Smad7 Is Induced by Norepinephrine and Protects Rat Hepatocytes ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 49, Issue of December 7, pp. 45636 –45641, 2001 Printed in U.S.A.

Smad7 Is Induced by Norepinephrine and Protects Rat Hepatocytes from Activin A-induced Growth Inhibition* Received for publication, June 8, 2001, and in revised form, August 27, 2001 Published, JBC Papers in Press, September 10, 2001, DOI 10.1074/jbc.M105302200

Chiho Kanamaru, Hiroshi Yasuda‡, Masafumi Takeda, Namiki Ueda, Junko Suzuki, Tomohiro Tsuchida, Hirosato Mashima, Hirohide Ohnishi, and Toshiro Fujita From the Department of Medicine, University of Tokyo School of Medicine, Tokyo 113– 8655, Japan

Activin A induces growth arrest of rat hepatocytes in vitro and in vivo. The ␣1-adrenergic agonist, norepinephrine (NE), enhances epidermal growth factor-stimulated DNA synthesis and inhibits activin A-induced growth inhibition, but the mechanisms of these actions are unclear. Smad proteins have recently been identified as intracellular signaling mediators of transforming growth factor-␤ family members. In the present study, we explored how NE modulates the Smad signaling pathway in rat cultured hepatocytes. We demonstrate that NE inhibits activin A-induced nuclear accumulation of Smad2/3 and that NE rapidly induces inhibitory Smad7 mRNA expression. Infection of Smad7 adenovirus into rat hepatocytes inhibited activin A-induced nuclear accumulation of Smad2/3, enhanced epidermal growth factor-stimulated DNA synthesis, and abolished the growth inhibitory effect of activin A. We also demonstrated that the induction of Smad7 by NE is dependent on nuclear factor-␬B (NF-␬B). The amount of active NF-␬B complex rapidly increased after NE treatment. Preincubation of the cells with an NF-␬B pathway inhibitor N-tosyl-L-phenylalanine chloromethyl ketone or infection of the cells with an adenovirus expressing an I␬B super-repressor (Ad5I␬B) abolished the NE-induced Smad7 expression. These results indicate a mechanism of transmodulation between the Smad and trimeric G protein signaling pathways in rat hepatocytes.

Cell proliferation does not generally occur in the adult mammalian liver. However, the hepatic parenchyma rapidly responds to surgical resection or chemical injury with coordinated waves of DNA synthesis. This makes liver regeneration a useful model for studying stimulated cell growth. Positive and negative growth regulators control hepatocyte proliferation (1, 2, 3). Tumor necrosis factor (TNF)-␣1 and interleukin-6 * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (to H. Y. and H. O.) 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. ‡ Current address: Dept. of Gastroenterology at Showa University Fujigaoka Hospital, 1-30 Fujigaoka Aoba-ku, Yokohama 227-8501 Japan. To whom correspondence should be addressed: Dept. of Medicine, University of Tokyo School of Medicine, 7-3-1, Hongo, Tokyo, 113-8655, Japan. Tel.: 81-3-3943-3104; Fax: 81-3-3943-3102; E-mail: [email protected]. 1 The abbreviations used are: TNF, tumor necrosis factor; EGF, epidermal growth factor-like growth factor; TGF, transforming growth factor; NE, norepinephrine; Smad, Sma- and Mad-related protein; R-Smad, receptorregulated Smad; Co-Smad, common mediator Smad; I-Smad, inhibitory Smad; NF, nuclear factor; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; IFN; interferon; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.

