h post-UV. In contrast, total Smad2 levels were not affected by either TGF- or UV treatment (Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   UV irradiation inhibits TGF--induced Smad2 phosphorylation. MLECs were UV-irradiated (20 mJ/cm2), and TGF- 1 (1 ng/ml) was added at the indicated times post-UV for 1 h. Phosphorylated Smad2 in nuclear extracts (open bars) and total Smad2 in whole cell extracts (filled bars) were quantified by Western blot analysis. The inset shows representative Western blots for phospho-Smad2 and total Smad2. Bar heights are the means ± S.E. for fold change in phospho-Smad2 and total Smad2 relative to levels in untreated control (Ctrl) cells. n = 4. *, p < 0.05 versus no UV, TGF--treated cells.

We next examined whether UV irradiation affects TGF--induced nuclear translocation of phospho-Smad2 and Smad3. As shown in Fig. 3, phospho-Smad2 and Smad3 were predominantly located in the cytoplasm of untreated cells or cells treated with UV alone (data not shown). Treatment of cells with TGF-1 induced nuclear translocation of phospho-Smad2 and Smad3. Pretreatment of cells with UV 8 h before the addition of TGF- 1 substantially blocked TGF--induced translocation of phospho-Smad2 and Smad3 (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   UV irradiation inhibits TGF--induced Smad2 and Smad3 nuclear translocation. MLECs were treated with vehicle or TGF- (1 ng/ml) for 1 h. Where indicated, cells were exposed to UV irradiation (20 mJ/cm2) 8 h before addition of TGF-. Phosphorylated Smad 2 and Smad 3 were visualized by indirect immunofluoresence using confocal laser microscopy. n = 3.

UV Irradiation Inhibits DNA Binding of Smad Proteins-- UV inhibition of Smad2 and Smad3 activation by TGF- would be expected to reduce Smad protein DNA binding. To investigate this possibility, we performed electrophoretic mobility shift assays with two well characterized Smad3/4 DNA binding probes, one containing the TGF- response element in the PAI-1 promoter (36), and a second probe, identified by random oligonucleotide screening, as a Smad3 DNA binding element (37). MLECs treated with TGF- alone exhibited enhanced protein binding to both probes (Fig. 4). In contrast, treatment of cells with UV irradiation before the addition of TGF- blocked protein binding to both probes in a time-dependent manner (Fig. 4). These results indicate that UV inhibits TGF--induced DNA binding of Smad proteins.

View larger version (66K): [in this window] [in a new window]   Fig. 4.   UV irradiation inhibits TGF--induced DNA binding of Smad proteins. MLECs were UV-irradiated (20 mJ/cm2), and TGF- (1 ng/ml) was added at the indicated times post-UV for 1 h. Nuclear extracts were prepared and analyzed by electrophoretic mobility shift assays using double-stranded oligonucleotides containing the Smad binding element in the PAI-1 promoter (left panel) and 4× synthetic Smad binding element (right panel) as probes. The open triangle indicates specific retarded complexes. The solid triangle indicates nonspecific bands. n = 6.

UV Irradiation Inhibits Binding of TGF-1 to Its Cell Surface Receptors-- The above data indicate that UV irradiation interferes with TGF- activation of Smad2 and Smad3. This interference could occur as a result of physical and/or functional loss of TGF- receptors. To investigate this possibility, we determined the effect of UV irradiation on TGF- binding to its cell surface receptors. Fig. 5A shows specific binding of ¹²⁵I-TGF- 1 to intact MLEC after UV irradiation. TGF- binding increased 35% at 1 h post-UV and returned to its initial level by 3 h post-UV. Between 5 and 24 h post-UV, there was a progressive decrease in the level of ¹²⁵I-TGF-1 binding. At 24 h post-UV, TGF-1 binding was reduced ~60% relative to non-irradiated control cells (100%) (Fig. 5A).

