c-Myb modulates transcription of the alpha -smooth muscle actin gene in activated hepatic stellate cells

Martina Buck, Dong Joon Kim, Karl Houglum, Tarek Hassanein, and Mario Chojkier

Department of Medicine, Veterans Affairs Medical Center, San Diego 92161; and Center for Molecular Genetics, University of California, San Diego, California 92037


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Expression of alpha -smooth muscle actin (alpha -SMA) defines the phenotype of activated (myofibroblastic) hepatic stellate cells. These cells, but not quiescent stellate cells, have a high level of alpha -SMA and c-Myb expression, as well as increased c-Myb-binding activities to the proximal alpha -SMA E box. Therefore, we analyzed the role of c-Myb in alpha -SMA transcription and stellate cell activation. Activated primary rat stellate cells displayed a high expression of the -724 and -271 alpha -SMA/luciferase (LUC) chimeric genes, which contain c-Myb binding sites (-223/-216 bp). alpha -SMA/LUC minigenes with mutation (-219/-217 bp), truncation (-224 bp), or deletion (-191 bp) of the c-Myb binding site were not efficiently transcribed. Transfection of wild-type c-Myb into quiescent stellate cells, which do not express endogenous c-Myb, induced a ~10-fold stimulation of -724 alpha -SMA/LUC expression. Conversely, expression of either a dominant-negative c-Myb basic domain mutant (Cys43 right-arrow Asp) or a c-Myb antisense RNA blocked transcription from the -724 alpha -SMA/LUC or -271 alpha -SMA/LUC in activated cells. Moreover, transfection of c-myb antisense, but not sense, RNA inhibited both expression of the endogenous alpha -SMA gene and stellate cell activation, whereas transfection of c-myb stimulated alpha -SMA expression in quiescent stellate cells. These findings suggest that c-Myb modulates the activation of stellate cells and that integrity of the redox sensor Cys43 in c-Myb is required for this effect.

liver fibrosis; oxidative stress


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

STELLATE CELLS PLAY a key role in the pathogenesis of hepatic fibrosis (14, 36). Although we (5) and others (13, 15) have reported that quiescent stellate cells produce little collagen type I, activated (myofibroblastic) stellate cells display a high level of collagen alpha 1(I) gene expression (13, 14, 36). Therefore, stellate cell activation is a critical step in hepatic fibrogenesis. Studies with primary cultures of adult rat stellate cells have provided evidence that cell type-specific growth regulatory mechanisms exist (41), but the cell-specific factors regulating stellate cell activation have only been partially identified (9). We found that oxidative stress is a common and indispensable step in the cascade of molecular events initiated by collagen type I matrix or transforming growth factor-alpha (TGF-alpha ), resulting in stellate cell activation (31).

The expression of alpha -smooth muscle actin (alpha -SMA) defines the activated phenotype of stellate cells (43). In this context, we have previously reported that c-Myb expression and/or binding activities to an oligonucleotide including the proximal E box of the alpha -SMA gene are increased in association with enhanced oxidative stress in activated stellate cells in culture (31), in animals treated with CCl4 (31, 32), and in patients with chronic hepatitis C (26). These findings strongly suggest that c-Myb is a molecular mediator of oxidative stress on stellate cell activation (26, 31, 32).

Although little is known about the mechanisms that modulate c-Myb activity, it has been suggested that oxidation of Cys43 could function as a molecular sensor for the redox state of the cell by affecting the DNA-binding affinity of c-Myb (38). In agreement with this hypothesis, we found that addition of purified redox protein Ref-1 (52, 53) to nuclear extracts from activated stellate cells inhibits their binding (presumably c-Myb) to the alpha -SMA-proximal E box (32). In this study, we show that c-Myb plays a major role in the transcription of the alpha -SMA gene in activated stellate cells. Moreover, c-myb antisense RNA blocked the development of the myofibroblastic phenotype and expression of the endogenous alpha -SMA gene induced in stellate cells by collagen type I matrix, whereas transfection of c-myb stimulated alpha -SMA expression in quiescent stellate cells.