are critical for initiating hepatocyte proliferation. While heparin-binding epidermal growth factor-like growth factor (EGF) (4), hepatocyte growth factor, and transforming growth factor (TGF)-␣ stimulate the growth of hepatocytes, members of the TGF-␤ superfamily, TGF-␤ and activin A, inhibit their growth. TGF-␤ induces apoptosis of hepatocytes and inhibits their proliferation in paracrine and autocrine manners (5, 6, 7, 8). Activin A is an autocrine negative regulator of DNA synthesis in hepatocytes (9, 10). It also induces cell death of parenchymal liver cells in vitro and in vivo (11). In addition to these growth factors and cytokines, adrenergic agonists can act as co-mitogens of rat hepatocytes; ␣1-adrenergic receptors were reported to be involved in the proliferation of rat hepatocytes in vitro (12, 13) and in vivo during liver regeneration (14). Norepinephrine (NE) enhances DNA synthesis in rat-cultured hepatocytes in the presence of EGF and insulin, and injection of the ␣1 antagonist, prazosin, attenuates the [3H]thymidine incorporation in the rat regenerating liver after partial hepatectomy. In addition to co-mitogenic action, NE was shown to reduce TGF␤- and activin A-induced growth inhibition and apoptosis of rat hepatocytes in culture (15, 16), but the precise mechanism underlying the reduction is not well understood. Smad proteins are a group of recently identified molecules that function as intracellular signaling mediators and modulators of TGF-␤ family members (17, 18). Smad proteins are involved in TGF-␤ and activin-induced growth inhibitions in many cell systems. Smads can be classified into three groups: receptor-regulated Smads (R-Smads), common mediator Smads (Co-Smads), and inhibitory Smads (I-Smads). Upon the binding of a ligand to a type II receptor, type II receptor kinases phosphorylate the GS domain of type I receptors, leading to activation of the type I receptor. The activated type I receptor kinases phosphorylate R-Smads differentially at two serine residues in the SSXS motif at their extreme C termini (19, 20). R-Smads include Smad1, -2, -3, -5 and -8. Smad1 and -5 mediate the signaling of bone morphogenetic protein 2 and 4, Smad2 and -3 mediate the signaling of TGF-␤ and activins, and Smad8 mediates the signaling of ALK-2 receptor kinases. The phosphorylated R-Smads form oligomeric complexes with a CoSmad, Smad4; the complexes then translocate into the nucleus. These complexes then activate the transcription of target genes. I-Smads Smad6 and Smad7) act in opposition to the signal-transducing R- and Co-Smads by forming stable associations with activated type-I receptors and thus preventing the phosphorylation of R-Smads (21–23). Interplays between TGF-␤/Smad signaling and other signaling systems, such as vitamin D (24) and EGF (25), have been recently reported. However, signaling pathways from G protein-coupled receptors to those of TGF-␤ family members are poorly defined. In this study, we examined how NE inhibits

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Smad7 Is Induced by Norepinephrine activin A-induced growth inhibition of rat hepatocytes. We demonstrate that NE inhibits activin A-induced nuclear accumulation of Smad proteins. We also show that NE induces the expression of an anti-Smad, Smad7. Overexpression of Smad7 enhances EGF-stimulated DNA synthesis and abolishes the growth inhibitory effects of activin A. We also demonstrate that the induction of Smad7 by NE is dependent on nuclear factor-␬B (NF-␬B). EXPERIMENTAL PROCEDURES