View larger version (51K): [in this window] [in a new window]   Fig. 5.   UV irradiation inhibits TGF-1 binding to intact MLECs. A, MLECs were UV-irradiated (20 mJ/cm2). Cells were washed to remove any endogenous TGF- at the indicated times post-UV and then incubated with 125I-TGF- 1 (0.2 nM) for 3 h at 4 °C. Unbound 125I-TGF-1 was removed by washing, and cells were collected and counted. Bar heights are the means ± S.E. of percent 125I-TGF-1 binding to non-irradiated control cells (set at 100%). n = 3. *, p < 0.05 versus non-irradiated control (Ctrl) cells. B, MLECs were irradiated with UV (20 mJ/cm2) and then affinity-labeled with 125I-TGF- 1 at the indicated times after UV exposure. 125I-TGF- 1 was then cross-linked to receptors by adding disuccinimidyl suberate, as described under "Experimental Procedures." 125I-TGF-1-receptor protein complexes (TRI, TRII, TRIII, arrowheads) were resolved by SDS-polyacrylamide gel electrophoresis and visualized by STORM PhosphorImager. n = 3.

We next determined the level of TGF-·TGF- receptor complexes by cross-linking ¹²⁵I-TGF- 1 to its receptors in intact MLEC. In non-irradiated and UV-irradiated cells, three major binding complexes were identified, with apparent molecular weights of 65,000, 85,000, and 200,000-250,000, shown previously to be TGF- complexed with TRI, TRII, and TRIII, respectively (46). Consistent with other studies (47, 48), TRIII receptors were most prominently labeled with ¹²⁵I-TGF- 1, whereas TRII and TRI yielded weaker-labeled complexes (Fig. 5B). Cells treated with UV exhibited significant time-dependent decreases of the cross-linked complexes for all three TGF- receptors relative to levels in non-irradiated control cells (Fig. 5B).

UV Irradiation Down-regulates TRII but Not TRI-- Reduced binding of TGF- to its receptors after UV irradiation could occur as a result of decreased expression of TGF- receptors. To investigate this possibility, we next examined the effect of UV on TGF- receptor mRNA and protein levels. UV irradiation reduced TRII mRNA (Fig. 6A) and protein levels (Fig. 6B) in a time-dependent manner, as measured by Northern and Western analysis, respectively. TRII mRNA was reduced within 3 h post-UV and was progressively reduced during the succeeding 13 h (Fig. 6A). By 16 h after UV, TRII mRNA was reduced ~50% relative to control. TRII protein expression was also reduced after UV exposure. TRII protein was maximally reduced (80%) at 16 h post-UV (Fig. 6B). In contrast, TRI mRNA levels were modestly increased between 1 and 3 h post-UV and then returned to their initial values (Fig. 6C). UV had no significant effect on TRI protein levels (Fig. 6D).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   UV irradiation reduces TRII but not TRI mRNA and protein. MLECs were UV-irradiated (20 mJ/cm2), and total RNA or membrane protein was prepared at the indicated times post-UV. A, TRII and 36B4 mRNA (a ribosomal protein used as an internal control for quantitation) were analyzed by Northern analysis. The inset shows representative Northern analysis. Bars heights indicate means ± S.E. of TRII mRNA levels (normalized to 36B4 mRNA levels) relative to levels in non-irradiated control cells (Ctrl, 100%). *, p < 0.05 versus Ctrl. n = 4. B, TRII protein in the membrane fraction of MLECs was quantified by Western analysis. The inset shows a representative Western blot. Bar heights indicate the means ± S.E. of receptor protein levels relative to receptor protein levels in non-irradiated control cells (Ctrl, 100%). *, p < 0.05 versus Ctrl. n = 6. C, TRI and 36B4 mRNA were analyzed by Northern analysis. Representative Northern blots for TRI and 36B4 mRNA are shown in the inset. Bar heights indicate means ± S.E. for fold change in TRI mRNA levels (normalized to 36B4 mRNA levels) relative to levels in non-irradiated control cells (Ctrl). *, p < 0.05 versus Ctrl. n = 3. D, TRI protein in the membrane fraction of MLEC cells was quantified by Western analysis. The inset shows representative Western blots. Bar heights indicate the means ± S.E. of receptor protein levels in UV-irradiated cells relative to receptor protein levels in non-irradiated control cells (Ctrl). n = 3.

