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Endocrinology, doi:10.1210/en.2003-1663
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Endocrinology Vol. 145, No. 6 2753-2759
Copyright © 2004 by The Endocrine Society

The Dependence of Transforming Growth Factor-ß-Induced Collagen Production on Autocrine Factor Activin A in Hepatic Stellate Cells

Wataru Wada, Hiroyuki Kuwano, Yoshihisa Hasegawa and Itaru Kojima

Institute for Molecular and Cellular Regulation (W.W., I.K.), Gunma University, Maebashi 371-8512; Department of General Surgical Science (W.W., H.K.), Gunma University Graduate School of Medicine; and School of Veterinary Medicine and Animal Science (Y.H.) Kitasato University, Towada 034-8628, Japan

Address all correspondence and requests for reprints to: Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan. E-mail: ikojima{at}showa.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was conducted to examine the role of activin A in the activation of cultured rat hepatic stellate cells (HSC). HSC expressed mRNA for the ßA-subunit of activin and the type I and II activin receptors. TGF-ß increased the mRNA expression of the ßA-subunit of activin as well as the release of the ßA dimer, activin A. Exogenous activin A activated HSC and increased the expression of {alpha}-smooth muscle actin and collagen. Exogenous follistatin, an antagonist of activin A, blocked not only the effect of activin A but also the effect of TGF-ß on the expression of type I collagen. Similarly, follistatin inhibited TGF-ß-induced secretion of collagen from HSC. Additionally, the effect of TGF-ß was markedly reduced in HSC overexpressing the dominant-negative type II activin receptor. In contrast, the effect of activin A on the collagen production was not affected in HSC overexpressing the dominant-negative type II TGF-ß receptor. In conclusion, an autocrine factor activin A mediates part of the action of TGF-ß on the production of collagen in HSC. The results also suggest that follistatin may be useful for the treatment of hepatic fibrosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AMONG MEMBERS OF the TGF-ß superfamily, activin A plays a critical role in organogenesis and modulates growth and differentiation of various types of cells (1, 2). Activin A also acts as a factor regulating tissue repair and modulates inflammation, tissue regeneration, and differentiation (3). In the liver, activin A is predominantly synthesized in hepatocytes and functions as an autocrine factor to inhibit proliferation (4). It also induces apoptosis of hepatocytes when administered in intact animal (5). In normal liver, the expression of activin A is relatively low but is up-regulated after partial hepatectomy (4), which may slow down the regeneration speed (6). It also participates in the maintenance of the constant liver mass because blocking the activin A action in intact liver, by either infusion of the activin antagonist follistatin (7) or transfection of the dominant-negative activin receptor gene (8), results in hepatocyte replication.

In addition to modulation of the hepatocyte functions, activin A also modifies the function of nonparenchymal cells and is involved in the pathogenesis of hepatic fibrosis. Thus, the expression of activin A is up-regulated in hepatic fibrosis and cirrhosis (9, 10). In vitro studies indicate that activin A activates hepatic stellate cells (HSC), which play a central role in the pathobiology of hepatic fibrosis (10, 11). This factor also stimulates the expression of extracellular matrix proteins, collagen, and fibronectin (11). It is well known that TGF-ß plays a crucial role in the pathophysiology of hepatic fibrosis (12, 13, 14). It stimulates phenotypic conversion of HSC to myofibroblasts and strongly induces the expression of matrix proteins. Thus, the effects of activin A resemble in many respects those of TGF-ß. The following questions thus arise: 1) Which is more potent, TGF-ß or activin A, in activating HSC? 2) Is there any difference between the actions of TGF-ß and activin A on HSC? and finally 3) What is the interaction between the actions of two factors? Regarding potency, it is generally accepted that TGF-ß is more potent than activin A in activating HSC at least in a culture system. Regarding the difference in their actions, Date et al. (11) showed that activin A induces the expression of fibronectin as potently as TGF-ß does, whereas the effect of activin A on collagen production is less than that of TGF-ß. Their results show that the actions of TGF-ß and activin are similar but not identical. With regard to the interaction of these two factors, Sugiyama et al. (10) showed that activin A and TGF-ß synergistically stimulate collagen production in HSC. Consequently, it is conceivable that both of these two factors are involved in the pathogenesis of hepatic fibrosis with TGF-ß being more important in promoting hepatic fibrosis. In view of the fact that activin A is also produced in HSC (9, 10), it is possible that activin A produced in HSC further modulates the activation process of HSC as an autocrine factor.