Materials—Recombinant human activin A was kindly provided by Dr. Y. Eto, Ajinomoto, Inc. (Kawasaki, Japan). Norepinephrine and mouse EGF were purchased from Sigma. [3H]Thymidine (25 Ci/mmol) was purchased from Amersham Pharmacia Biotech. Anti-Smad2/3, anti-Smad4, anti-NF-␬B p65 and anti-I␬B␣ antibodies were purchased from Santa Cruz (Santa Cruz, CA). Rat Smad6 cDNA was kindly provided by Dr. T. Kato (Fukushima Medical College), and AdSmad7 adenovirus was kindly provided by Dr. Miyazono (Japanese Foundation for Cancer Research). Ad5IkB adenovirus was kindly provided by Drs. Y. Iimuro (Hyogo Medical Collage) and D. A. Brenner (Univ. of North Carolina at Chapel Hill). Smad7 cDNA was kindly provided by Dr. P. ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden). Cell Culture and Adenovirus Infection—Rat hepatocytes obtained from male Wistar rats (170 –200 g) were used. Rat parenchymal liver cells were prepared by the method of Berry and Friend (26). Cells were plated on a collagen-coated dish in William’s E medium containing 5% fetal calf serum, 1 nmol/liter insulin, 10 nmol/liter dexamethasone, streptomycin, and penicillin. The cells were allowed to attach for 3 h, and then the medium was changed to serum-free William’s E medium containing 1 nmol/liter insulin, 0.05% bovine serum albumin, 10 nmol/ liter dexamethasone, streptomycin, and penicillin. All of the culture incubations were carried out at 37 °C under a humidified condition of 95% air and 5% CO2. In all subsequent experiments, primary hepatocytes were infected with recombinant adenovirus 2 h after plating at a dose of 10 –50 plaque-forming units per cell in hormonally defined media. After 1 h, the medium was changed. Since ␣1-adrenergic receptor is reported to be dominant during first 0 – 8 h in rat cultured hepatocytes (27), we harvested the cells 5 h after infection with Ad5I␬B. Measurement of DNA Synthesis—DNA synthesis was assessed by measuring [3H]thymidine incorporation into trichloroacetic acid-precipitable material. [3H]Thymidine was added to the culture medium from 48 to 72 h, and [3H]thymidine incorporation was measured as described by McNiel et al. (28). As previously reported (9), activin A completely inhibits DNA synthesis between 0 –36 h after plating, and we added activin A at 24 h. RNA Extraction and RNase Protection Assay—Total RNA was extracted from cultured hepatocytes using TRIzol Reagent (Life Technologies, Inc.). The RNase protection assay was performed using the RPA II kit (Ambion Inc., Austin, TX) according to the manufacturer’s instructions. As templates, we used the 306-base pair fragment corresponding to nucleotides 1183–1488 of rat Smad6 cDNA and the 154base pair fragment corresponding to nucleotides 999 –1152 of rat Smad7 cDNA (GenBank™ AF042499). Ten ␮g of total RNA was allowed to hybridize with 5 ⫻ 105 cpm per each 32P-labeled riboprobe at 45 °C for 16 h, followed by digestion with RNase A and RNase T1 at 37 °C. The resultant protected hybrids were isolated by ethanol precipitation and separated on a 5% polyacrylamide/8 M urea denaturing gel. The dried gel was exposed to Kodak XAR-2 film (Eastman Kodak, Rochester NY) at ⫺80 °C to an autoradiogram. Immunoblotting—Cells were solubilized in a lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 100 units/ml aprotinin. Proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. Visualization was performed with an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech). Immunofluorescence Microscopy—Rat hepatocytes were cultured on collagen-coated glass coverslips at a density of 2 ⫻ 104 cells/35 mm. Cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with PBS containing 0.1% (v/v) Triton X-100, and incubated sequentially with Blocking Ace (Yukijirushi Co., Tokyo, Japan). The cells were then incubated with anti-Smad2/3 antibody (Santa-Cruz) at room temperature for 60 min, extensively rinsed with PBS, and exposed to secondary fluorescent antibody at room temperature for 60 min. The samples were examined with a Nikon E-600 microscope (Nikon, Tokyo, Japan). Images were captured and digitized

