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Serum Growth Factors and Proinflammatory Cytokines Are Potent Inducers of Activin Expression in Cultured Fibroblasts and Keratinocytes
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
228, 106–113 (1996)
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Serum Growth Factors and Proinflammatory Cytokines Are Potent Inducers of Activin Expression in Cultured Fibroblasts and Keratinocytes GRISELDIS HU¨BNER
AND
SABINE WERNER1
Max-Planck-Institut fu¨r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany
Recently we demonstrated a large induction of activin expression in fibroblasts and keratinocytes after cutaneous injury in mice. To identify possible mediators of activin induction during skin repair, we have now analyzed the regulation of this factor in cultured keratinocytes and fibroblasts. Here we show that activin A mRNA and protein levels are low in quiescent keratinocytes and fibroblasts but expression is strongly induced upon serum treatment. The stimulatory effect of serum on activin expression is likely to be a combinatorial effect of different growth factors, since platelet-poor plasma serum and several purified serum growth factors also stimulated activin expression, although to a lesser extent than complete serum. Furthermore, we found increased expression of activin in keratinocytes and fibroblasts after addition of the proinflammatory cytokines interleukin 1b and tumor necrosis factor a. Taken together, our data suggest that serum growth factors which are released upon hemorrhage as well as proinflammatory cytokines derived from neutrophils and macrophages might be responsible for induction of activin expression after injury. q 1996 Academic Press, Inc.
INTRODUCTION
The activins belong to the transforming growth factor b (TGFb) superfamily [1] and were originally discovered as proteins of gonadal origin which regulate the release of follicle-stimulating hormone by pituitary cells [2, 3]. In the meantime, many biological activities of activin have been identified. Activin induces differentiation of hematopoietic cells [4, 5], osteoblasts [6], and endocrine cells [7, 8], and inhibits proliferation of gonadal cell lines [9], hepatocytes [10], endothelial cells [11], and lung epithelial cells [12]. Furthermore, activin modulates cell proliferation in fibroblasts [13] and vascular smooth muscle cells [14]. In 1 To whom reprint requests and correspondence should be addressed at Max-Planck-Institut fu¨r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany. Fax: 089-8578-2814.
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0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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addition, activin constitutes an inhibitor of neuronal cell differentiation [15, 16] and plays a role in mesoderm induction during amphibian development [17–19]. Like the other members of the TGFb superfamily, activins consist of two disulfide-linked polypeptide chains. They are homo- or heterodimers of a bA- and/ or a bB-chain. Three different isoforms of activin have been identified: activin A (bAbA), activin B (bBbB), and activin AB (bAbB). Additional isoforms might arise from a putative bC-chain, which has recently been cloned from human cells [20]. Inhibins which antagonize the activin actions are heterodimers consisting of a common a-subunit and a b-subunit: inhibin A (abA) and inhibin B (abB) [21, 22]. Biological responses to activin could only be observed after dimerization of two different receptor types which form a heteromeric signaling complex [23–28]. Both types of activin receptors are transmembrane serine/threonine kinases: type I receptors (ARI and ARIB , also called activin receptor-like kinases Alk-2 and Alk-4, respectively) and type II receptors (ARII and ARIIB) [for review see 24]. A soluble activin binding protein, follistatin, has also been characterized. Its in vivo function is currently under debate, but it inhibits activin action in vitro [29–32]. The TGFbs have long been known to play a role in the skin and during wound healing. TGFb is an important differentiation factor for keratinocytes [33, 34]. It stimulates fibroblast proliferation and induces extracellular matrix production by epithelial, mesenchymal, and endothelial cells [for review, 35]. Furthermore, exogenous TGFb stimulates angiogenesis and wound reepithelialization [36–38]. By contrast, little is known about a possible function of the other family members during cutaneous wound repair. The phenotypes of recently generated transgenic mice suggested a role of activin in skin development. Mice missing the activin bA-chain lacked whiskers and whisker follicles were abnormal [39]. Mice deficient in follistatin showed disturbed whisker development and hyperkeratotic skin [40]. Apart from skin development, activin seems to play an important role in wound repair. Recently we demonstrated a large induction of activin but not inhibin mRNA expression within the first day after skin
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injury in mice [41]. Expression levels were high during the first 7 days and activin bA expression returned to the basal level 2 weeks after wounding, whereas activin bB expression was still high. Activin bA mRNA was found at highest levels in the granulation tissue below the wound and activin bB mRNA was particularly abundant in the hyperproliferative epithelium at the wound edge and in the migrating epithelial tongue. To identify possible mediators of the activin induction during skin repair, we have now analyzed the regulation of activin expression in cultured keratinocytes and fibroblasts. Our results suggest a role of serum growth factors in the early induction of activin expression after injury, whereas proinflammatory cytokines could be responsible for the prolonged activin expression at later stages of the wound healing process. MATERIALS AND METHODS Cell culture. The human keratinocyte cell line HaCaT [42], human primary embryonal fibroblasts, and a murine Balb/c 3T3 fibroblast cell line were used for cell culture experiments. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS) (human cells) or with 10% newborn calf serum (NCS) (Balb/c 3T3). For activin induction experiments they were grown to confluency without changing the medium. Cells were rendered quiescent by a 16-h incubation in serum-free DMEM (HaCaT) or in DMEM with 1% NCS (Balb/c 3T3). Confluent human embryonal fibroblasts were starved for 3 days in DMEM with 0.5% FCS followed by a 16-h incubation in serum-free DMEM. Cells were then incubated for varying periods in fresh DMEM containing serum, purified growth factors, or cytokines, harvested before and at different time points after treatment with these reagents, and used for RNA isolation. Each experiment was repeated at least twice. FCS, NCS, and DMEM were purchased from Gibco/BRL, complete adult human serum and platelet-poor plasma serum were obtained from a healthy adult volunteer and were prepared as described [43]. Growth factors and cytokines were from Boehringer Mannheim Biochemicals and cycloheximide was from Sigma Biochemicals. They were used in the following final concentrations: bFGF (10 ng/ml), EGF (20 ng/ml), IL-1b (100 U/ml), IL-6 (100 U/ml), KGF (10 ng/ml), PDGF-BB (10 ng/ml), TGFb1 (1 ng/ml), TNFa (300 U/ml), cycloheximide (10 mg/ml). DNA templates. RNase protection assay templates for murine activin/inhibin a-, bA-, bB-chains, murine activin receptors ARI (Alk2), ARIB (Alk-4), ARII, ARIIB , and murine follistatin were recently described [41]. The human activin bA template was generated by PCR using primers corresponding to the published human sequences. The amplified fragment was a 358-bp fragment corresponding to the 3*-end of the activin bA-chain cDNA [44]. RNA isolation and RNase protection analysis. Isolation of RNA was performed as described [45]. Twenty micrograms of total RNA from cultured cells was used for RNase protection analysis. RNase protection assays were carried out as recently described [46]. All experiments were repeated with a different set of RNAs from an independent cell culture experiment. Generation of activin-specific antibodies. A peptide corresponding to the carboxy-terminal region of activin A (CGGYYDDGQNIIKKDIQ) was synthesized, coupled to keyhole limpet hemocyanin (Sigma Biochemicals), and used for immunization of rabbits. Antisera were tested by immunoblotting for their reactivity with recombinant activin A as well as for cross-reactivity with recombinant TGFb1. Western blot analysis of activin proteins. Five milliliters of DMEM per 10-cm petri dish were conditioned by quiescent HaCaT
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cells or human embryonal fibroblasts in the presence or the absence of growth factors and cytokines. After addition of these factors, conditioned medium from three petri dishes was harvested at different time points and centrifuged for 10 min at 3000 rpm to remove cell debris. Proteins were precipitated with 6% trichloroacetic acid, washed with acetone, and resuspended in PBS. After addition of Laemmli sample buffer, proteins were denatured at 957C, separated on a 15% SDS–polyacrylamide gel, and transferred to nitrocellulose membranes. Activin proteins were detected using a 1:500 dilution of a polyclonal antiserum directed against the carboxy-terminal region of activin A (see above) and an alkaline phosphatase detection system (Promega). Each experiment was repeated at least twice.