    METHODS
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INTRODUCTION
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Cell cultures. Stellate cells were prepared from male Sprague-Dawley rats (400-500 g) by in situ perfusion and single-step density Nycodenz gradient (Accurate Chemical & Scientific, Westbury, NY), as described previously (5, 11, 22, 23, 25). Cells were plated on collagen type I or EHS matrix (Matrigel; Collaborative Biomedical Products, Bedford, MA) tissue culture dishes, according to the experimental design, with the initial seeding of fat-storing cells at a density of 2 × 105/cm2. Matrigel's major components are laminin, collagen IV, proteoglycans, entactin, and nidogen. It also contains TGF-beta , fibroblast growth factor, and tissue plasminogen activator. Cells were cultured under an atmosphere of 5% CO2/95% air in tissue culture dishes using DMEM containing 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 10% FBS (GIBCO BRL, Gaithersburg, MD) and 10% FCS (Omega, Tarzana, CA). Medium was changed every 48 h for all conditions. Stellate cells were identified by their typical autofluorescence at 328-nm excitation wavelength, staining of lipid droplets by Oil Red, and immunohistochemistry with a monoclonal antibody against desmin (2). Greater than 95% of the cells were stellate cells. Freshly isolated stellate cells were transfected with the mammalian vectors expressing the protein or reporter of interest using lipofectin (GIBCO BRL), as described by the manufacturer. To increase the transfectability of activated cells, a transfection-enhancing reagent (Life Technologies, Gaithersburg, MD) was added in conjunction with lipofectamine as recommended by the manufacturer. The efficiency of transfection was determined using the pRSV-beta -galactosidase vector (7).

The rat alpha -SMA/luciferase (LUC) chimeric reporter genes contained -724/+46 bp (-724 alpha -SMA/LUC), -271/+46 bp (-271 alpha -SMA/LUC), -271/+46 bp [-232/-231 mut] (-271 [-232/-231 mut] alpha -SMA/LUC), -271/+46 bp [-219/-217 mut] (-271 [-219/-217 mut] alpha -SMA/LUC), -230/+46 bp (-230 alpha -SMA/LUC), -271/-230/+46 bp (-271/-230 alpha -SMA/LUC), -224/+46 bp (-224 alpha -SMA/LUC), or -191/+46 bp (-191 alpha -SMA/LUC) from the 5' region of the rat alpha -SMA gene (4) inserted into pGL3-Basic (Promega, Madison, WI), as described previously (23). The wild-type c-Myb and c-Myb Asp43 mutant clones (38) were inserted into the pcDNA3.1 mammalian expression vector (Invitrogen, San Diego, CA). The c-Myb antisense was derived by reversing the wild-type c-Myb insert. The total amount of transfected DNA was 2 µg. The transfection efficiency was 23 ± 5% for day 0 quiescent cells and 28 ± 8% for activated cells growing on a collagen type I matrix as described previously (11, 23, 25). Cells were either harvested for LUC assays at 24-48 h (23) or fixed at 72-96 h after transfection according to the experimental design.

Immunohistochemistry. Cells fixed with acetone and methanol (60:40) at -20°C for 20 min were immunostained as described previously (6, 7, 23). Monoclonal alpha -SMA and anti-rabbit beta -galactosidase antibodies were obtained from Sigma (St. Louis, MO) and Cappel (Durham, NC). Oregon green and Texas red secondary fluorochromes were obtained from Molecular Probes (Eugene, OR). Fluorescent labels were visualized using a triple-channel Nikon microscope as described previously (6, 7, 23, 25). The number of SMA-positive cells was expressed as a percentage of total transfected cells. At least 100 transfected cells were analyzed per experimental point, and a minimum of two observers analyzed each immunohistochemical experiment independently, as described previously (7, 25). Negative control samples were processed in parallel under the same conditions but with omission of the first antibody. Hoechst 33342 was used as a nuclear counterstain.

Statistical analysis. All results are expressed as means ± SE. Student's t-test was used to evaluate differences of the means between groups, with P < 0.05 considered significant.