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using a Spot CCD camera (Diagnostic Instrument, Sterling Height, MI). Nuclear Extract Preparation—Nuclear extracts were prepared by the method of Dignam et al. (29). After the appropriate treatment, the cells were washed twice with ice-cold PBS, scraped, and resuspended in 400 ␮l of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM PMSF). After 15 min, Nonidet P-40 was added to a final concentration of 0.6%. Nuclei were pelleted and resuspended in 50 ␮l of buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM PMSF). After incubation at 4 °C for 30 min, the lysates were centrifuged, and the supernatants containing the nuclear proteins were transferred to new vials. The protein concentration of the extract was measured using a Bio-Rad Protein Determination kit (Hercules, CA). Electrophoretic Mobility Shift Assay (EMSA)—Double-strand NF-␬B oligonucleotides (Promega, Madison, WI) were 32P-end labeled using 3000 Ci/mmol [␥-32P]ATP and polynucleotide kinase. The probe was incubated with 10 ␮g of the nuclear protein along with 1 ␮g of poly(dIdC)-poly(dI-dC) (Amersham Pharmacia Biotech) in each reaction for 30 min at room temperature in a buffer containing 20 mM Hepes (pH 7.9), 60 mM KCl, 0.2 mM EDTA, 5 mM MgCl2, 8% glycerol, 0.1 mM PMSF, and 10 ␮g/ml aprotinin. For the antibody supershift assay, 2 ␮g of NF-␬B p65 antibody (Santa Cruz) was added to the samples 30 min before incubation with the labeled probe, and all samples were further incubated at room temperature for 30 min before electrophoresis. DNAprotein complexes were resolved on a 4% nondenaturing acrylamide gel, dried, and visualized by autoradiography. Statistical Analyses and Expression of Results—The experiments presented under “Results” are representative of three or more similar experiments. The results were expressed as means ⫾ S.D., and statistical analysis was carried out using Student’s t test for paired data. Differences at p ⬍ 0.05 were considered to be significant. RESULTS

NE Enhances EGF-stimulated DNA Synthesis and Inhibits Activin A-induced Growth Inhibition of Rat Hepatocytes—NE is a co-mitogen of hepatocyte proliferation. Activin A potently inhibits the growth of primary cultured rat hepatocytes (9). It has recently been reported that NE reverses the effect of activin A on DNA synthesis (16). Consistent with previous data, NE significantly enhanced EGF-stimulated DNA synthesis in rat hepatocytes (Fig. 1A). Activin A inhibited EGF-stimulated DNA synthesis in rat hepatocytes in a dose-dependent manner. The addition of NE 24 h prior to the addition of activin A to the culture medium nearly completely reversed the growth inhibitory effect of activin A (Fig. 1B). Inhibition of Activin A-induced Smad2/3 Nuclear Accumulation in Rat Hepatocytes by NE—Next, we examined whether NE interferes with activin A/Smad signaling by preventing the nuclear translocation of Smad2/3. Most untreated rat hepatocytes showed granular cytoplasmic staining for Smad2/3, and only nuclear bodies were stained in their nuclei (Fig. 2A, a; 14% of cells showed nuclear staining). After exposure to activin A alone for 30 min, nuclear staining of Smad2/3 was exclusively detectable in 90% of the cells (Fig. 2A, c). In contrast, incubation with NE alone had no significant effect on the subcellular localization of Smad2/3 (Fig. 2A, b; 12% of cells showed nuclear staining). The addition of NE 24 h prior to adding activin A suppressed the activin A-induced nuclear translocation of Smad2/3 in rat hepatocytes (Fig. 2A, d; 28% of the cells showed nuclear staining). We also examined the level of activin A-induced Smad4 nuclear localization by Western blot analysis of nuclear extracts (Fig. 2B). In the activin A-treated cells, an increase in Smad4 nuclear localization was observed. When the cells were treated with NE 24 h prior to adding activin A, the accumulation of nuclear Smad4 was markedly reduced. Thus, NE treatment modulated an early event in the activin A/Smad signaling pathway. NE Stimulated Inhibitory Smad7 Expression—Down-regulation of TGF-␤ signaling is affected, in part, by a feedback mechanism involving induction of expression of the inhibitory Smads, Smad6 and Smad7 (21–23). Ectopic expression of

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Smad7 Is Induced by Norepinephrine