RESULTS
We have recently demonstrated a large induction of activin expression after skin injury. Activin mRNA could be localized in epithelial and mesenchymal cells during wound healing. To identify the factors which could mediate this induction in the wound, we have studied the regulation of activin expression in cultured murine fibroblasts, human fibroblasts, and human keratinocytes. Because fibroblasts of the granulation tissue are the primary producers of activin bA mRNA in the wound tissue of mice [41], we first investigated the regulation of activin expression in a murine fibroblast cell line. Regulation of Activin mRNA Expression in Fibroblasts Induction of activin mRNA expression by serum. Since hemorrhage is one of the early events after skin injury, we first tested the potency of serum to stimulate activin expression in fibroblasts. As shown in Fig. 1A, activin bA mRNA could hardly be detected in confluent, quiescent murine Balb/c 3T3 fibroblasts. Upon addition of 10% newborn calf serum (NCS), a large induction of activin bA mRNA expression was observed. Maximal expression levels were reached between 2.5 and 5 h after stimulation with serum (Fig. 1A). Expression of activin bA mRNA dropped after 5 h and reached basal levels 24 h after serum addition. A similar induction of activin bA by serum was seen in human embryonal fibroblasts (data not shown). By contrast, no expression of the bB- and a-chains was found in Balb/c 3T3 murine fibroblasts (data not shown). To determine if the strong induction of activin bA expression in Balb/c 3T3 murine fibroblasts is the result of an additive action of different growth factors, the effect of complete adult human serum was compared to platelet-poor plasma serum (PPPS). As shown in Fig. 1B, complete human serum induced activin expression much more strongly than PPPS. Therefore, most of the factors which are responsible for activin A induction seem to be derived from platelets. To determine whether the induction of activin A mRNA expression requires de novo protein synthesis, the effect of cycloheximide was analyzed. Addition of
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FIG. 1. Induction of activin bA mRNA expression by serum in cultured murine Balb/c 3T3 fibroblasts. Induction of activin bA mRNA expression by newborn calf serum (NCS) (A), complete human serum (HS) (B), and platelet-poor plasma serum (PPPS) (B). Cells were rendered quiescent by serum starvation and stimulated with 10% serum for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 1000 cpm of the hybridization probe was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA was used as a negative control. The gels were exposed for 12 h. An ethidium bromide stain of 1 mg RNA from the same batch of RNA is shown below.
FIG. 2. Effect of serum on the expression of activin receptors and follistatin in cultured murine Balb/c 3T3 fibroblasts. Serum starved cells were treated with 10% newborn calf serum (NCS) for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin receptor and follistatin mRNA expression by RNase protection assays. 1000 cpm of the hybridization probes was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA were used as a negative control. The gels were exposed for 16 h (ARI, follistatin), 48 h (ARII, ARIB), or 4 days (ARIIB). The same set of RNAs was used for the protection assays of Figs. 1 and 2.
this protein synthesis inhibitor completely blocked the induction of activin expression by serum (data not shown), demonstrating that other proteins have to be synthesized before activin expression can occur. Serum dependent expression of activin receptors and follistatin. To investigate the mRNA expression of activin receptors and follistatin in Balb/c 3T3 fibroblasts upon treatment with serum, RNase protection assays were performed using the same RNA preparations as for expression analysis of activin a- and b-chains (Fig. 2). All known activin binding proteins were expressed in confluent, quiescent Balb/c 3T3 fibroblasts. Considering the different exposure times of the autoradiograms, we can conclude that ARI (Alk-2) and ARII were expressed at higher levels compared to ARIB (Alk-4), ARIIB , and follistatin. A slight induction of mRNA expression upon treatment with serum was observed for ARI, ARIB , and for follistatin. ARIIB was expressed at extremely low levels and expression even dropped upon serum treatment of the cells. Induction of activin A expression by purified serum growth factors. To identify the serum components which are responsible for the stimulatory effect on activin bA expression, we analyzed the effect of purified serum growth factors on Balb/c 3T3 fibroblasts. As shown in Fig. 3, transforming growth factor b1 (TGFb1) induced activin expression in a dose- and time-dependent manner. A dose of 1 ng/ml of TGFb1
was sufficient to elicit a maximal response. A first elevated signal was observed 2 h after treatment of the cells with this growth factor, and maximal induction was seen after 8 h. In addition to TGFb1, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) also stimulated activin bA expression in confluent, quies-
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FIG. 3. TGFb1 induces activin bA mRNA expression in cultured murine Balb/c 3T3 fibroblasts. Serum-starved cells were treated for 5 h with different concentrations of TGFb1 (left panel) and for different time periods with 1 ng/ml TGFb1 (right panel) as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 1000 cpm of the hybridization probe was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA was used as a negative control. The gels were exposed for 12 h.
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FIG. 5. Proinflammatory cytokines induce activin bA mRNA expression in cultured murine Balb/c 3T3 fibroblasts. Serum-starved cells were stimulated with IL-1b (100 U/ml), IL-6 (100 U/ml), and TNFa (300 U/ml) for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 50 mg tRNA was used as a negative control. The gels were exposed for 48 h.
FIG. 4. Synergistic effect of EGF and PDGF on activin bA mRNA expression in cultured murine Balb/c 3T3 fibroblasts. Serum-starved cells were stimulated with EGF (20 ng/ml) and PDGF (10 ng/ml) alone or together for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 50 mg tRNA was used as a negative control. The gels were exposed for 24 h.
cent Balb/c 3T3 fibroblasts (Fig. 4 and Table 1). Interestingly, when cells were treated with a combination of EGF and PDGF, an additive effect on activin expression was seen. However, the observed signal did not reach the intensity of the signal obtained after treatment of the cells with serum. This provides further evidence for a combinatorial effect of several different growth factors in the regulation of activin expression. Similar to Balb/c 3T3 fibroblasts, serum growth factors also induced activin A expression in primary human embryonal fibroblasts (Table 1).