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To assess the role of c-Myb on the transcription from the alpha -SMA gene, we first characterized the cis-regulatory region within the alpha -SMA gene 5' flanking sequences that is necessary for high expression of the alpha -SMA in activated stellate cells. Primary rat stellate cells cultured on a collagen type I matrix became activated (23, 25, 31) and were transfected on day 4 with alpha -SMA chimeric reporter minigenes containing -724 bp, -271 bp, -230 bp, -271/-230 bp, -224 bp, or -191 bp of the rat alpha -SMA promoter (4) and expressing LUC (Fig. 1A). To obtain optimal reporter expression, cells were harvested 48 h after transfection. In these activated stellate cells, transcription from alpha -SMA/LUC reporter genes was very high when the chimeric gene contained -724 bp or -271 bp of the 5' flanking region of the alpha -SMA promoter [including both E boxes and the TGTTTATC motif (distal to -224 bp); Fig. 1B]. Truncation of the region containing the distal alpha -SMA E box to -224 bp (including the proximal E box) or -191 bp (including the CArG A and B boxes) eliminated the transcription inducibility of the alpha -SMA gene (Fig. 1B), which is characteristic of the early phases of stellate cell activation (9, 43). The core binding for c-Myb, CATAAGCA (-223/-216), which is distal to the proximal E box, is disrupted with the truncation at -224 bp. However, conservation of the c-Myb (and other cooperative transcription factors) cognate DNA with the truncation at -230 bp (only an additional 6 bp) leads to a much higher alpha -SMA/LUC expression (Fig. 1B). Moreover, mutation of the c-Myb binding site (A-219 right-arrow T; C-217 right-arrow T) (Fig. 1A) markedly inhibited transcription from the -271 alpha -SMA/LUC reporter gene (Fig. 1B). Furthermore, a -271 bp/-230 bp cis element (271/230 alpha -SMA/LUC), containing the distal E box but neither the c-Myb-binding region nor the proximal E box, had only background LUC expression. In addition to a role for the c-Myb cis element in the transcription from the alpha -SMA promoter, the TGTTTATC motif (-233 to -226), immediately distal to the c-Myb binding site, also contributes to the alpha -SMA transcription in activated stellate cells. Mutation of this motif (T<UNL>AC</UNL>TTATC) (-271 bp [232/231 mut] alpha -SMA/LUC) markedly decreases the expression of alpha -SMA/LUC (Fig. 1B).


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Fig. 1.   Expression of alpha -smooth muscle actin (SMA)/luciferase (LUC) minigenes in activated stellate cells. Primary rat hepatic stellate cells were isolated and cultured on collagen type I matrix for 6 days as described in METHODS. A: 5' flanking regions of alpha -SMA/LUC minigenes are shown as open rectangles. E boxes are depicted as closed rectangles. Arrow denotes start of transcription. LUC reporter gene sequences are indicated in gray. B: vectors depicted in A were transfected on day 4 as described in METHODS (11, 23, 25). Luciferase assay was performed as described by kit manufacturer and corrected for cellular protein content (11, 23). Results are means ± SE of quadruplicate samples and representative of 3 independent experiments. The -724 bp alpha -SMA/LUC and -271 bp alpha -SMA were highly expressed compared with control (pcDNA), -271/-230 bp alpha -SMA/LUC, -271 [219/217 mut] alpha -SMA/LUC, -224 bp alpha -SMA/LUC, and -191 bp alpha -SMA/LUC. P < 0.01 for 724 alpha -SMA/LUC and 271 alpha -SMA/LUC; P < 0.05 for 271 [232/231 mut] alpha -SMA/LUC and 230 alpha -SMA/LUC.