FIG. 1. A, increase in DNA synthesis upon incubation of primary culture of rat hepatocytes with NE. Hepatocytes were incubated with or without (control) 1 nmol/liter EGF and 0.1 ␮mol/liter insulin in the presence or absence of 10 ␮mol/liter NE. [3H]Thymidine was included in the culture medium from 48 h to 72 h. Values are expressed as the mean ⫾ S.D. Each result is representative of three independent experiments with similar results performed in triplicate. *p ⬍ 0.05 versus control. B, effect of NE on activin-induced inhibition of [3H]thymidine incorporation. Hepatocytes were incubated with 1 nmol/liter EGF and 0.1 ␮mol/liter insulin and various concentrations of activin A in the presence or absence of 10 ␮mol/liter NE. NE was included in the culture medium from 0 h to 72 h, and activin A was included from 24 h to 72 h. Values are expressed as the mean ⫾ S.D. Each result is representative of three independent experiments with similar results performed in triplicate.

Smad7 inhibited the TGF-␤- and activin-induced growth inhibition (30, 31). Interferon (IFN)-␥ was recently shown to inhibit TGF-␤/Smad signaling through induction of Smad7 expression (32). We reasoned, therefore, that Smad6 or Smad7 may play a role in the NE-induced suppression of activin A/Smad signaling. As shown in Fig. 3, NE treatment increased the mRNA level of Smad7 in rat hepatocytes. This increase was observed within 1 h and reached a maximum level at 3 h of exposure. However, NE did not affect the expression of Smad6, another anti-Smad (data not shown). Increased Smad7 Expression Enhanced EGF-stimulated DNA Synthesis and Inhibited Activin A-induced Growth Inhibition of Rat Hepatocytes—To examine whether Smad7 expression by itself could inhibit activin A-induced growth inhibition, we infected rat hepatocytes with an adenovirus expressing Smad7 (AdSmad7). We used an adenovirus expressing ␤-galactosidase (AdLacZ) as an infection control. We determined the infection efficiency by in situ staining with 5-bromo-4-chloro-

FIG. 2. A, effect of NE on activin A-induced Smad2/3 nuclear accumulation in rat hepatocytes. Cells were incubated with (b, d) or without (a, c) 10 ␮mol/liter NE for 24 h, and then either 10 nmol/liter activin A was included in the culture medium for 1 h (c, d) or the cells were left untreated (a, b). Smad2/3 immunofluorescence was examined. Phase contrast images of the same field are on the right side. Insets, percentage of cells with exclusively nuclear staining with Smad2/3. To score nuclear staining, 200 cells were examined in each experiment. B, Western blot analysis of activin A-induced Smad4 nuclear accumulation. Control cells or NE-treated cells were either left untreated or treated for 1 h with 10 nmol/liter activin A. Nuclear extracts were analyzed by Western blot analysis using anti-Smad4 antibody.

FIG. 3. RNase protection assay of Smad7 mRNA expression in response to NE treatment of primary culture of rat hepatocytes. Ten ␮g of total RNA was allowed to hybridize to the cRNA probe for rat Smad7, and 2 ␮g of total RNA for ␤-actin. Cells were treated with 10 ␮mol/liter of NE, and RNA was harvested at the indicated time. Results are representative of four independent experiments with similar results.

3-indolyl ␤-D-galactopyranoside (X-gal). At a multiplicity of infection of 10, we found that more than 95% of the AdLacZinfected rat hepatocytes expressed ␤-gal (data not shown). Northern blot analysis showed that while the AdLacZ-infected cells expressed an undetectable level of Smad7 mRNA, the AdSmad7-infected cells showed increased expression of Smad7 mRNA (Fig. 4A). To evaluate the effect of overexpression of Smad7 on activin A/Smad signaling in rat hepatocytes, we examined the level of activin A-induced Smad2/3 nuclear localization. Without activin treatment, both the AdLacZ-infected