Activin A expression is stimulated by proinflammatory cytokines. Recently we demonstrated expression of proinflammatory cytokines in polymorphonuclear leukocytes and macrophages after skin injury [47]. To determine a possible role of these factors for activin regulation, we analyzed their effect on activin expression in vitro. As shown in Fig. 5, interleukin 1b (IL1b), tumor necrosis factor a (TNFa), and interleukin 6 (IL-6) acted as inducers of activin expression in confluent, quiescent murine 3T3 fibroblasts. Activin A signals appeared 5 h after treatment of the cells with these cytokines and were still high after 8 h. Of all three cytokines, TNFa induced the strongest response. However, none of these cytokines was as potent as complete serum. As is shown in Table 1, a similar induction of activin bA expression by proinflammatory cytokines occurred in primary human embryonal fibroblasts. Regulation of Activin mRNA Expression in Keratinocytes
TABLE 1 Induction of Activin A by Growth Factors and Cytokines
FCS/NCS TGFb1 KGF bFGF EGF PDGF EGF / PDGF IL-1b TNFa IL-6
Balb/c fibroblasts
Human embryonal fibroblasts
HaCaT keratinocytes
///// //// n.d. /// /// /// ///// /// //// //
/// /// n.d. n.d. // // //// /// //// ///
//// / / n.d. /// 0 n.d. / /// 0
Note. bFGF (basic fibroblast growth factor), EGF (epidermal growth factor), FCS (fetal calf serum), IL-1b (interleukin 1-b), IL-6 (interleukin 6), KGF (keratinocyte growth factor), NCS (new born calf serum), PDGF (platelet-derived growth factor), TGFb1 (transforming growth factor b1), TNFa (tumor necrosis factor a), n.d. (not done).
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Induction of activin mRNA expression by serum growth factors and proinflammatory cytokines. Although fibroblasts are the major source for activin A in the wound [41], low levels of activin A mRNA were also found in the epidermis of mouse tail skin (G.H., unpublished data). Therefore, the regulation of activin bA expression was also investigated in the human keratinocyte cell line HaCaT. As shown in Fig. 6A, no expression of activin A mRNA was found in confluent, quiescent HaCaT cells. Within 5 h after addition of fetal calf serum (FCS), activin expression was strongly induced, but declined after 8 h. The activin induction by serum growth factors in HaCaT cells was long lasting and increased levels of activin mRNA were still observed 24 h after treatment of the cells. In contrast to the bA-chain, activin bB expression could not be detected in quiescent or serum-stimulated HaCaT cells (data not shown). EGF was an even more potent inducer of activin bA expression than serum (Fig. 6A). Already 2 h after
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FIG. 6. Serum, purified growth factors, and proinflammatory cytokines stimulate activin bA mRNA expression in cultured HaCaT keratinocytes. (A) Serum (10% fetal calf serum), EGF (20 ng/ml), KGF (10 ng/ml), and (B) TGFb1 (1 ng/ml) and TNFa (300 U/ml) induce activin bA mRNA expression. Cells were rendered quiescent by serum starvation and stimulated with serum, growth factors, and cytokines for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assays. 1000 cpm of the hybridization probes was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA was used as a negative control. The gels were exposed for 24 h.
treatment of the cells with this growth factor, the first elevated bA signal was detected. Furthermore, EGF caused a more prolonged expression of activin bA and— in contrast to serum—maximal levels of bA mRNA were still observed 8 h after addition of EGF. Keratinocyte growth factor (KGF), which is produced by fibroblasts and acts as a strong mitogen for keratinocytes [48], also induced activin bA mRNA expression. Interestingly, there was a large difference in activin bA expression after treatment of keratinocytes with EGF and KGF. Although both growth factors are potent mitogens for keratinocytes, KGF caused a much weaker induction than EGF. In contrast to EGF and KGF, the platelet-derived mitogen PDGF had no effect on activin expression. To determine a possible correlation between activin induction and stimulation of cell proliferation, we analyzed the effect of TGFb1 on keratinocytes. This factor is a major differentiation factor for keratinocytes [33, 34], but inhibits keratinocyte proliferation in vitro and in vivo [49–52]. As shown in Fig. 6B, TGFb1 stimulated activin expression in HaCaT cells (Fig. 6B), although it was a weaker inducer than EGF. Thus, induction of activin expression is not dependent on the mitogenic activity of a growth factor. Similar as in fibroblasts, the proinflammatory cytokines TNFa and to a lesser extent IL-1b also induced activin expression in HaCaT cells (Fig. 6B and Table 1). Interestingly, TNFa, which inhibits keratinocyte growth in vitro [53], caused a different kinetics of activin expression in HaCaT keratinocytes than the other factors. TNFa already caused maximal induction of activin expression 2 h after treatment of the cells and
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transcript levels were still high after 8 h. By contrast, IL-6 which acts as mitogen for keratinocytes [54] had no effect on activin A expression in these cells (data not shown). The effects of all tested factors on activin expression in fibroblasts and keratinocytes are summarized in Table 1. Induction of Activin mRNA Expression Correlates with Protein Secretion into the Culture Medium To determine if the induction of activin bA mRNA expression is followed by synthesis and secretion of immunoreactive protein, total protein was precipitated from conditioned culture medium at different time points after treatment of the cells with serum growth factors or cytokines. The presence of activin was then determined by Western blot analysis using an activinspecific antiserum which does not cross-react with TGFb1. Activin A protein could be detected in the medium within 6–24 h after treatment of confluent, quiescent HaCaT keratinocytes and human embryonal fibroblasts with serum growth factors and cytokines (Figs. 7A–7C). The additional bands of higher molecular weight are likely to be a result of nonspecific crossreactivity of the antiserum, since they are also present in the control lanes. These data demonstrate that bA mRNA expression correlates with protein synthesis and subsequent secretion of mature activin into the cell culture medium. DISCUSSION
Recently, we demonstrated a large induction of activin expression after cutaneous injury in mice [41].
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FIG. 7. TNFa and EGF induce activin A protein expression and secretion in fibroblasts and keratinocytes. HaCaT keratinocytes (A, B) and human embryonal fibroblasts (C) were rendered quiescent by serum starvation and stimulated with TNFa (300 U/ml) or EGF (20 ng/ml) for different time periods as indicated. Proteins were precipitated from 6 ml of conditioned medium with 6% trichloroacetic acid and analyzed by immunoblotting for the presence of activin A protein. 10 ng of recombinant activin A or TGFb1 was used as control. The upper band in the activin lane represents full-length recombinant activin, whereas the lower band is likely to be a degradation product.