To further assess the role of activin A in the pathogenesis of hepatic fibrosis, the present study was conducted to identify the interaction of activin A and TGF-ß in stimulation of extracellular matrix production in HSC. Special attention was paid to elucidate the role of the autocrine factor activin A in the action of TGF-ß in HSC. Results show that the effect of TGF-ß is greatly dependent on the autocrine factor activin A. Hence, the role of activin A in the pathogenesis of hepatic fibrosis is much greater than ever thought, and results suggest the efficacy of follistatin, an activin antagonist, for the treatment of hepatic fibrosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Anti-{alpha}-smooth muscle actin ({alpha}-SMA) antibody was purchased from Sigma (St. Louis, MO). Platelet-derived growth factor-BB (PDGF) was obtained from Pepro Tech (Los Angeles, CA). Anti-type I and II activin receptor antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-ßA antibody was described previously (15). Recombinant human activin A (4) and follistatin (6) were generously provided by Dr. Y. Eto of the Ajimonoto Inc. (Kawasaki, Japan).

Isolation of HSCs
HSC were prepared as described by Knock et al. (16). Purity of HSC assessed by the autofluorescence of the cells by UV-excited fluorescence microscopy was above 90%. There was virtually no contamination of parenchymal cells. HSC were cultured in DMEM containing 5% fetal calf serum (Invitrogen Life Technologies, Grand Island, NY), 100 U/ml penicillin, and 100 mg/ml streptomycin under an atmosphere containing 5% CO2 at 37 C. Cells were plated at a density of 7.5 x 105 cells per well in 1.5 ml of culture medium on 35-mm diameter plastic dishes for 3 d. To obtain quiescent cells, cells were then cultured in serum-free medium for 3 d.

Northern blotting and RT-PCR
Total RNA was isolated from HSC using TRIzol reagent (Invitrogen Life Technologies). Northern blotting was done as described previously (17).

First-strand cRNA was made from total RNA using a ReverTra Ace System (Toyobo, Tokyo, Japan). Primers used for rat ßA-subunit, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: ßA-subunit: sense, 5'-GGACCTAACTCTTCAGCCAGAGATG-3'; antisense: 5'-TCTCAAAATGGAGTGTCTTCCTGG-3', GAPDH: sense, 5'-CATGACCACAGTCCATGCCATC-3' antisense, 5' CACCCTGTTGCTGTAGCCATATTC-3'. The reactions were performed in a DNA Thermal Cycler (PerkinElmer, Norwalk, CT) with the following cycle conditions: denaturation at 94 C for 1 min, annealing at 52 C for 45 sec, and extension at 72 C for 45 sec. The number of cycles for ßA and GAPDH was 30 and 18, respectively. PCR for TGF-ß was performed using a PCR kit for rat TGF-ß (Maximbio, San Francisco, CA).

Immunoblot analysis
Cells were washed twice with PBS, suspended in Laemmli buffer, and heated to 100 C for 10 min. After centrifugation, the supernatant was collected, and the protein concentration was determined by using a protein assay kit (Bio-Rad, Hercules, CA). Twenty micrograms of protein from each sample were separated by SDS-PAGE (10–20% gradient gel) under reduced conditions and transferred to a polyvinylidene difluoride membrane (Nihon Millipore, Yonezawa, Japan) by electroblotting. To reduce nonspecific antibody binding, the membrane was blocked with 5% BSA and 0.1% NaN3 dissolved in Tris-saline for 1 h at 37 C, then incubated overnight with primary antibody and washed with Tris-PBS. After incubation with peroxidase-labeled secondary antibody for 1 h at room temperature, the membrane was washed with Tris-PBS and analyzed by exposure to x-ray film using ECL Western blotting detection reagent (Amersham Life Science, Buckinghamshire, UK).

Measurement of DNA synthesis
DNA synthesis was assessed by measuring [3H]thymidine incorporation. Quiescent HSC were culture in medium containing 2 nM activin A or 1 nM PDGF for 48 h and [3H]thymidine was included in the last 12 h. [3H]Thymidine incorporated into acid-precipitated materials was measured as described previously (4).

Measurement of activin A and TGF-ß
Concentration of activin A was measured by bioassay as described previously by Eto et al. (18). Concentration of TGF-ß was determined by using a kit from DRG International (Mountainside, NJ).

Measurement of collagen production
Collagen secreted into culture medium was measured by using Sircol Sirius red dye (Biocolar Ltd., Newtown Abbey, Northern Ireland) as described by Williams et al. (19).