Because c-Myb expression is induced during the early stages of stellate cell activation and c-Myb binds with high affinity to an oligonucleotide including the proximal E box (GCAGCT -218 to -213 bp) of the alpha -SMA promoter (5'-GAT<OVL>CATAAGCA</OVL>GCTGAACTGCC-3') (32), we investigated whether c-Myb is capable of stimulating transcription from the alpha -SMA promoter in quiescent stellate cells. Day 4 primary rat stellate cells, growing on an EHS matrix to prevent their spontaneous activation and the expression of the endogenous c-Myb (11, 25, 31), were transfected with a vector expressing wild-type c-Myb. Nuclear expression of c-Myb as determined by immunofluorescence (31) (data not shown) was sufficient to increase the basal transcription from the cotransfected -724 alpha -SMA/LUC reporter gene by ~10-fold (216 ± 52 vs. 2,376 ± 850 U/mg protein; P < 0.05) (Fig. 2). In addition, a c-Myb basic domain mutant (Cys43 right-arrow Asp) that binds cognate DNA with reduced affinity (38) behaved as a dominant negative when expressed in activated stellate cells (growing on a collagen type I matrix), markedly decreasing the -724 alpha -SMA/LUC reporter expression (2,670 ± 29 vs. 320 ± 130 U/mg protein; P < 0.05) (Fig. 3A).


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Fig. 2.   c-Myb stimulates expression of alpha -SMA/LUC minigene in quiescent stellate cells. Primary rat hepatic stellate cells were freshly isolated and cultured on an EHS matrix for 6 days as described in METHODS (11, 23, 25, 31). On day 4, cells were transfected with -724 alpha -SMA/LUC and vectors expressing pcDNA (control) or c-Myb as described previously (11, 23, 25). Values are means ± SE for quadruplicate samples and representative of 4 independent experiments. P < 0.05 for c-Myb.



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Fig. 3.   c-Myb is critical for expression of alpha -SMA/LUC minigene in activated stellate cells. Primary rat hepatic stellate cells were isolated and cultured on collagen type I matrix for 6 days as described in METHODS (25, 31). A: on day 4, cells were transfected with vectors expressing -724 alpha -SMA/LUC and/or pcDNA (control), c-Myb mutant (Cys43 right-arrow Asp), or c-Myb antisense (AS). Results are means ± SE of triplicate samples and representative of 3 independent experiments. P < 0.05 for c-Myb mutant and c-Myb AS. B: day 4 cells were transfected with vectors expressing -271 alpha -SMA/LUC -271 [-219/-217 mut] alpha -SMA/LUC, and/or pcDNA (control) or c-myb. Values are means ± SE for triplicate samples. P < 0.01 for 271 alpha -SMA/LUC plus c-myb and P < 0.05 for 271 alpha -SMA/LUC.

These results suggest that c-Myb is both sufficient and necessary to stimulate, perhaps in concert with other transcription activators (21, 28, 31), a high level of transcription from the alpha -SMA promoter in activated stellate cells. We have previously reported that c-myb antisense oligonucleotides inhibited the activation of stellate cells induced by TGF-alpha in a conditioned medium (31). However, Burgess et al. (8) suggested that the antiproliferative activity of antisense c-myb-specific oligonucleotides, at least in smooth muscle cells (SMC), is not due to a hybridization-dependent antisense mechanism. Therefore, to circumvent this confounding issue (8), we transfected day 4 activated primary rat stellate cells, growing on a collagen type I matrix, with a vector expressing antisense c-myb RNA and assessed the expression of the -724 alpha -SMA/LUC reporter. As we reported previously for c-myb antisense oligonucleotides (31), c-myb antisense RNA also blocked c-Myb expression. In agreement with the results obtained by expressing a dominant-negative c-myb mutant (Fig. 3A), expression of the antisense, but not sense, c-myb RNA inhibited by approximately fivefold the -724 alpha -SMA/LUC reporter activity (Fig. 3A) and by approximately threefold expression from the -271 alpha -SMA/LUC (data not shown). These experiments strongly support the notion that c-Myb is required for optimal expression of the alpha -SMA gene. Furthermore, these effects were selective for the -724 bp and -271 bp 5' flanking sequences, since c-myb antisense RNA did not modify the (already modest) expression of the -224 alpha -SMA/LUC reporter (data not shown). As expected, overexpression of c-myb in activated stellate cells, stimulated (approximately threefold) transcription from the -271 alpha -SMA/LUC minigene but not from the same construct with point mutations of the c-Myb binding site within the proximal E box (-271 [-219/-217 mut] alpha -SMA/LUC) (Fig. 3B).