Reduction of the TRII mRNA after UV irradiation could reflect inhibition of TRII gene transcription. To examine this possibility, we transiently transfected MLECs with two different TRII promoter/luciferase constructs, a longer construct (1640 to +62) and a shorter construct (137 to +62). Both promoter constructs were active in non-irradiated cells (Fig. 7). However, the shorter TRII promoter/luciferase construct (137 to +62) was markedly more active than the longer promoter constructs, consistent with the observed presence of a negative regulatory element between 1240 to 504 (40). UV irradiation inhibited the activity of both TRII promoter constructs nearly 70% (Fig. 7).

View larger version (11K): [in this window] [in a new window]   Fig. 7.   UV irradiation inhibits TRII promoter activity. MLEC were transiently transfected with TRII gene promoter (1640 to +62 or 137 to +62)/luciferase constructs. Twenty-four hours after transfection, cells were irradiated with UV (20 mJ/cm2). Cell lysates were prepared 48 h after transfection and normalized by -galactosidase activity. Promoter activities were determined by luciferase assay. Bar heights represent the means ± S.E. *, p < 0.05 versus non-irradiated cells. n = 3.

UV Irradiation Induces Smad7 mRNA and Protein-- Recent evidence indicates that cytokines and growth factors can induce Smad7, which inhibits TGF- signaling (49). Since UV irradiation activates cytokine and growth factor receptors in a variety of cell types (25, 50), we examined whether UV inhibition of the TGF- responsiveness involves induction of Smad7 in addition to loss of TRII. As shown in Fig. 8A, Smad7 mRNA increased 2-fold between 1 and 3 h post-UV and then returned to basal levels between 8 and 24 h post-UV. Smad7 protein levels rose steadily between 1 and 8 h post-UV and remained elevated (3-fold) for at least 24 h post-UV (Fig. 8B). We also examined the effect of UV on Smad7 gene promoter activity by transiently transfecting MLECs with long (4200 to +112) and short (408 to +112) Smad7 promoter/luciferase constructs. As shown in Fig. 8C, the two promoter constructs were equally active in non-irradiated cells and equally induced (2-fold) after UV irradiation. These data indicate that the induction of Smad7 by UV irradiation is mediated at least in part by increased transcription.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   UV irradiation induces Smad7 mRNA, protein expression, and promoter activity. MLECs were exposed to UV irradiation (20 mJ/cm2). At the indicated times post-UV, cells were collected, and either total RNA or protein were extracted. A, Smad7 and 36B4 (used as an internal control) mRNA levels were analyzed by Northern analysis. The inset shows representative Northern blot for Smad7 and 36B4 (normalized to 36B4 mRNA levels). The bars indicate the means ± S.E. for fold change in Smad7 mRNA levels relative to levels in non-irradiated control cells (Ctrl). *, p < 0.05 versus control. n = 3. B, Smad7 protein was quantified by Western analysis. The inset shows a representative Western blot for Smad7 protein. The bar heights indicate the means ± S.E. for fold change in Smad7 protein relative to levels in non-irradiated control cells (Ctrl). *, p < 0.05 versus control. n = 3. C, UV irradiation increases Smad7 promoter activity. MLECs were transiently transfected with Smad7 promoter (4200 to +112 or 408 +112)/luciferase constructs. Twenty-four hours after transfection, cells were irradiated with UV (20 mJ/cm2). Cell lysates were prepared 48 h after transfection and normalized by -galactosidase activity. Promoter activities were determined by luciferase assay. The bar heights represent the means ± S.E. *, p < 0.05 versus non-irradiated cells, n = 3.