FIG. 4. A, rat hepatocytes were infected with either AdSmad7 or AdLacZ adenovirus at a multiplicity of infection of 10 and were harvested after 24 h. Total RNA (20 ␮g/lane) from AdLacZ-infected or AdSmad7-infected cells was subjected to Northern blot analysis using a 32 P-labeled Smad7 or ␤-actin cDNA probe (500,000 cpm/ml). B, cells were infected with AdLacZ (a, c) or AdSmad7 (b, d). After 24 h, cells were treated with (c, d) 10 nmol/liter activin A for 1 h or left untreated (a, b). Smad2/3 immunofluorescence was examined. Phase contrast images of the same field are on the right side. Insets, percentage of cells with exclusively nuclear staining with Smad2/3. To score nuclear staining, 200 cells were examined in each experiment.

cells (Fig. 4B, a) and AdSmad7-infected cells (Fig. 4B, b) showed granular cytoplasmic staining for Smad2/3. In the AdLacZ-infected cells, activin A treatment led to an increase in Smad2/3 nuclear localization (Fig. 4B, c; 92% of the cells showed nuclear staining). In the AdSmad7-infected cells, the accumulation of nuclear Smad2/3 in response to activin A was markedly diminished (Fig. 4B, d; 22% of the cells showed nuclear staining). Thus, overexpression of Smad7 inhibited an initial event in the activin A/Smad signaling pathway. We then investigated the effect of Smad7 overexpression on the proliferation of hepatocytes. As shown in Fig. 5A, infection of AdSmad7 significantly enhanced EGF-stimulated DNA synthesis in rat hepatocytes compared with that in the AdLacZ-infected cells. Although the AdLacZ-infected cells exhibited potent growth inhibition in response to treatment with activin A, the growth inhibitory effect of activin A was nearly abolished in the AdSmad7-infected cells (Fig. 5B). Smad7 overexpression seems to be sufficient to mimic the NE-mediated enhancement of EGF-stimulated DNA synthesis and inhibition of activin A-induced growth inhibition of rat hepatocytes. NE Induced Smad7 Expression through NF-␬B Activation— Recently, TNF␣-induced Smad7 expression was shown to be mediated through NF-␬B transcription factor (33). In addition to cytokines such as TNF␣ and IFN␤, several agonists working through G protein-coupled receptors can activate NF-␬B (34 –

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FIG. 5. A, effect of overexpression of Smad7 or LacZ on DNA synthesis in primary culture of rat hepatocytes. Cells were incubated with or without 1 nmol/liter EGF and 0.1 ␮mol/liter insulin. [3H]Thymidine was included in the culture medium from 48 h to 72 h. Values are expressed as the mean ⫾ S.D. *p ⬍ 0.05 versus AdLacZ-infected cells. B, effect of Smad7 overexpression on activin A-induced growth inhibition of rat hepatocytes. AdSmad7- or AdLacZ-infected cells were cultured with 1 nmol/liter EGF and 0.1 ␮mol/liter insulin and various concentrations of activin A. Activin A was included in the culture medium from 24 h to 72 h, and [3H]thymidine was included from 48 h to 72 h. Values are expressed as the mean ⫾ S.D. Each result is representative of three independent experiments with similar results performed in triplicate.

42). We reasoned, therefore, that the induction of Smad7 by NE might depend on NF-␬B. We examined whether NE treatment increased NF-␬B binding activity, as assessed by EMSA. As shown in Fig. 6, the amount of active NF-␬B complex increased upon NE treatment for 30 min or 1 h in comparison with that before treatment. Competition with 100⫻ molar excess of cold NF-␬B probe confirmed the specificity. The NF-␬B complex contained p65, as demonstrated by the supershift with p65specific antibody. We then investigated the effect of the NF-␬B pathway inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) that prevents the degradation of I␬B, on the induction of Smad7 expression by NE. As shown in Fig. 7A, NF-␬B binding was nearly completely abolished in the presence of TPCK. We next performed RNase protection analysis to determine the effect of TPCK on the induction of Smad7 expression by NE. Rat hepatocytes were preincubated in the presence or absence of TPCK before the addition of NE. As shown in Fig. 7B, NE-induced Smad7 expression was inhibited in the presence of TPCK. We further evaluated the role of NF-␬B on NE-induced Smad7 expression using an adenovirus

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FIG. 6. Effect of NE on NF-␬B DNA binding activity in rat hepatocytes. Cells were treated with 10 ␮mol/liter NE and were harvested at the indicated times. Aliquots of 10 ␮g of nuclear protein from each sample were subjected to EMSA. To examine specific NF-␬B subunits, 2 ␮g of the specific antibody against p65 was added to the reaction before the addition of the labeled oligonucleotide. Data are representative of six independent experiments with similar results.