In addition, we assessed whether expression of the c-myb antisense RNA could also inhibit expression of the endogenous alpha -SMA gene in stellate cells and, therefore, their activation (9, 13, 43). Day 0 stellate cells growing on a collagen type I matrix were transfected with vectors expressing beta -galactosidase with either pcDNA (control) or c-myb antisense RNA. On day 4, cells were fixed and analyzed in a triple-channel microscope by immunohistochemistry with specific antibodies against beta -galactosidase and alpha -SMA, as described previously (23, 31, 32). Nuclei were stained in blue with Hoechst 33342. As expected, control stellate cells transfected with pcDNA (together with the transfection indicator beta -galactosidase; in red) became activated, judged by their myofibroblastic phenotype and their expression of alpha -SMA (in green) (Fig. 4A, beta -galactosidase). The transfected beta -galactosidase is shown in yellow because of the superimposition of the green alpha -SMA over the red beta -galactosidase. Approximately 70% of the control cells transfected with pcDNA adopted an activated phenotype and expressed alpha -SMA (Fig. 4B). In contrast, the majority of cells expressing the c-myb antisense RNA (together with the cotransfected beta -galactosidase) had the phenotype of quiescent stellate cells (nonmyofibroblastic; in red) (Fig. 4A). More importantly, expression of c-myb antisense RNA was sufficient to block the induction of the endogenous alpha -SMA gene, which is expected in day 4 primary stellate cells growing on a collagen type I matrix (13, 22, 25, 31, 32). Only <10% of the cells transfected with c-myb antisense RNA expressed alpha -SMA and had the activated phenotype (Fig. 4B). Omission of the first antibody resulted in negative immunofluorescence for both alpha -SMA- and beta -galactosidase-positive samples, as described previously (6, 7, 23, 25, 31).


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Fig. 4.   c-myb AS inhibits activation of stellate cells. Rat hepatic stellate cells were freshly isolated and cultured on a collagen type I matrix as described previously (22, 31). A: on day 0, cells were cotransfected as described in METHODS, with vectors (1 µg of each) expressing either pcDNA and beta -galactosidase (control) or c-myb antisense and beta -galactosidase. Day 4 cells were fixed with acetone and methanol (60:40), and immunofluorescence for alpha -SMA and beta -galactosidase was performed using specific primary antibodies as described in METHODS (23, 25, 31). Hoechst 33342 was used as a nuclear counterstain (in blue). Endogenous alpha -SMA expression (in green) and myofibroblastic phenotype of activated cells were observed in majority of control (beta -galactosidase) cells. Transfected perinuclear beta -galactosidase is shown in yellow (because of superimposition of green alpha -SMA and red beta -galactosidase). Cells transfected with c-myb AS remained nonmyofibroblastic and expressed cotransfected beta -galactosidase (in red) but not endogenous alpha -SMA. B: number of cells expressing endogenous alpha -SMA among transfected cells (as indicated by coexpression of beta -galactosidase) was determined for control (pcDNA) and c-myb AS as described previously (31). Values are means ± SE of triplicate samples. P < 0.05 for c-myb AS.

In addition, day 1 quiescent stellate cells growing on EHS were transfected with the indicator green fluorescent protein (in green) and either control beta -galactosidase or c-myb. On day 4, cells were stained for alpha -SMA (in red). Although only ~2% of control cells expressed alpha -SMA (Fig. 5), ~40% of cells transfected with c-myb were alpha -SMA-positive (Fig. 5). However, unlike activated stellate cells growing on collagen type I (Fig. 4B), c-myb-transfected cells growing on EHS displayed a diffuse rather than a fibrillar alpha -SMA phenotype.