To examine the functional consequences of this induction of Smad7 by UV irradiation, we determined the relationship between Smad7 protein levels and inhibition of TGF--induced gene expression. For these studies, MLECs were transfected with varying amounts of Smad7 expression vector, and the levels of Smad7 protein and TGF--induced luciferase reporter activity were determined. Transfection of MLECs with increasing amounts of Smad7 cDNA resulted in dose-dependent increases in Smad7 protein expression and inhibition of TGF--induced luciferase activity (Fig. 9). Transfection of MLEC cells with 2 µg of Smad7 cDNA resulted in an approximate 3-fold induction of Smad7 protein and 30% inhibition of TGF--induced luciferase activity (Fig. 9). As described above (Fig. 8B), UV irradiation resulted in a 3-fold induction of Smad7 protein at 8-24 h post-UV. Together, these data indicate that UV induction of Smad7 could account for ~30% of the total inhibitory effect of UV on TGF--induced gene expression.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Smad7 overexpression inhibits TGF--induced gene transcription. MLECs stably expressing a TGF--inducible PAI promoter luciferase construct were transfected with the indicated concentrations of Smad7 expression vector. Twenty-four hours after transfection, cells were treated with TGF-1 (1 ng/ml), and luciferase activity was determined 48 h after transfection. The bar heights (left axis, open bars) indicate the means ± S.E. of luciferase activity relative to levels in non-transfected cells (control (Ctrl)). The bar heights (right axis, hatched bars) indicate the means ± S.E. for fold change in Smad7 protein relative to levels in non-transfected cells (control). *, p < 0.05 versus control. n = 3.

Overexpression of TRII Overcomes UV Inhibition of TGF--induced Gene Expression-- The finding that UV-induced Smad7 only modestly inhibits TGF- responsiveness suggests that the majority of UV inhibition results from down-regulation of TRII. To examine this possibility, we determined whether maintaining TRII levels by forced overexpression of TRII could overcome UV inhibition of TGF--induced gene expression. Overexpression of TRII had no significant effect on TGF--induced (or basal, data not shown) PAI-1 promoter activity (Fig. 10), indicating that TRII levels are not limiting for TGF- responsiveness in non-irradiated cells. As expected, exposure of mock-transfected cells to UV before the addition of TGF- repressed TGF-1-induced reporter gene 80%. In contrast, in cells overexpressing TRII, UV did not inhibit TGF- responsiveness. Complete prevention of UV-induced loss of TGF--induced gene expression was observed with 4 µg of TRII expression vector. These data demonstrate that down-regulation of TRII by UV is critical for UV inhibition of TGF- responsiveness.

View larger version (13K): [in this window] [in a new window]   Fig. 10.   Overexpression of TRII overcomes UV inhibition of TGF- induced gene expression. MLECs stably expressing a PAI promoter-luciferase reporter gene were transiently transfected with 2 or 4 µg of TRII expression vector. Twenty-four hours after transfection, cells were irradiated with UV (20 mJ/cm2) and then 8 h later treated with TGF- 1 (1 ng/ml) for 16 h. Cells lysates were normalized to cell numbers, and luciferase activity was determined by luciferase assay. The bar heights are the means ± S.E. of fold change in luciferase activity relative to activity in control (Ctrl) cells. n = 3. *, p < 0.05 versus mock-transfected, non-treated control cells; , p < 0.05 versus mock-transfected, no UV + TGF--treated cells. UV irradiation had no significant effect on luciferase activity in cells over-expressing T RII.