Fibroblasts in the granulation tissue were the major source for activin bA mRNA, but low levels were also detected in the epidermis. To identify the factors which might be responsible for the activin induction during wound repair, we analyzed the effect of growth factors and cytokines on activin expression in cultured fibroblasts and keratinocytes. Because activin induction occurs very early after injury (during the first 15 to 24 h), serum growth factors derived from the blood upon hemorrhage were likely candidates for activin induction. Here we show that activin bA expression is very low in cultured quiescent fibroblasts and keratinocytes, but is strongly induced upon stimulation with serum or purified serum growth factors such as TGFb1, EGF, or PDGF, whereby the increase in activin bA mRNA correlates with secretion of immunoreactive activin A protein into the culture
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medium. Since the observed induction of activin bA expression by platelet-poor plasma serum and purified growth factors is lower compared to the effect seen with complete serum, we presume that the strong activin induction is a combinatorial effect of many different growth factors. Indeed, a combination of PDGF and EGF caused a much stonger induction than a single factor. Induction of activin by serum growth factors has also been demonstrated for other cell types, such as smooth muscle cells [55], intestinal epithelial cells [56], capillary endothelial cells [11], and differentiated cell lines derived from P19 embryonal carcinoma cells [57]. Since activin bB mRNA and the inhibin a-chain were not expressed in Balb/c 3T3 fibroblasts, only activin A can be produced by these cells. In addition to activin, all known activin binding proteins were expressed in confluent, quiescent Balb/c 3T3 fibroblasts, suggesting autocrine effects of activin in these cells. Similar to the in vivo situation, ARI and ARII were significantly more abundant than ARIB and ARIIB in Balb/c 3T3 fibroblasts. In contrast to activin, expression of the receptors was not induced upon serum stimulation. A lack of activin receptor induction was also observed during wound healing [41], demonstrating that the wounding response is mediated via the ligand but not via the receptor. Surprisingly we could not detect bB mRNA in quiescent or serum-treated HaCaT cells. This seems to be contradictory to the in vivo situation, where bB mRNA was found at high levels in the hyperproliferative epithelium at the wound edge in mice [41]. However, data from in situ hybridization and immunhistochemistry (G.H., unpublished data) revealed the presence of bB mRNA and activin protein exclusively in differentiated keratinocytes in the suprabasal layers, suggesting that keratinocytes must first receive a differentiation signal before activin induction can occur. Such a signal might be specific for the in vivo situation. Alternatively, the lack of bB mRNA expression might be due to speciesspecific differences between human and mouse keratinocytes. Another early event in wound repair is the infiltration of the wound area by polymorphonuclear leukocytes as a first defense against bacterial invasion [58]. Recently, we could demonstrate a strong expression of proinflammatory cytokines by these cells in mouse excisional skin wounds as soon as 12 h after injury [47]. The migration of macrophages into the wound area occurs approximately 24 h after injury. Like neutrophils, activated macrophages also produce proinflammatory cytokines [59–62], whereby the temporal and spatial expression pattern of these factors correlates with activin expression in the wound [41, 47]. Therefore, we analyzed the effect of proinflammatory cytokines on activin expression in cultured fibroblasts and keratinocytes. Interestingly, we also found a significant increase in activin A mRNA and protein by
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these cytokines. The strongest inducer was TNFa which caused a significantly faster induction of activin expression in HaCaT keratinocytes compared to the other factors, suggesting that activin induction is a direct effect of this cytokine. Indeed, preliminary data from our laboratory revealed an inhibitory effect of cycloheximide on activin induction after EGF treatment, whereas activin expression was superinduced by treatment of the cells with both cycloheximide and TNFa. TNFa is produced both by neutrophils and macrophages [47, 63]. In contrast to neutrophils, macrophages stay in the wound area beyond the initial inflammatory phase and produce cytokines until the healing process is completed. Furthermore, they are an important source of PDGF [64–66], another potent inducer of activin expression in fibroblasts. Therefore, macrophages could be responsible for the prolonged activin expression at later stages of the healing process. Apart from serum growth factors and cytokines, KGF is another growth factor which is highly abundant in the healing wound [46]. KGF is produced by fibroblasts and—like EGF—is a strong mitogen for keratinocytes [42, 48, 67]. Interestingly, KGF stimulated activin expression only weakly, whereas EGF was the most potent activator of activin bA expression in HaCaT keratinocytes. This finding suggests, that EGF and KGF activate different intracellular signaling pathways, resulting in different degrees of activin bA induction. Differential effects of EGF and KGF on gene expression in keratinocytes seems to be a more general feature, since we recently identified novel KGF-regulated genes which do not respond to EGF (S. Frank, S. Werner, unpublished data). In conclusion, we have identified several positive regulators of activin expression in fibroblasts and keratinocytes. Since these growth factors and cytokines are present in the wound at early and later stages of the repair process, they might also be responsible for the strong activin expression during wound healing in vivo. Growth factors derived from serum upon hemorrhage and proinflammatory cytokines secreted by neutrophils are likely to initiate the large induction of activin expression after skin injury, whereas cytokines derived from macrophages could be responsible for the prolonged expression at later stages of the repair process. These results provide further evidence for a role of activin in the wound healing scenario. Although the function of activin in the repair process is currently unknown, preliminary data from in vitro studies suggest a role of this factor in extracellular matrix production and keratinocyte differentiation (G.H., unpublished data). Future studies using transgenic animal models will help to clarify the role of activin in tissue repair. We thank Dr. P. H. Hofschneider for support, Irene Dick for help
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with the tissue culture experiments, Dr. D. Huylebroeck, University of Leuven, for the Alk-2 cDNA, and Dr. A. J. M. van den Eijndenvan Raaij, Hubrecht Laboratory, Utrecht, for follistatin and inhibin cDNAs. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (WE 1983/1-2). G. H. is a recipient of a predoctoral fellowship from the Boehringer Ingelheim foundation. S. W. is a Hermann-and-Lilly-Schilling Professor of Medical Research.
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46. Werner, S., Peters, K. G., Longaker, M. T., Fuller-Pace, F., Banda, M. J., and Williams, L. T. (1992) Proc. Natl. Acad. Sci. USA 89, 6896–6900. 47. Hu¨bner, G., Brauchle, M., Smola, H., Madlener, M., Fa¨ssler, R., and Werner, S. (1996) Cytokine 8, 548–556. 48. Rubin, J. S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S., and Aaronson, S. A. (1989) Proc. Natl. Acad. Sci. USA 86, 802–806. 49. Shipley, G. D., Pittelkow, M. R., Wille, J. J., Scott, R. E., and Moses, H. L. (1986) Cancer Res. 46, 2068–2071. 50. Coffey, R., J. J., Bascom, C. C., Sipes, N. J., Graves-Deal, R., Weissman, B. E., and Moses, H. L. (1988) Mol. Cell. Biol. 8, 3088–3093. 51. Sellheyer, K., Bickenbach, J. R., Rothnagel, J. A., Bundman, D., Longley, M. A., Krieg, T., Roche, N. S., Roberts, A. B., and Roop, D. R. (1993) Proc. Natl. Acad. Sci. USA 90, 5237–5241. 52. Glick, A. B., Kulkarni, A. B., Tennenbaum, T., Hennings, H., Flanders, K. C., O‘Reilly, M., Sporn, M. B., Karlsson, S., and Yuspa, K. C. (1993). Proc. Natl. Acad. Sci. USA 90, 6076–6080. 53. Symington, F. W. (1989) J. Invest. Dermatol. 92, 798–805. 54. Grossman, R. M., Krueger, J., Yourish, D., Granelli-Piperno, A., Murphy, D. P., May, L. T., Kupper, T. S., Sehgal, P. B., and Gottlieb, A. B. (1989) Proc. Natl. Acad. Sci. USA 86, 6367– 6371. 55. Kanzaki, M., Nobusawa, R., Mogami, H., Yasuda, H., Kawamura, N., and Kojima, I. (1995) Mol. Cell. Endocrinol. 108, 11– 16. 56. Kawamura, N., Nobusawa, R., Mashima, H., Kanzaki, M., Shibata, H., and Kojima, I. (1995) Dig. Dis. Sci. 40, 2280–2285. 57. van der Kruijssen, C. M. M., Feijen, A., Huylebroeck, D., and van den Eijnden-van Raaij, A. J. M. (1993) Exp. Cell Res. 207, 407–412. 58. Clark, R. A. F. (1991) in Physiology, Biochemistry, and Molecular Biology of the Skin (Goldsmith, L. A., Ed.), pp. 576–601, Oxford Univ. Press, Oxford. 59. Dinarello, C. A. (1991) in The Cytokine Handbook (Thomson, A. W., Ed.), pp. 47–82, Academic Press, New York. 60. Leibovich, S. J., Polverini, P. J., Shepard, H. M., Wiseman, D. M., Shively, V., and Nuseir, M. (1987) Nature 329, 630–632. 61. Madtes, D. K., Raines, E. W., Sakariassen, K. S., Assoian, R. K., Sporn, M. B., Bell, G. I., and Ross, R. (1988) Cell 53, 285–293. 62. Tosato, G., Seamon, K. B., Goldman, N. D., Sehgal, P. B., May, L. T., Washington, G. C., Jones, K. D., and Pike S. E. (1988) Science 239, 502–504. 63. Feiken, E., Romer, J., Eriksen, J., and Lund, L. R. (1995) J. Invest. Dermatol. 105, 120–123. 64. Shimokado, K., Raines, E. W., Madtes, D. K., Barrett, T. B., Benditt, E. P., and Ross, R. (1985) Cell 43, 277–286. 65. Martinet, Y., Bittermann, P. B., Mornex, J.-F., Grotendorst, G. R., Martin, G. R., and Crystal, R. G. (1986) Nature 319, 158– 160. 66. Rappolee, D. A., Mark, D., Banda, M. J., and Werb, Z. (1988) Science 241, 708–712. 67. Rheinwald, J. G. (1980) Methods Cell. Biol. 21A, 229–254.