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Fig. 5.   c-myb Stimulates alpha -SMA expression in stellate cells. Stellate cells were freshly isolated and cultured in an EHS matrix (22, 31). A: on day 1, cells were cotransfected as described in METHODS with vectors (1 µg of each) expressing green fluorescent protein (GFP) and either beta -galactosidase (control) or c-myb. Day 4 cells were fixed, and immunofluorescence for alpha -SMA was performed as in Fig. 4. Hoechst 33342 was used as nuclear counterstain (in blue). Endogenous alpha -SMA expression is shown in red, whereas transfection indicator GFP is shown in green. B: number of cells expressing endogenous alpha -SMA among transfected cells (as indicated by coexpression of GFP) was determined for control (beta -galactosidase) and c-myb as described (31). Values are means ± SE of triplicate samples. P < 0.05 for c-myb.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Expression of alpha -SMA and a myofibroblastic phenotype defines stellate cell activation (43). In this study, we have characterized some of the molecular mechanisms involved in the activation of stellate cells, an important step in hepatic fibrogenesis (13, 14).

We have previously reported that quiescent primary rat stellate cells, cultured on an EHS matrix, are activated by the generation of free radicals using ascorbic acid/FeSO4 as well as by malondialdehyde, a product of lipid peroxidation (31). In addition, enhanced hepatic oxidative stress in animals treated with CCl4 (31, 32) or in patients with chronic hepatitis C (26) was associated with stellate cell activation. Complementary results supporting the role of oxidative stress in stellate cell activation include the finding that stellate cell activation induced by collagen type I matrix or TGF-alpha can be blocked by antioxidants, such as D-alpha -tocopherol or butylated hydroxytoluene (31). Furthermore, a pilot study suggests that D-alpha -tocopherol can prevent stellate cell activation in patients with chronic hepatitis C (26).

Several studies indicate that c-Myb plays an important role in cell differentiation and proliferation (34, 35). For example, regulation of c-myb expression is critical for the growth and differentiation of the progeny of hematopoietic cells (1, 17, 50). c-Myb protein binds to a consensus cognate DNA (16) through three homeo domain-like regions (44) and activates the transcription of target genes (3, 29, 51). The molecular mechanism responsible for stellate cell alpha -SMA expression and activation in primary cultures growing on collagen type I (25, 31) in animals treated with CCl4 (22, 31) and in patients with chronic hepatitis C (26) seems to be associated with increased c-myb expression (26, 31, 32) and binding of nuclear proteins to the proximal alpha -SMA E box (31, 32).

The appropriate expression of the alpha -SMA gene requires the interaction of cell type-specific sequences within the promoter and transcriptional factors (18-20, 37, 45, 49). Although in SMC -125 bp of the 5' flanking region are sufficient to confer high expression of the alpha -SMA gene, at least -271 bp are required in skeletal myotubes (45), which also express alpha -SMA. Critical cis-acting elements within the -125 bp of the alpha -SMA gene in SMC include the CArG boxes, which bind serum-response factors (45) and are necessary for ANG II inducibility (18). A mesenchymal transcription factor, MHox, mediates the ANG II stimulation of alpha -SMA expression (20). In addition, stimulation of alpha -SMA gene expression by TGF-beta in SMC requires the interaction of the CArG A (-62 bp) and B (-112 bp) boxes, together with the TGF-beta control element (-42 bp) (19).

Here we demonstrated that, unlike in SMC, high-level transcription of alpha -SMA/LUC chimeric genes in activated stellate cells requires more than -224 bp within its 5' flanking region. This -271 bp 5' region includes the E boxes, the TGTTTATC motif, and a c-Myb binding site (37, 45). In contrast, the presence of -224 bp (including the proximal E box as well as the CArG boxes) is not sufficient for efficient transcription from the alpha -SMA promoter in activated stellate cells. Studies including truncations, deletions, and mutations suggest which are the relevant cis elements within the -724 bp of the 5' region of the alpha -SMA gene in activated stellate cells. Most likely, the -224 bp truncation disrupts the c-Myb binding site (-223/-216) because addition of only 6 bp (-230 alpha -SMA/LUC) restores, to a substantial extent, transcription from the alpha -SMA promoter. Indeed, mutation of the c-Myb binding site (-219/-217 bp mut), within the proximal E box, markedly inhibits expression from the -271 alpha -SMA/LUC reporter gene in activated stellate cells. We found that the TGTTTATC motif also plays a role in alpha -SMA transcription in activated primary rat stellate cells because a mutation of this site (T<OVL>AC</OVL>TTATC) impairs expression of the alpha -SMA/LUC. Constructs displaying the distal E box and adjacent cis elements (-271/-230 alpha -SMA/LUC) had only a background level of expression.