Transfection of HSC by using adenovirus vector
Adenovirus vectors containing the truncated type II activin receptor (AdextActRII) and truncated type II TGF-ß receptor (AdextTRII) were prepared as described previously (8). Recombinant adenovirus carrying the LacZ gene, which encodes the Escherichia coli ß-galactosidase (AdexLacZ) was provided by Dr. T. Takeuchi (Gunma University, Gunma, Japan) and was used as a control (8).

HSC were transfected with AdextActRII, AdextTRII, or AdexLacZ at a titer of 10 multiplicity of infection for 2 h at 37 C and then cultured for 24 h in serum-free medium before the addition of the ligand. Efficacy of transfection determined by ß-galactosidase expression was more than 95%.

Statistical analysis
Statistical analysis was done using Student’s t test. The P value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of activin A and type I and II activin receptors
We first examined whether or not activin A is an autocrine factor of HSC. We measured the expression of the ßA-subunit of activin and activin receptors. As shown in Fig. 1AGo, quiescent HSC expressed mRNA for the ßA-subunit of activin and the type I and II activin receptors. Note that the expression of the ßB-subunit of activin was not detected in HSC at least in our experimental condition. The type I and II activin receptors and the ßA-subunit were also detected by Western blotting (Fig. 1BGo). We then studied the agents that stimulated the expression of the ßA-subunit of activin. We found that TGF-ß was a potent inducer of the expression of the ßA-subunit. As shown in Fig. 1CGo, TGF-ß increased the mRNA for the ßA-subunit. Figure 1DGo depicts the dose-response relationship for the TGF-ß-induced production of activin A in HSC. The maximal effect was obtained by 100 pM TGF-ß.



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  		FIG. 1. Expression of mRNA for the ßA-subunit of activin, and the type I and II activin receptors in HSC. A, The expression of mRNA for the type I (1 ) and II (2 ) activin receptors and the ßA-subunit of activin (3 ) was measured by RT-PCR. B, The expression of the type I (ActRI) and II (ActRII) activin receptors and the ßA-subunit was measured by Western blotting. C, Effect of TGF-ß on the expression of the ßA-subunit. Quiescent HSC were incubated for various periods with 100 pM TGF-ß and mRNA for the ßA-subunit of activin was measured by Northern blotting. D, Production of activin A induced by TGF-ß. Quiescent HSCs were incubated with various doses of TGF-ß for 24 h and activin A released into the medium was measured. Values are the mean ± SE for four independent experiments each performed in triplicate.

 
Effect of activin A on the activation of HSC
We then examined the effect of exogenous activin A on HSC activation. We measured changes in the expression of {alpha}-SMA. As depicted in Fig. 2AGo, activin A increased the expression of {alpha}-SMA in a dose-dependent manner, and the maximal effect of activin A was obtained at 1 nM (Fig. 2BGo). Activin A also increased the secretion of collagen (Fig. 2CGo). The maximal effect of activin A was again observed at 1 nM. Activin also increased [3H]thymidine incorporation in HSC (Fig. 2DGo).



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  		FIG. 2. Effect of activin A on the activation of HSC. A, Quiescent HSC were cultured for 2 d with various doses of activin A and the expression of {alpha}-SMA was measured by Western blotting. The expression of {alpha}-tubulin was measured as an internal control. B, Expression of {alpha}-SMA was quantified by densitometry. The ratio of {alpha}-SMA to {alpha}-tubulin was measured. Values are the mean ± SE for three independent experiments. C, Effect of activin A on the secretion of collagen. Quiescent HSC were cultured for 2 d and collagen secreted into the medium was measured. Values are the mean ± SE for four independent experiments each done in triplicate. D, Effect of activin A on DNA synthesis in HSC. Quiescent HSCs were cultured for 2 d with 2 nM activin A or 1 nM PDGF and [3H]thymidine incorporation was measured. Values are the mean ± SE for four independent experiments each done in triplicate. *, P < 0.05 vs. none; **, P < 0.01 vs. none.