Received April 30, 1996 Revised version received July 2, 1996
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0305
Serum Growth Factors and Proinflammatory Cytokines Are Potent Inducers of Activin Expression in Cultured Fibroblasts and Keratinocytes GRISELDIS HU¨BNER
AND
SABINE WERNER1
Max-Planck-Institut fu¨r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany
Recently we demonstrated a large induction of activin expression in fibroblasts and keratinocytes after cutaneous injury in mice. To identify possible mediators of activin induction during skin repair, we have now analyzed the regulation of this factor in cultured keratinocytes and fibroblasts. Here we show that activin A mRNA and protein levels are low in quiescent keratinocytes and fibroblasts but expression is strongly induced upon serum treatment. The stimulatory effect of serum on activin expression is likely to be a combinatorial effect of different growth factors, since platelet-poor plasma serum and several purified serum growth factors also stimulated activin expression, although to a lesser extent than complete serum. Furthermore, we found increased expression of activin in keratinocytes and fibroblasts after addition of the proinflammatory cytokines interleukin 1b and tumor necrosis factor a. Taken together, our data suggest that serum growth factors which are released upon hemorrhage as well as proinflammatory cytokines derived from neutrophils and macrophages might be responsible for induction of activin expression after injury. q 1996 Academic Press, Inc.
INTRODUCTION
The activins belong to the transforming growth factor b (TGFb) superfamily [1] and were originally discovered as proteins of gonadal origin which regulate the release of follicle-stimulating hormone by pituitary cells [2, 3]. In the meantime, many biological activities of activin have been identified. Activin induces differentiation of hematopoietic cells [4, 5], osteoblasts [6], and endocrine cells [7, 8], and inhibits proliferation of gonadal cell lines [9], hepatocytes [10], endothelial cells [11], and lung epithelial cells [12]. Furthermore, activin modulates cell proliferation in fibroblasts [13] and vascular smooth muscle cells [14]. In 1 To whom reprint requests and correspondence should be addressed at Max-Planck-Institut fu¨r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany. Fax: 089-8578-2814.
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addition, activin constitutes an inhibitor of neuronal cell differentiation [15, 16] and plays a role in mesoderm induction during amphibian development [17–19]. Like the other members of the TGFb superfamily, activins consist of two disulfide-linked polypeptide chains. They are homo- or heterodimers of a bA- and/ or a bB-chain. Three different isoforms of activin have been identified: activin A (bAbA), activin B (bBbB), and activin AB (bAbB). Additional isoforms might arise from a putative bC-chain, which has recently been cloned from human cells [20]. Inhibins which antagonize the activin actions are heterodimers consisting of a common a-subunit and a b-subunit: inhibin A (abA) and inhibin B (abB) [21, 22]. Biological responses to activin could only be observed after dimerization of two different receptor types which form a heteromeric signaling complex [23–28]. Both types of activin receptors are transmembrane serine/threonine kinases: type I receptors (ARI and ARIB , also called activin receptor-like kinases Alk-2 and Alk-4, respectively) and type II receptors (ARII and ARIIB) [for review see 24]. A soluble activin binding protein, follistatin, has also been characterized. Its in vivo function is currently under debate, but it inhibits activin action in vitro [29–32]. The TGFbs have long been known to play a role in the skin and during wound healing. TGFb is an important differentiation factor for keratinocytes [33, 34]. It stimulates fibroblast proliferation and induces extracellular matrix production by epithelial, mesenchymal, and endothelial cells [for review, 35]. Furthermore, exogenous TGFb stimulates angiogenesis and wound reepithelialization [36–38]. By contrast, little is known about a possible function of the other family members during cutaneous wound repair. The phenotypes of recently generated transgenic mice suggested a role of activin in skin development. Mice missing the activin bA-chain lacked whiskers and whisker follicles were abnormal [39]. Mice deficient in follistatin showed disturbed whisker development and hyperkeratotic skin [40]. Apart from skin development, activin seems to play an important role in wound repair. Recently we demonstrated a large induction of activin but not inhibin mRNA expression within the first day after skin
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injury in mice [41]. Expression levels were high during the first 7 days and activin bA expression returned to the basal level 2 weeks after wounding, whereas activin bB expression was still high. Activin bA mRNA was found at highest levels in the granulation tissue below the wound and activin bB mRNA was particularly abundant in the hyperproliferative epithelium at the wound edge and in the migrating epithelial tongue. To identify possible mediators of the activin induction during skin repair, we have now analyzed the regulation of activin expression in cultured keratinocytes and fibroblasts. Our results suggest a role of serum growth factors in the early induction of activin expression after injury, whereas proinflammatory cytokines could be responsible for the prolonged activin expression at later stages of the wound healing process. MATERIALS AND METHODS Cell culture. The human keratinocyte cell line HaCaT [42], human primary embryonal fibroblasts, and a murine Balb/c 3T3 fibroblast cell line were used for cell culture experiments. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS) (human cells) or with 10% newborn calf serum (NCS) (Balb/c 3T3). For activin induction experiments they were grown to confluency without changing the medium. Cells were rendered quiescent by a 16-h incubation in serum-free DMEM (HaCaT) or in DMEM with 1% NCS (Balb/c 3T3). Confluent human embryonal fibroblasts were starved for 3 days in DMEM with 0.5% FCS followed by a 16-h incubation in serum-free DMEM. Cells were then incubated for varying periods in fresh DMEM containing serum, purified growth factors, or cytokines, harvested before and at different time points after treatment with these reagents, and used for RNA isolation. Each experiment was repeated at least twice. FCS, NCS, and DMEM were purchased from Gibco/BRL, complete adult human serum and platelet-poor plasma serum were obtained from a healthy adult volunteer and were prepared as described [43]. Growth factors and cytokines were from Boehringer Mannheim Biochemicals and cycloheximide was from Sigma Biochemicals. They were used in the following final concentrations: bFGF (10 ng/ml), EGF (20 ng/ml), IL-1b (100 U/ml), IL-6 (100 U/ml), KGF (10 ng/ml), PDGF-BB (10 ng/ml), TGFb1 (1 ng/ml), TNFa (300 U/ml), cycloheximide (10 mg/ml). DNA templates. RNase protection assay templates for murine activin/inhibin a-, bA-, bB-chains, murine activin receptors ARI (Alk2), ARIB (Alk-4), ARII, ARIIB , and murine follistatin were recently described [41]. The human activin bA template was generated by PCR using primers corresponding to the published human sequences. The amplified fragment was a 358-bp fragment corresponding to the 3*-end of the activin bA-chain cDNA [44]. RNA isolation and RNase protection analysis. Isolation of RNA was performed as described [45]. Twenty micrograms of total RNA from cultured cells was used for RNase protection analysis. RNase protection assays were carried out as recently described [46]. All experiments were repeated with a different set of RNAs from an independent cell culture experiment. Generation of activin-specific antibodies. A peptide corresponding to the carboxy-terminal region of activin A (CGGYYDDGQNIIKKDIQ) was synthesized, coupled to keyhole limpet hemocyanin (Sigma Biochemicals), and used for immunization of rabbits. Antisera were tested by immunoblotting for their reactivity with recombinant activin A as well as for cross-reactivity with recombinant TGFb1. Western blot analysis of activin proteins. Five milliliters of DMEM per 10-cm petri dish were conditioned by quiescent HaCaT