FIG. 8. Rat hepatocytes were infected with either Ad5I␬B or AdLacZ at a multiplicity of infection of 30 and were harvested after 5 h. A, expression of HA-I␬B␣ S32A/S36A was assessed by Western blotting using an anti-I␬B␣ antibody. B, effect of the I␬B super-repressor on NE-induced NF-␬B DNA binding in rat hepatocytes. Infected cells were further treated with 10 ␮mol/liter NE for 30 min. C, effect of the I␬B super-repressor on NE-induced Smad7 mRNA expression. Infected cells were further treated with 10 ␮mol/liter NE for 1 h. Ten ␮g of total RNA was allowed to hybridize to the cRNA probe for rat Smad7 and 2 ␮g of total RNA for ␤-actin, and the RNase protection assay was performed. Results are representative of three independent experiments with similar results.

FIG. 7. A, effect of NF-␬B inhibitor on NE-induced NE-␬B DNA binding in rat hepatocytes. Cells were preincubated with 100 ␮mol/liter TPCK for 30 min, followed by treatment with 10 ␮mol/liter NE for 30 min. B, effect of NF-␬B inhibitor on NE-induced Smad7 mRNA expression. Cells were preincubated with 100 ␮mol/liter TPCK for 30 min, followed by treatment with 10 ␮mol/liter NE for 3 h. Ten ␮g of total RNA was allowed to hybridize to the cRNA probe for rat Smad7 and 2 ␮g of total RNA for ␤-actin, and the RNase protection assay was performed. Results are representative of four independent experiments with similar results.

expressing an I␬B super-repressor that contained serine-toalanine mutations in residues 32 and 36 (43). Using an antiI␬B␣ antibody, the HA-tagged I␬B␣ was identified by its slower mobility compared with the endogenous I␬B by Western blotting (Fig. 8A). As shown in Fig. 8B, induction of NF-␬B DNA binding by NE was markedly abolished by prior infection with Ad5I␬B. As shown in Fig. 8C, NE-induced Smad7 expression was inhibited by prior infection with Ad5I␬B. These results strongly suggested that NE induced Smad7 expression through an NF-␬B-dependent pathway in rat hepatocytes. DISCUSSION

We report a mechanism of suppression of activin A signaling by NE in rat hepatocytes. Our results suggest that activation of

NF-␬B by NE induces inhibitory Smad7 expression. Overexpression of exogenous Smad7 abrogated activin-induced growth inhibition. Smad7 was originally identified as a shear stress-inducible gene in endothelial cells (44). Subsequently, it was found that its expression can be induced by TGF␤ or activin itself, and Smad7 is therefore postulated to be a mediator that participates in a negative feedback loop. Accordingly, ectopic expression of Smad7 inhibited the TGF-␤- and activininduced growth inhibition and apoptosis of various cell lines including hepatoma cells (30, 31). Recent reports have demonstrated that IFN-␥ (32), TNF-␣ (33, and CD40 stimulation (45) inhibit TGF-␤/Smad signaling through induction of Smad7 expression. Together with our finding presented here, control of Smad7 expression seems to provide a general mechanism as a physiological molecular switch to suppress TGF␤/activin activity. NE may play a particularly important role in hepatocyte proliferation. NE enhances the mitogenic effects of various growth stimulators of rat hepatocytes (3). In partially hepatectomized rats, the plasma NE level increases as early as 2 h after surgery and remains elevated for 2 days. Surgical hepatic denervation or injection of an ␣1 antagonist, prazosin, reduced the [3H]thymidine incorporation in the rat regenerating liver after partial hepatectomy (14). As shown in Fig. 4, overexpression of Smad7 enhanced EGF-stimulated DNA synthesis and nearly completely abolished activin A-induced growth inhibition of rat hepatocytes. Thus, our results indicate that the co-mitogenic effect of NE may be due, at least in part, to