 
Effect of follistatin on activin A- and TGF-ß-induced collagen expression
Activated HSC produce types I, III, and IV collagen (20). We then assessed whether or not activin A produced in HSC modifies the effect of TGF-ß. To block the action of activin A, we used follistatin, an activin-binding protein that blocks the effect of activin A (19). As shown in Fig. 3AGo, follistatin completely inhibited the effect of exogenous activin A on the mRNA expression of the type I collagen. Follistatin also blocked the effect of exogenous activin A on the secretion of collagen (Fig. 3BGo). More importantly, the effect of TGF-ß on the expression of collagen was markedly attenuated by follistatin (Fig. 3AGo). Similarly, TGF-induced secretion of collagen was significantly inhibited by follistatin (Fig. 3BGo).



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  		FIG. 3. Effect of follistatin on activin A- and TGF-ß-induced collagen production. A, Northern blotting of the type I collagen. Quiescent SHC were incubated for 2 d with 1 nM activin A or 100 pM TGF-ß in the presence or absence of 4 nM follistatin. The mRNA for the type I collagen was measured by Northern blotting. B, Secretion of collagen into the medium. Quiescent SHCs were incubated as indicated above and secreted collagen was measured. Values are the mean ± SE for four independent experiments each done in triplicate. *, P < 0.01.

 
Effect of TGF-ß in HSC expressing the dominant-negative activin receptor
To confirm that the effect of follistatin was due to the blocking of the activin action, we examined the effect of activin A and TGF-ß in HSC overexpressing the dominant-negative type II activin receptor (tActRII). We transfected tActRII by using the adenovirus vector. As shown in Fig. 4AGo, activin A was not able to induce the production of collagen in HSC overexpressing tActRII. In these cells, TGF-ß did not increase collagen secretion significantly. Note that in cells overexpressing LacZ, a control gene to monitor the efficacy of transfection, both activin A and TGF-ß significantly increased collagen secretion. We also examined the effect of activin A and TGF-ß in HSC overexpressing the dominant-negative type II TGF-ß receptor (tTRII). As shown in Fig. 4BGo, TGF-ß could not increase collagen secretion in HSC overexpressing tTRII. However, activin A was still able to increase collagen secretion in these cells. Note that activin A and TGF-ß elicited their effects in cells transfected with LacZ.



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  		FIG. 4. Effect of activin A and TGF-ß in HSC overexpressing dominant-negative activin and TGF-ß receptors. A, HSC were transfected with AdexAxtRII (a) or AdexLacZ (b). Cells were then incubated with 1 nM activin A and 100 pM TGF-ß for 48 h and collagen released into the medium was measured. Values are the mean ± SE for four independent experiments. *, P < 0.05 vs. none. B, HSC were transfected with AdextATRII (a) or AdexLacZ (b). Cells were incubated with 1 nM activin A and 100 pM TGF-ß for 48 h and collagen released into the medium was measured. Values are the mean ± SE for four independent experiments. *, P < 0.05 vs. none.

 
Effect of activin A on the expression of TGF-ß
We then examined the effect of activin A on the expression of TGF-ß. Activin A increased the mRNA expression of TGF-ß (Fig. 5AGo). Likewise, activin A increased the release of TGF-ß into culture medium (Fig. 5BGo). The amount of TGF-ß released in response to activin A was much less than that of activin A released in response to TGF-ß.



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  		FIG. 5. Production of TGF-ß in HSC. A, Effect of activin A on the mRNA expression of TGF-ß1. Quiescent HSC were incubated for various periods with 1 nM activin A and mRNA for TGF-ß1 was measured by RT-PCR. B, Quiescent HSC were incubated for 24 h with various concentrations of activin A and TGF-ß released into the medium was measured. Note that the sum of TGF-ß1, ß2, and ß3 was quantified. Values are the mean ± SE for four independent experiments each done in quadruplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results clearly show that activin A is an autocrine factor secreted from HSC: the ßA-subunit and two types of receptors were expressed in HSC; and HSC responded to exogenous activin A, and the secretion of collagen was augmented. These results confirm the previous reports (9, 10) and provided an experimental system to study the regulation of the production of the autocrine factor. With regard to the significance of this autocrine factor, the most intriguing finding is that follistatin, an antagonist of activin A, markedly reduced collagen production induced by TGF-ß. Although follistatin was shown to inhibit the effect of bone morphogenic protein as well (21), follistatin may have elicited its action in HSC by blocking the effect of activin A because the effect of TGF-ß was also attenuated in HSC overexpressing the dominant-negative type II activin receptor. We interpret these findings to mean that part of the action of TGF-ß is mediated by activin A secreted from HSC. Consistent with this notion, TGF-ß increased the expression of the ßA-subunit of activin as well as the release of dimeric protein, activin A, into the culture medium. Sugiyama et al. (10) showed that activin A and TGF-ß act synergistically to induce the expression of collagen. Hence, it is quite likely that activin A produced in HSC in response to TGF-ß may amplify the effect of TGF-ß on the production of matrix proteins. When this amplifying mechanism is blocked, the TGF-ß action is significantly reduced. Taken together, TGF-ß and activin A not only elicit a similar effect in HSC, but these two factors interact intimately and coordinately induce their action (Fig. 6Go). Although the TGF-ß action was dependent on the production of the autocrine factor activin A, the activin A action was not affected even if the effect of TGF-ß was blocked by transfection of the dominant-negative TGF-ß receptor. Given that activin A up-regulates the expression of TGF-ß and increases the release of TGF-ß, it is possible that TGF-ß produced in HSC modifies the action of activin A. This was, however, not the case. The failure of TGF-ß to modify the activin effect was perhaps due to the limited amount of TGF-ß released from HSC. Presumably, the release of TGF-ß in response to activin A was not enough to amplify the action of activin A. We cannot rule out the possibility that activation of TGF-ß produced in HSC was not sufficient in the culture system. If so, it is possible that TGF-ß produced in HSC in vivo is activated in the vicinity of HSC and becomes high enough to modify the effect of activin A.