Transfection of a vector expressing wild-type c-Myb in quiescent stellate cells, which express negligible quantities of nuclear c-Myb (31), was sufficient to induce a ~10-fold stimulation of the -724 alpha -SMA/LUC reporter gene. As expected, transfection of c-myb stimulated expression of the -271 alpha -SMA/LUC but not of the -271 [-219/-217 mut] alpha -SMA/LUC. Conversely, expression of a c-myb antisense RNA in activated stellate cells prevented, to a substantial extent, transcription from the -724 alpha -SMA/LUC and -271 alpha -SMA/LUC chimeric genes. These studies indicate that c-Myb plays an important role in the transcription of the alpha -SMA gene in stellate cells. In chicken, alpha -SMA gene expression also involves a conserved sequence motif at -225 to -233 bp (TGTTTATC) (37), which is included in the -724 alpha -SMA/LUC and -271 alpha -SMA/LUC constructs that are fully expressed in activated stellate cells. Therefore, it is conceivable that the c-Myb binding site (-223/-216) interacts with the adjacent TGTTTATC motif through the formation of a transcriptional unit involving c-Myb. Indeed c-Myb is able to cooperate with other transcription factors, including CCAAT/enhancer binding protein-beta (28), which transactivates the collagen alpha 1(I) enhancer (21).

As reported for SMC and myotubes (45), alpha -SMA-expressing cells, a construct containing more than -547 bp of the alpha -SMA promoter was also transcriptionally active in myofibroblastic stellate cells growing on collagen type I matrix. However, the -724 alpha -SMA/LUC construct was inactive in quiescent stellate cells growing on an EHS matrix. It remains to be determined whether repression of the alpha -SMA gene occurs in quiescent stellate cells through the MCAT motif as reported for other cell types (49).

Although little is known about the mechanisms that modulate c-myb expression, it has been suggested that oxidation of Cys43 could function as a molecular sensor for the redox state of the cell by affecting the DNA-binding affinity of c-Myb (38). The modulation of AP-1 proteins involving oxidative stress is mediated by the nuclear redox factor Ref-1 (52), which also functions as a DNA repair enzyme (53). Ref-1 stimulates DNA-binding activity of several transcription factors, including c-Myb, and may itself be under a posttranslational control that is sensitive to the redox state of the cell (53). The redox activity of Ref-1 is mediated through a conserved Cys amino acid motif (KCR) that is present in Fos, c-Jun, and related proteins. In c-Myb, redox changes probably affect the motif KQCR (which includes Cys43) within the DNA binding domain. In agreement with this novel hypothesis (38, 53), we reported that oxidative stress affects the DNA-binding activity and expression of c-Myb (31). Although the molecular mechanisms remain to be elucidated, the increased expression of c-Myb could be achieved, for instance, by positive autoregulation of c-myb (39) through the redox modulation of c-Myb protein (53). We found that both the reducing agent dithiothreitol and the redox enzyme Ref-1 prevent the binding of c-Myb in nuclear extracts of activated stellate cells to the alpha -SMA-proximal E box (31, 32), suggesting a redox mechanism that may involve c-Myb, as proposed in cell-free systems (38, 52, 53).