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cells or human embryonal fibroblasts in the presence or the absence of growth factors and cytokines. After addition of these factors, conditioned medium from three petri dishes was harvested at different time points and centrifuged for 10 min at 3000 rpm to remove cell debris. Proteins were precipitated with 6% trichloroacetic acid, washed with acetone, and resuspended in PBS. After addition of Laemmli sample buffer, proteins were denatured at 957C, separated on a 15% SDS–polyacrylamide gel, and transferred to nitrocellulose membranes. Activin proteins were detected using a 1:500 dilution of a polyclonal antiserum directed against the carboxy-terminal region of activin A (see above) and an alkaline phosphatase detection system (Promega). Each experiment was repeated at least twice.
RESULTS
We have recently demonstrated a large induction of activin expression after skin injury. Activin mRNA could be localized in epithelial and mesenchymal cells during wound healing. To identify the factors which could mediate this induction in the wound, we have studied the regulation of activin expression in cultured murine fibroblasts, human fibroblasts, and human keratinocytes. Because fibroblasts of the granulation tissue are the primary producers of activin bA mRNA in the wound tissue of mice [41], we first investigated the regulation of activin expression in a murine fibroblast cell line. Regulation of Activin mRNA Expression in Fibroblasts Induction of activin mRNA expression by serum. Since hemorrhage is one of the early events after skin injury, we first tested the potency of serum to stimulate activin expression in fibroblasts. As shown in Fig. 1A, activin bA mRNA could hardly be detected in confluent, quiescent murine Balb/c 3T3 fibroblasts. Upon addition of 10% newborn calf serum (NCS), a large induction of activin bA mRNA expression was observed. Maximal expression levels were reached between 2.5 and 5 h after stimulation with serum (Fig. 1A). Expression of activin bA mRNA dropped after 5 h and reached basal levels 24 h after serum addition. A similar induction of activin bA by serum was seen in human embryonal fibroblasts (data not shown). By contrast, no expression of the bB- and a-chains was found in Balb/c 3T3 murine fibroblasts (data not shown). To determine if the strong induction of activin bA expression in Balb/c 3T3 murine fibroblasts is the result of an additive action of different growth factors, the effect of complete adult human serum was compared to platelet-poor plasma serum (PPPS). As shown in Fig. 1B, complete human serum induced activin expression much more strongly than PPPS. Therefore, most of the factors which are responsible for activin A induction seem to be derived from platelets. To determine whether the induction of activin A mRNA expression requires de novo protein synthesis, the effect of cycloheximide was analyzed. Addition of
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FIG. 1. Induction of activin bA mRNA expression by serum in cultured murine Balb/c 3T3 fibroblasts. Induction of activin bA mRNA expression by newborn calf serum (NCS) (A), complete human serum (HS) (B), and platelet-poor plasma serum (PPPS) (B). Cells were rendered quiescent by serum starvation and stimulated with 10% serum for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 1000 cpm of the hybridization probe was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA was used as a negative control. The gels were exposed for 12 h. An ethidium bromide stain of 1 mg RNA from the same batch of RNA is shown below.
FIG. 2. Effect of serum on the expression of activin receptors and follistatin in cultured murine Balb/c 3T3 fibroblasts. Serum starved cells were treated with 10% newborn calf serum (NCS) for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin receptor and follistatin mRNA expression by RNase protection assays. 1000 cpm of the hybridization probes was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA were used as a negative control. The gels were exposed for 16 h (ARI, follistatin), 48 h (ARII, ARIB), or 4 days (ARIIB). The same set of RNAs was used for the protection assays of Figs. 1 and 2.
this protein synthesis inhibitor completely blocked the induction of activin expression by serum (data not shown), demonstrating that other proteins have to be synthesized before activin expression can occur. Serum dependent expression of activin receptors and follistatin. To investigate the mRNA expression of activin receptors and follistatin in Balb/c 3T3 fibroblasts upon treatment with serum, RNase protection assays were performed using the same RNA preparations as for expression analysis of activin a- and b-chains (Fig. 2). All known activin binding proteins were expressed in confluent, quiescent Balb/c 3T3 fibroblasts. Considering the different exposure times of the autoradiograms, we can conclude that ARI (Alk-2) and ARII were expressed at higher levels compared to ARIB (Alk-4), ARIIB , and follistatin. A slight induction of mRNA expression upon treatment with serum was observed for ARI, ARIB , and for follistatin. ARIIB was expressed at extremely low levels and expression even dropped upon serum treatment of the cells. Induction of activin A expression by purified serum growth factors. To identify the serum components which are responsible for the stimulatory effect on activin bA expression, we analyzed the effect of purified serum growth factors on Balb/c 3T3 fibroblasts. As shown in Fig. 3, transforming growth factor b1 (TGFb1) induced activin expression in a dose- and time-dependent manner. A dose of 1 ng/ml of TGFb1
was sufficient to elicit a maximal response. A first elevated signal was observed 2 h after treatment of the cells with this growth factor, and maximal induction was seen after 8 h. In addition to TGFb1, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) also stimulated activin bA expression in confluent, quies-
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FIG. 3. TGFb1 induces activin bA mRNA expression in cultured murine Balb/c 3T3 fibroblasts. Serum-starved cells were treated for 5 h with different concentrations of TGFb1 (left panel) and for different time periods with 1 ng/ml TGFb1 (right panel) as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 1000 cpm of the hybridization probe was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA was used as a negative control. The gels were exposed for 12 h.