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  		FIG. 6. Role of autocrine factors, activin A, and TGF-ß in HSC activation. A, Activin A activates HSC. Activin A also increases the production of TGF-ß, but because the amount of TGF-ß produced in HSC is not large enough, TGF-ß does not amplify the effect of activin A. B, TGF-ß activates HSC. TGF-ß also increases the production of activin A, which acts synergistically with TGF-ß and amplifies the TGF-ß action. C, When TGF-ß is added together with follistatin, activin A released from HSC is trapped by follistatin. The amplification mechanism is blocked and the effect of TGF-ß is attenuated significantly.

 
The present results indicate the importance of activin A produced in HSC in the activation of HSC. Activin A is also produced in parenchymal cells surrounding HSC (4, 9). Considering the large mass of parenchymal cells, the amount of activin released from these cells may be considerable. Collectively, activin A may activate HSC in the liver by autocrine and paracrine mechanisms.

The present results demonstrate the efficacy of activin antagonist follistatin to inhibit the production of collagen induced by activin A and TGF-ß. This finding is rather unexpected but is intriguing in view of the therapeutic potential of follistatin. Both TGF-ß and activin A are involved in the pathogenesis of hepatic fibrosis (9, 10, 12, 13, 14). Blocking the action of either of these factors should be effective in treating hepatic fibrosis. To date, many studies have been conducted to establish therapeutic methods to manipulate the actions of TGF-ß. For example, the soluble extracellular domain of the TGF-ß receptor was shown to be effective to treat hepatic fibrosis (22). Also, an inhibitor of the type I TGF-ß receptor kinase was developed (23). Indeed, it would be more effective if one could block the effect of both TGF-ß and activin A simultaneously. To this end, inhibitors of the common intracellular signaling molecule involved in the action of two ligands, for example, Smad proteins (23), could be a good target for such treatment. In this regard, the present results indicate that follistatin is a potential candidate to inhibit simultaneously the actions of TGF-ß and activin A on HSC. Indeed, follistatin may be potentially useful for the treatment of hepatic fibrosis. We recently reported that follistatin markedly reduced TGF-ß-induced production of extracellular matrix proteins in renal myofibroblasts (24) and fibroblast-like synoviocytes (25), which are involved in the pathogenesis of renal fibrosis and rheumatoid arthritis, respectively. In addition, Ohnishi et al. (26) reported recently that follistatin inhibits TGF-ß-mediated collagen production in pancreatic stellate cells. These results indicate that involvement of activin A in TGF-ß-induced collagen production may not be a phenomenon specific to HSC. The result also suggest the usefulness of follistatin to control the TGF-ß-mediated fibrosis in various tissues.


   Acknowledgments
 
The authors are grateful to Mayumi Odagiri for secretarial assistance during the preparation of the manuscript.


   Footnotes
 
Abbreviations: AdexLacZ, Recombinant adenovirus carrying the LacZ gene; AdextActRII, truncated type II activin receptor; AdextTRII, truncated type II TGF-ß receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSC, hepatic stellate cells; {alpha}-SMA, {alpha}-smooth muscle actin; PDGF, platelet-derived growth factor.

Received December 8, 2003.

Accepted for publication February 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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