Because of the previous suggestion that oxidative stress may modulate alpha -SMA-proximal E box-binding activities in activated stellate cells through c-Myb (32), we assessed whether the c-Myb basic domain (including Cys43) is a molecular target necessary for activation. Day 0 stellate cells were cotransfected with a c-Myb Asp43 mutant and the alpha -SMA/LUC reporter gene. Expression of c-Myb Asp43, lacking the redox sensor Cys43 (38), did markedly reduce transcription from the -724 alpha -SMA/LUC in activated stellate cells. These results suggest that modification of Cys43 within the DNA-binding domain of c-Myb is critical in stimulating alpha -SMA gene expression from its promoter. The signal-transduction pathway targeting c-Myb Cys43 is likely to involve either an oxidative modification, such as an aldehyde adduct (6, 10, 24, 42), or a nitrosylation of Cys43 (12, 48), because the Cys43 mutation would be refractory to either pathway. In this context, oxidative stress pathways are known to stimulate, at least in skeletal muscle (6), liver, and brain (M. Buck, unpublished observations), nitric oxide synthase expression and activity resulting in the synthesis of NO, which interacts with superoxide to generate peroxynitrite (33, 48), a compound highly reactive with sulfur-containing amino acids, such as Cys (12). However, NO, apparently in the absence of oxidative stress, downregulates, at least in rat lung fibroblasts, alpha -SMA expression (54).

What is the physiological relevance of c-Myb activity in the expression of the endogenous alpha -SMA gene during stellate cell activation? We have previously suggested that c-Myb plays a critical role in the expression of alpha -SMA, on the basis of experiments in which c-myb antisense phosphorothioate oligonucleotides prevented alpha -SMA expression and the activation of stellate cells induced by TGF-alpha (31). However, this argument may not be valid because the antiproliferative activity of c-myb-specific oligonucleotides, at least in SMC, is not due to hybridization-dependent antisense mechanisms (8). Rather, a stretch of four contiguous guanosine residues, which is present in the antisense c-myb used by us (31) and others (46, 47), may be responsible for the sequence-specific but nonantisense antiproliferative effects of these oligonucleotides.

Because proliferation and activation of stellate cells are usually linked (23, 25, 31, 32) and c-Myb stimulates both the proliferation and activation of these cells (31), we studied the effects of c-myb antisense RNA on stellate cells. Our results indicate that expression of c-myb antisense RNA markedly inhibits stellate cell activation (and presumably proliferation), whereas transfection of c-myb into quiescent stellate cells stimulates alpha -SMA expression. It is conceivable that the antagonistic effects of phosphorylated Ser133 CREB and oxidatively modified c-Myb Cys43 on the stellate cell cycle (25, 31) are mediated through cyclin/cdk repression/derepression. For example, CREB-binding protein (27) is known to bind preferentially to the oncoprotein c-Myb or to CREB, depending on the cell cycle state of the cell (30, 40).

In summary, this study, in conjunction with our previous results (26, 31, 32), strongly suggests that c-Myb is a key transcription activator of the alpha -SMA gene in activated stellate cells by interacting with the proximal E box (and possibly other distal cis elements) and behaving as a redox sensor through its DNA-binding domain. These findings may facilitate development of therapeutic approaches to prevent stellate cell activation in chronic liver diseases.


    ACKNOWLEDGEMENTS

We are indebted to Drs. G. K. Owens and C. A. McNamara for providing the genomic rat alpha -SMA DNA, -271 alpha -SMA, and -271 [232/231 mut] alpha -SMA and to Dr. O. S. Gabrielsen for the c-Myb and c-Myb Asp43 mutant constructs. We thank Tao Li for technical assistance and Amy King for the preparation of this manuscript.


    FOOTNOTES

This study was supported by the National Institutes of Health Grants DK-38652, DK-46971, and GM-47165 and by grants from the Department of Veterans Affairs. D. J. Kim was supported by a grant from Il Song Foundation (South Korea), and M. Buck was supported by fellowships from the National Cancer Institute and the American Liver Foundation.

M. Buck's present address is The Salk Institute for Biological Studies, Molecular Biology and Virology Lab, P. O. Box 85800, San Diego, CA 92037.

D. J. Kim's present address is Department of Internal Medicine, Chunchon Sacred Heart Hospital, College of Medicine, Hallym University, 153 Kyo-Dong, Chunchon-Si, Kangwon-Do, 200-060 Korea.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Chojkier, Dept. of Medicine and Center for Molecular Genetics, Univ. of California, San Diego 9-111D, San Diego, CA 92161.

Received 3 March 1999; accepted in final form 3 November 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 278(2):G321-G328
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