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FIG. 5. Proinflammatory cytokines induce activin bA mRNA expression in cultured murine Balb/c 3T3 fibroblasts. Serum-starved cells were stimulated with IL-1b (100 U/ml), IL-6 (100 U/ml), and TNFa (300 U/ml) for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 50 mg tRNA was used as a negative control. The gels were exposed for 48 h.
FIG. 4. Synergistic effect of EGF and PDGF on activin bA mRNA expression in cultured murine Balb/c 3T3 fibroblasts. Serum-starved cells were stimulated with EGF (20 ng/ml) and PDGF (10 ng/ml) alone or together for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assay. 50 mg tRNA was used as a negative control. The gels were exposed for 24 h.
cent Balb/c 3T3 fibroblasts (Fig. 4 and Table 1). Interestingly, when cells were treated with a combination of EGF and PDGF, an additive effect on activin expression was seen. However, the observed signal did not reach the intensity of the signal obtained after treatment of the cells with serum. This provides further evidence for a combinatorial effect of several different growth factors in the regulation of activin expression. Similar to Balb/c 3T3 fibroblasts, serum growth factors also induced activin A expression in primary human embryonal fibroblasts (Table 1).
Activin A expression is stimulated by proinflammatory cytokines. Recently we demonstrated expression of proinflammatory cytokines in polymorphonuclear leukocytes and macrophages after skin injury [47]. To determine a possible role of these factors for activin regulation, we analyzed their effect on activin expression in vitro. As shown in Fig. 5, interleukin 1b (IL1b), tumor necrosis factor a (TNFa), and interleukin 6 (IL-6) acted as inducers of activin expression in confluent, quiescent murine 3T3 fibroblasts. Activin A signals appeared 5 h after treatment of the cells with these cytokines and were still high after 8 h. Of all three cytokines, TNFa induced the strongest response. However, none of these cytokines was as potent as complete serum. As is shown in Table 1, a similar induction of activin bA expression by proinflammatory cytokines occurred in primary human embryonal fibroblasts. Regulation of Activin mRNA Expression in Keratinocytes
TABLE 1 Induction of Activin A by Growth Factors and Cytokines
FCS/NCS TGFb1 KGF bFGF EGF PDGF EGF / PDGF IL-1b TNFa IL-6
Balb/c fibroblasts
Human embryonal fibroblasts
HaCaT keratinocytes
///// //// n.d. /// /// /// ///// /// //// //
/// /// n.d. n.d. // // //// /// //// ///
//// / / n.d. /// 0 n.d. / /// 0
Note. bFGF (basic fibroblast growth factor), EGF (epidermal growth factor), FCS (fetal calf serum), IL-1b (interleukin 1-b), IL-6 (interleukin 6), KGF (keratinocyte growth factor), NCS (new born calf serum), PDGF (platelet-derived growth factor), TGFb1 (transforming growth factor b1), TNFa (tumor necrosis factor a), n.d. (not done).
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Induction of activin mRNA expression by serum growth factors and proinflammatory cytokines. Although fibroblasts are the major source for activin A in the wound [41], low levels of activin A mRNA were also found in the epidermis of mouse tail skin (G.H., unpublished data). Therefore, the regulation of activin bA expression was also investigated in the human keratinocyte cell line HaCaT. As shown in Fig. 6A, no expression of activin A mRNA was found in confluent, quiescent HaCaT cells. Within 5 h after addition of fetal calf serum (FCS), activin expression was strongly induced, but declined after 8 h. The activin induction by serum growth factors in HaCaT cells was long lasting and increased levels of activin mRNA were still observed 24 h after treatment of the cells. In contrast to the bA-chain, activin bB expression could not be detected in quiescent or serum-stimulated HaCaT cells (data not shown). EGF was an even more potent inducer of activin bA expression than serum (Fig. 6A). Already 2 h after
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FIG. 6. Serum, purified growth factors, and proinflammatory cytokines stimulate activin bA mRNA expression in cultured HaCaT keratinocytes. (A) Serum (10% fetal calf serum), EGF (20 ng/ml), KGF (10 ng/ml), and (B) TGFb1 (1 ng/ml) and TNFa (300 U/ml) induce activin bA mRNA expression. Cells were rendered quiescent by serum starvation and stimulated with serum, growth factors, and cytokines for different time periods as indicated. 20 mg total cellular RNA from these cells was analyzed for activin bA mRNA expression by RNase protection assays. 1000 cpm of the hybridization probes was added to the lanes labeled ‘‘probe.’’ 50 mg tRNA was used as a negative control. The gels were exposed for 24 h.
treatment of the cells with this growth factor, the first elevated bA signal was detected. Furthermore, EGF caused a more prolonged expression of activin bA and— in contrast to serum—maximal levels of bA mRNA were still observed 8 h after addition of EGF. Keratinocyte growth factor (KGF), which is produced by fibroblasts and acts as a strong mitogen for keratinocytes [48], also induced activin bA mRNA expression. Interestingly, there was a large difference in activin bA expression after treatment of keratinocytes with EGF and KGF. Although both growth factors are potent mitogens for keratinocytes, KGF caused a much weaker induction than EGF. In contrast to EGF and KGF, the platelet-derived mitogen PDGF had no effect on activin expression. To determine a possible correlation between activin induction and stimulation of cell proliferation, we analyzed the effect of TGFb1 on keratinocytes. This factor is a major differentiation factor for keratinocytes [33, 34], but inhibits keratinocyte proliferation in vitro and in vivo [49–52]. As shown in Fig. 6B, TGFb1 stimulated activin expression in HaCaT cells (Fig. 6B), although it was a weaker inducer than EGF. Thus, induction of activin expression is not dependent on the mitogenic activity of a growth factor. Similar as in fibroblasts, the proinflammatory cytokines TNFa and to a lesser extent IL-1b also induced activin expression in HaCaT cells (Fig. 6B and Table 1). Interestingly, TNFa, which inhibits keratinocyte growth in vitro [53], caused a different kinetics of activin expression in HaCaT keratinocytes than the other factors. TNFa already caused maximal induction of activin expression 2 h after treatment of the cells and
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transcript levels were still high after 8 h. By contrast, IL-6 which acts as mitogen for keratinocytes [54] had no effect on activin A expression in these cells (data not shown). The effects of all tested factors on activin expression in fibroblasts and keratinocytes are summarized in Table 1. Induction of Activin mRNA Expression Correlates with Protein Secretion into the Culture Medium To determine if the induction of activin bA mRNA expression is followed by synthesis and secretion of immunoreactive protein, total protein was precipitated from conditioned culture medium at different time points after treatment of the cells with serum growth factors or cytokines. The presence of activin was then determined by Western blot analysis using an activinspecific antiserum which does not cross-react with TGFb1. Activin A protein could be detected in the medium within 6–24 h after treatment of confluent, quiescent HaCaT keratinocytes and human embryonal fibroblasts with serum growth factors and cytokines (Figs. 7A–7C). The additional bands of higher molecular weight are likely to be a result of nonspecific crossreactivity of the antiserum, since they are also present in the control lanes. These data demonstrate that bA mRNA expression correlates with protein synthesis and subsequent secretion of mature activin into the cell culture medium. DISCUSSION
Recently, we demonstrated a large induction of activin expression after cutaneous injury in mice [41].
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FIG. 7. TNFa and EGF induce activin A protein expression and secretion in fibroblasts and keratinocytes. HaCaT keratinocytes (A, B) and human embryonal fibroblasts (C) were rendered quiescent by serum starvation and stimulated with TNFa (300 U/ml) or EGF (20 ng/ml) for different time periods as indicated. Proteins were precipitated from 6 ml of conditioned medium with 6% trichloroacetic acid and analyzed by immunoblotting for the presence of activin A protein. 10 ng of recombinant activin A or TGFb1 was used as control. The upper band in the activin lane represents full-length recombinant activin, whereas the lower band is likely to be a degradation product.
Fibroblasts in the granulation tissue were the major source for activin bA mRNA, but low levels were also detected in the epidermis. To identify the factors which might be responsible for the activin induction during wound repair, we analyzed the effect of growth factors and cytokines on activin expression in cultured fibroblasts and keratinocytes. Because activin induction occurs very early after injury (during the first 15 to 24 h), serum growth factors derived from the blood upon hemorrhage were likely candidates for activin induction. Here we show that activin bA expression is very low in cultured quiescent fibroblasts and keratinocytes, but is strongly induced upon stimulation with serum or purified serum growth factors such as TGFb1, EGF, or PDGF, whereby the increase in activin bA mRNA correlates with secretion of immunoreactive activin A protein into the culture
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medium. Since the observed induction of activin bA expression by platelet-poor plasma serum and purified growth factors is lower compared to the effect seen with complete serum, we presume that the strong activin induction is a combinatorial effect of many different growth factors. Indeed, a combination of PDGF and EGF caused a much stonger induction than a single factor. Induction of activin by serum growth factors has also been demonstrated for other cell types, such as smooth muscle cells [55], intestinal epithelial cells [56], capillary endothelial cells [11], and differentiated cell lines derived from P19 embryonal carcinoma cells [57]. Since activin bB mRNA and the inhibin a-chain were not expressed in Balb/c 3T3 fibroblasts, only activin A can be produced by these cells. In addition to activin, all known activin binding proteins were expressed in confluent, quiescent Balb/c 3T3 fibroblasts, suggesting autocrine effects of activin in these cells. Similar to the in vivo situation, ARI and ARII were significantly more abundant than ARIB and ARIIB in Balb/c 3T3 fibroblasts. In contrast to activin, expression of the receptors was not induced upon serum stimulation. A lack of activin receptor induction was also observed during wound healing [41], demonstrating that the wounding response is mediated via the ligand but not via the receptor. Surprisingly we could not detect bB mRNA in quiescent or serum-treated HaCaT cells. This seems to be contradictory to the in vivo situation, where bB mRNA was found at high levels in the hyperproliferative epithelium at the wound edge in mice [41]. However, data from in situ hybridization and immunhistochemistry (G.H., unpublished data) revealed the presence of bB mRNA and activin protein exclusively in differentiated keratinocytes in the suprabasal layers, suggesting that keratinocytes must first receive a differentiation signal before activin induction can occur. Such a signal might be specific for the in vivo situation. Alternatively, the lack of bB mRNA expression might be due to speciesspecific differences between human and mouse keratinocytes. Another early event in wound repair is the infiltration of the wound area by polymorphonuclear leukocytes as a first defense against bacterial invasion [58]. Recently, we could demonstrate a strong expression of proinflammatory cytokines by these cells in mouse excisional skin wounds as soon as 12 h after injury [47]. The migration of macrophages into the wound area occurs approximately 24 h after injury. Like neutrophils, activated macrophages also produce proinflammatory cytokines [59–62], whereby the temporal and spatial expression pattern of these factors correlates with activin expression in the wound [41, 47]. Therefore, we analyzed the effect of proinflammatory cytokines on activin expression in cultured fibroblasts and keratinocytes. Interestingly, we also found a significant increase in activin A mRNA and protein by
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these cytokines. The strongest inducer was TNFa which caused a significantly faster induction of activin expression in HaCaT keratinocytes compared to the other factors, suggesting that activin induction is a direct effect of this cytokine. Indeed, preliminary data from our laboratory revealed an inhibitory effect of cycloheximide on activin induction after EGF treatment, whereas activin expression was superinduced by treatment of the cells with both cycloheximide and TNFa. TNFa is produced both by neutrophils and macrophages [47, 63]. In contrast to neutrophils, macrophages stay in the wound area beyond the initial inflammatory phase and produce cytokines until the healing process is completed. Furthermore, they are an important source of PDGF [64–66], another potent inducer of activin expression in fibroblasts. Therefore, macrophages could be responsible for the prolonged activin expression at later stages of the healing process. Apart from serum growth factors and cytokines, KGF is another growth factor which is highly abundant in the healing wound [46]. KGF is produced by fibroblasts and—like EGF—is a strong mitogen for keratinocytes [42, 48, 67]. Interestingly, KGF stimulated activin expression only weakly, whereas EGF was the most potent activator of activin bA expression in HaCaT keratinocytes. This finding suggests, that EGF and KGF activate different intracellular signaling pathways, resulting in different degrees of activin bA induction. Differential effects of EGF and KGF on gene expression in keratinocytes seems to be a more general feature, since we recently identified novel KGF-regulated genes which do not respond to EGF (S. Frank, S. Werner, unpublished data). In conclusion, we have identified several positive regulators of activin expression in fibroblasts and keratinocytes. Since these growth factors and cytokines are present in the wound at early and later stages of the repair process, they might also be responsible for the strong activin expression during wound healing in vivo. Growth factors derived from serum upon hemorrhage and proinflammatory cytokines secreted by neutrophils are likely to initiate the large induction of activin expression after skin injury, whereas cytokines derived from macrophages could be responsible for the prolonged expression at later stages of the repair process. These results provide further evidence for a role of activin in the wound healing scenario. Although the function of activin in the repair process is currently unknown, preliminary data from in vitro studies suggest a role of this factor in extracellular matrix production and keratinocyte differentiation (G.H., unpublished data). Future studies using transgenic animal models will help to clarify the role of activin in tissue repair. We thank Dr. P. H. Hofschneider for support, Irene Dick for help
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with the tissue culture experiments, Dr. D. Huylebroeck, University of Leuven, for the Alk-2 cDNA, and Dr. A. J. M. van den Eijndenvan Raaij, Hubrecht Laboratory, Utrecht, for follistatin and inhibin cDNAs. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (WE 1983/1-2). G. H. is a recipient of a predoctoral fellowship from the Boehringer Ingelheim foundation. S. W. is a Hermann-and-Lilly-Schilling Professor of Medical Research.
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Received April 30, 1996 Revised version received July 2, 1996
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