UV Induction of c-Jun/AP-1 Does Not Correlate with Inhibition of TGF- Responsiveness-- Accumulating evidence indicates that transcription factor AP-1 or its component c-Jun can both positively and negatively modulate TGF-/Smad-dependent gene expression (51-53). In addition, we and others show that UV irradiation induces c-Jun and AP-1 activity (23, 25, 50, 54-56). These observations raise the possibility that UV induction of c-Jun/AP-1 could contribute to reduced TGF- responsiveness. To examine this issue, we determined the kinetics of UV induction of c-Jun protein and AP-1 DNA binding in MLECs. UV irradiation induced c-Jun protein 3.0 ± 0.7-fold (n = 3) within 1 h. Maximum induction (5.8 ± 0.9-fold, n = 3) occurred within 5 h post-UV. c-Jun protein levels returned to base line by 24 h post-UV (1.3 ± 0.3-fold, n = 3). Electrophoretic mobility shift assays using oligonucleotides containing the consensus AP-1 binding element as probe revealed nearly identical kinetics of induction (data not shown). These kinetics of c-Jun/AP-1 activation substantially differ from the kinetics of UV inhibition of TGF- responsiveness described above. At early times post-UV (1-5 h), c-Jun/AP-1 is maximally induced, whereas TGF- responsiveness is not altered. At later times post-UV (8-24 h) c-Jun/AP-1 activation subsides, whereas TGF- responsiveness is inhibited. These data indicate that UV inhibition of TGF- responsiveness does not correlate with c-Jun/AP-1 activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Accumulating evidence indicates that UV irradiation activates cytokine and growth factor signal transduction pathways (25, 50). In contrast, we report here that UV irradiation impairs TGF-/Smad signaling. This impairment results primarily from reduction of TRII expression and, to a lesser extent, from induction of Smad7. This UV-induced loss of TRII and increase in Smad7 impairs TGF- signal transduction as manifested by inhibition of TGF--induced Smad2/3 activation and nuclear translocation, inhibited formation of Smad/DNA complexes, and inhibition of TGF--induced gene expression.

TGF- exerts its cellular effects by interacting with specific cell surface receptors. Binding of TGF- to TRII results in the formation of a heteromeric complex between TRI and TRII, which in turn results in the phosphorylation of TRI by TRII. Activation of TRI results in propagation of signals through phosphorylation of Smad2 and Smad3. Overexpression of either truncated TRI or TRII, which lack cytoplasmic kinase domains, results in suppression of TGF- responsiveness in lung fibroblasts (10, 47), indicating that both TRI and TRII are indispensable for TGF- signaling (57, 58). TRII is essential for binding of TGF- to the receptor complex, and TRI is necessary for downstream signal transduction induced by TGF- binding to TRII (12). Binding of TGF- to TRII is the first critical step in the TGF--signaling cascade because TRI does not bind TGF- in the absence of TRII (58-60).

Our results indicate that UV irradiation of cells targets the first step of the TGF-/Smad-signaling cascade by causing substantial loss of TRII protein. UV irradiation caused simultaneous loss of TRII mRNA and protein. TRII promoter/luciferase activity is inhibited after UV irradiation, suggesting that down-regulation of TRII mRNA results, at least in part, from decreased gene transcription.

The TRII gene promoter has been identified and partially characterized (40, 61). It contains both positive and negative regulatory elements in addition to a core element required for basal activity. Both receptor constructs that we utilized contain a strongly negative element spanning nucleotides 100 to 67. It is possible that this element may mediate UV inhibition of promoter activity, although the cis-acting factors that regulate this element are not known. Recently, Choi et al. (11) identified a novel Ets-related transcription factor that activates transcription of the TRII gene by binding to a response element between +13 and +24 and multiple Ets-binding sites in the TRII promoter. Whether this Ets-related transcription factor is involved in UV down-regulation of TRII gene transcription remains to be determined. Although our data indicate that UV irradiation represses TRII gene transcription, they do not rule out the possibility of additional post-transcriptional regulation. Reduced levels of TRII protein could result from reduced synthesis attributable to reduced transcription and/or to increased degradation. UV irradiation may cause internalization of TRII, which could accelerate degradation of TRII protein. Future studies are needed to clarify the precise molecular mechanism(s) by which UV irradiation reduces TRII expression.

In contrast to its effect upon TRII, UV irradiation did not alter TRI protein levels. However, UV transiently induced TRI mRNA, indicating that UV regulates TRI and TRII differently. Although cDNA sequences of TRI and TRII share partial homology, there are significant differences in their promoter regions (40, 62, 63), consistent with our finding that these two genes are differentially regulated by UV irradiation. The 5'-flanking region of the TRI gene promoter is extremely GC-rich and contains multiple Sp1 sites, which are essential for basal and maximal promoter activity (62, 63).