Smad7 Is Induced by Norepinephrine suppression of the autocrine activin A signaling via induction of Smad7. Autocrine activin has been reported to modulate hepatocyte proliferation in vitro and in vivo. Addition of the activin-binding protein, follistatin, to the culture enhances EGF-stimulated DNA synthesis in rat hepatocytes (9), and administration of follistatin potentiates liver regeneration after partial hepatectomy (10). Moreover, injection of follistatin into the portal vein initiates DNA synthesis even in the quiescent rat liver (46). Thus, NE can act as an inhibitor of autocrine activin signaling in rat hepatocytes. NF-␬B is a dimeric, ubiquitously expressed transcription factor that plays a critical role in regulating inducible gene expression in immune and inflammatory responses (47). In most cells, NF-␬B proteins exist in the cytoplasm in an inactive complex bound to the I␬B family of inhibitory proteins. Various stimuli can induce rapid phosphorylation, ubiquitinylation, and degradation of I␬B, resulting in nuclear translocation of NF-␬B proteins and transcriptional activation. The critical role of NF-␬B in the induction of Smad7 is supported by previous results. Using RelA-defective fibroblasts, Bitzer et al. (33) demonstrated that the induction of Smad7 by TNF␣ requires NF␬B. The anti-apoptotic effect of CD40 is associated with the transcriptional activation of Smad7 in an NF-␬B-dependent manner in B cells (45). It has been shown that the 5⬘-flanking region of the Smad7 gene contains multiple potential NF-␬B binding sites (48, 49). An increasing number of agonists working through G protein-coupled receptors have been found to activate NF-␬B. These agonists include serotonin (working through 5-HT1A receptor) (34), platelet-activating factor (35), thrombin (36, 37), substance P (38), endothelin (39), bradykinin (40, 41), and lysophosphatidic acid (42). We demonstrated that NE stimulates NF-␬B activation in rat hepatocytes and that inhibition of NF-␬B pathway by a proteosome inhibitor or an I␬B super-repressor abolished the Smad7 expression induced by NE. Dajani et al. reported that protein kinase C inhibitor GF109203X attenuates co-mitogenic effect of NE on rat hepatocytes (50). There is a possibility that the Gq/protein kinase C pathway couples with the ␣1-adrenergic receptor to cause NF-␬B activation. Using type 1 TNF␣ receptor-deficient mice, Yamada et al. (51) reported that TNF-␣/NF-␬B pathway is required for initiation of liver regeneration after partial hepatectomy. In addition to co-mitogenic action, NE is possibly involved in initiation of liver regeneration through NF-␬B activation. Together with these findings, our results indicate a novel link between Smad and the trimeric G protein signaling pathway in rat hepatocytes: NE rapidly increases the expression of an anti-Smad, Smad7, through NF-␬B activation, causing the inhibition of activin-mediated growth inhibition. Our results also suggest the possible involvement of NE in modulating hepatocyte proliferation through NF-␬B activation and Smad7 induction. Acknowledgments—We thank Dr. Y. Eto (Ajinomoto, Inc.) for the recombinant human activin A, Dr. Kato (Fukushima Medical College) for the rat Smad6 cDNA, Dr. Miyazono (Japanese Cancer Foundation) for the AdSmad7 adenovirus, Dr. P. ten Dijke (Ludwig Institute for Cancer Research) for the mouse Smad7 cDNA, and Drs. Y. Iimuro (Hyogo Medical Collage) and D. A. Brenner (Univ. of North Carolina at Chapel Hill) for the Ad5I␬B adenovirus. REFERENCES 1. 2. 3. 4.

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