Smad7 antagonizes TGF-/Smad signaling by interacting with TRI to interfere with phosphorylation and activation of Smad2 and -3. TGF- induces Smad7 expression, which serves as a negative feedback loop to limit the intensity and duration of the TGF- response (21, 22). UV transiently induced Smad7 mRNA and elevated Smad7 protein levels for at least 24 h post-UV. There was a significant correlation between Smad7 protein levels and inhibition of TGF--induced gene expression. We estimate that UV induction of Smad7 (3-fold) could account for 30% of the observed inhibition of TGF--induced gene expression.

Smad7 gene promoter activity was increased after UV irradiation, suggesting that up-regulation of Smad7 mRNA was due at least in part to increased gene transcription. Recently, Bitzer et al. (49) reported that pro-inflammatory cytokines such as tumor necrosis factor- and interleukin-1 suppress TGF-/Smad signaling through up-regulation of Smad7 gene transcription. This induction of Smad7 is mediated by the NF-B/RelA pathway. UV irradiation activates NF-B and induces proinflammatory cytokines such as interleukin-1 and tumor necrosis factor- (64-71). Interestingly, we have recently observed that interleukin-1 and tumor necrosis factor- reduce expression of TRII and induce expression of Smad7.² These data raise the possibility that UV induction of NF-B and/or NF-B-regulated cytokines may coordinately reduce TRII and induce Smad7. We are currently investigating this possibility.

Finally, we utilized Smad7 and TRII expression vectors to determine the relative contributions of Smad7 induction and TRII down-regulation to UV inhibition of TGF- responsiveness. Our data indicate that UV-induced Smad7 accounts for ~30% of the total inhibitory effect of UV on TGF- responsiveness. In contrast, overexpression of TRII completely prevented UV inhibition of TGF- responsiveness, indicating that down-regulation of TRII plays a major role in UV inhibition of TGF- signaling.

In addition, we examined the possibility that UV induction of c-Jun/AP-1 could contribute to reduced TGF- responsiveness since UV irradiation induces c-Jun/AP-1 and overexpression of c-Jun inhibits Smad3-dependent transcription (51, 52). However, we found there was no correlation between the kinetics of UV induction of c-Jun/AP-1 and the kinetics of UV inhibition of TGF- responsiveness. Furthermore, inhibition of TGF--induced gene expression by c-Jun/AP-1 does not involve inhibition of Smad2/3 nuclear translocation (51), whereas we found that UV irradiation blocked Smad2/3 nuclear translocation, presumably due to down-regulation of TRII. These results indicate that UV-induced c-Jun/AP-1 is not a major contributing factor to UV-inhibition of TGF- responsiveness in MLEC. Alternatively, c-Jun/AP-1 may cooperate with Smad2/3 to induce TGF--regulated genes (53, 72). If so, then UV activation may make c-Jun/AP-1 unable to function as a co-factor in TGF--induced gene expression. Our data indicate that this scenario is unlikely because return of c-Jun/AP-1 to its basal state 24 h post-UV did not restore TGF- responsiveness.

In summary, UV irradiation blocks cellular responsiveness to TGF- through two mechanisms that impair TGF- receptor function. The primary mechanism is down-regulation of TRII, and the secondary mechanism is induction of Smad7.

	ACKNOWLEDGEMENTS

We thank YueXian Hu for technical assistance, Laura VanGoor for the preparation of graphic material, Ted Hamilton for statistical analysis, and Anne Chapple for editorial assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^§ 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@umich.edu.

Published, JBC Papers in Press, April 24, 2001, DOI10.1074/jbc.M010835200

² T. Quan, T. He, J. J. Voorhees, and G. J. Fisher, unpublished data.

	ABBREVIATIONS

The abbreviations used are: TGF-, transforming growth factor-; MLEC, mink lung epithelial cell; PAI-1, plasminogen activator inhibitor-1; TRI, TGF- receptor I; TRII, TGF- receptor II; PBS, phosphate-buffered saline; BSA, bovine serum albumin; AP-1, activator protein-1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES