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Activin A Induces Terminal Differentiation of Cultured Human Keratinocytes
Activin A Induces Terminal Differentiation of Cultured Human Keratinocytes Mariko Seishima,*† Mari Nojiri,* Chikako Esaki,* Kozo Yoneda,‡ Yuzuru Eto,§ and Yasuo Kitajima* *Department of Dermatology, Gifu University School of Medicine, Gifu, Japan; †Department of Dermatology, Ogaki Municipal Hospital, Ogaki City, Japan; ‡Department of Dermatology, Akita University School of Medicine, Akita, Japan; §Central Research Laboratories, Ajinomoto, Kawasaki-ku, Kawasaki, Japan
Activin A, a member of the TGFβ-superfamily, is well known to play important roles in the growth and differentiation of various target cells. We have previously demonstrated that activin A is produced at an early stage of cultivation at both the protein and the mRNA levels in cultured human keratinocytes. In this study, the effects of activin A on differentiation and proliferation of human keratinocytes were examined. Activin A (^1 nM) induced cornified envelope formation and the synthesis of loricrin, keratin 1, involucrin, and transglutaminase 1. In addition, transglutaminase
activity and mRNA level of transglutaminase 1 were increased by activin A. [3H]Thymidine incorporation and cell number were reduced by activin A (^1 nM) compared with control, suggesting an inhibitory effect of activin A on cell proliferation. On the basis of these findings, it is likely that activin A contributes to differentiation and suppression of proliferation in human keratinocytes. Key words: loricrin/proliferation/ transforming growth factor β-superfamily/transglutaminase 1. J Invest Dermatol 112:432–436, 1999
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development and hyperkeratotic skin (Matzuk et al, 1995b). These findings suggest that activin A plays an important role in keratinocyte functions; however, the role of activins in keratinocytes has not been fully determined. The aim of this study was to investigate the effects of activin A on proliferation and differentiation in cultured human keratinocytes.
ctivin A, a member of the TGFβ-superfamily that was originally purified from ovarian fluids, stimulates secretion of follicle-stimulating hormone from the anterior pituitary gland (Ling et al, 1986; Vale et al, 1986). Erythroid differentiation factor was independently isolated from human monocytic leukemia cells (Eto et al, 1987). Molecular cloning of cDNA has shown that activin A and erythroid differentiation factor are identical. Activins have been identified as homo- or heterodimers of the βA and βB chain and have been classified into three types: activin A (βAβA), activin B (βBβB), and activin AB (βAβB). Activins induce differentiation of hematopoietic cells (Eto et al, 1987), osteoblasts (Ogawa et al, 1992), and endocrine cells (Sugino et al, 1988), and inhibit proliferation of gonadal cell lines (Gonzalez-Manchon and Vale, 1989), hepatocytes (Yasuda et al, 1993), and endothelial cells (McCarthy and Bicknell, 1993). We have previously shown that mRNA of activin A (βA), but not βB, is detected in normal human epidermis and in keratinocytes at an early culture stage (Seishima et al, 1996). In contrast to our results, Roberts et al showed that activin βA is expressed in the developing hair bulb and also in the dermal layer of embryonic skin, but not in adult skin (Roberts et al, 1991). It has been reported that activin A expression is enhanced in keratinocytes by cutaneous injury (Hu¨bner et al, 1996) and by the addition of interleukin 1β, tumor necrosis factor α, or serum (Hu¨bner and Werner, 1996). On the other hand, mice lacking the activin βA-chain in whiskers and whisker follicles are abnormal (Matzuk et al, 1995a). Furthermore, mice deficient in follistatin, an activin binding protein, show disturbed whisker
MATERIALS AND METHODS Cell culture and cell number count Normal human keratinocytes from foreskin (Kurabo, Osaka, Japan) were grown in Eagle’s minimum essential medium (with 0.09 mM Ca2) supplemented with antibiotics (100 units penicillin per ml, 100 mg streptomycin per ml) and 10% fetal calf serum (HyClone Laboratories, Logan, UT) on 60 mm dishes (Corning Plastics) at 37°C with 5% CO2/95% air in a humidified incubator. To adjust the Ca21 level in the medium to 0.09 mM, fetal calf serum treated with 8% Chelex 100 (Bio-Rad Laboratories, Richmond, CA) and 0.09 mM Ca2 were added to Ca21 and Mg21 free-minimum essential medium (Whittaker Bioproducts, Walkerville, MD) (Li et al, 1996). Cells were used after one passage and the medium was changed every 2–3 d. After reaching confluence, the cells were treated with Hank’s balanced salt solution containing 0.02% trypsin and 0.02% ethylenediamine tetraacetic acid for 15 min at 37°C, and were then resuspended in the culture medium. The cells were seeded onto 35 mm dishes at a density of 13105 per ml of culture medium in the presence of 0.09 mM (low) or 1.8 mM (normal concentration) of calcium. After incubation with activin A at different concentrations (0, 0.1, 1, 10, or 100 nM) for 24 h, the cells were washed with phosphate-buffered saline (PBS) and were dissociated by treatment with 0.25% trypsin and 0.1% ethylenediamine tetraacetic acid solution at 37°C for 10 min. After centrifugation at 1000 3 g for 10 min, the cells were resuspended in 10 mM Tris-HCl buffer, pH 7.5 and then the cell number was counted under a microscope. Subconfluent and monolayer culture was observed after 36 h incubation without activin A.
Manuscript received November 19, 1998; revised December 18, 1998; accepted for publication December 22, 1998. Reprint requests to: Dr. Mariko Seishima, Department of Dermatology, Ogaki Municipal Hospital, Minaminokawa-cho 4-86, Ogaki City, Japan 503-8502.
Measurement of DNA synthesis DNA synthesis was assessed by measuring the incorporation of [3H]thymidine. After keratinocytes were cultured with activin A at different concentrations for 24 h, the cells were labeled with 1.0 µCi [3H]thymidine per ml for 4 h. Cultures were washed
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three times with PBS and then lyzed with 2N NaOH at 37°C for 20 min and neutralized with 2N HCl. Acid-insoluble material was precipitated by addition of four volumes of ice-cold trichloroacetic acid and was collected on GF/A glass-fiber filters (Whatman, Ann Arbor, MI). After washing three times with 10% trichloroacetic acid and once with ethanol, the filters were dried at room temperature for measurement of radioactivity with a liquid scintillation counter (Beckman LS 7500). Transglutaminase activity assay Tranglutaminase (TGase) activity was assayed by measuring the formation of ε-amino-γ-glutamyl bonds between [3H]putrescine and casein (Yuspa et al, 1980; Seishima et al, 1993). After incubation with 0.1, 1, 10, or 100 nM of activin A for 24 h, cells were scraped off and sonicated in 10 mM Tris HCl buffer (pH 7.4) containing 10 mM dithreitol, 0.5 mM ethylenediamine tetraacetic acid, and 0.1% Triton X-100. The homogenates were centrifuged at 105,000 3 g for 60 min and the resultant supernatants were used for the TGase activity assay. One hundred microliters of cell lysate was incubated with 100 liters of 50 mM Tris-HCl buffer (pH 7.4) containing casein (20 g per ml) and [3H]putrescine (5 mM). After incubation for 30 min at 37°C, the reactions were terminated by adding the same volume of ice-cold 10% trichloroacetic acid and the mixture was kept on ice for 30 min. The acid-insoluble material was collected on GF/A glass-fiber filters. After three washes with 5 ml of 5% ice-cold trichloroacetic acid containing 1% putrescine, the filters were dried at room temperature. The dried filters were mixed with scintillation fluid and the radioactivity was determined with a liquid scintillation counter. Scoring of cornified envelopes The scoring of cornified envelope formation was performed essentially as described by Sun and Green (1976). The cells incubated with activin A at various concentrations for 24 h were washed with PBS, and were dissociated by treatment with 0.25% trypsin and 0.1% ethylenediamine tetraacetic acid solution at 37°C for 10 min. After centrifugation at 1000 3 g for 10 min, the cells were resuspended in 10 mM Tris-HCl buffer, pH 7.5, containing 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol at a density of 1–2 3 106 cells per ml. After the cell suspensions had been kept at room temperature for 10 min, sodium dodecyl sulfate- and mercaptoethanol-resistant cornified envelopes were counted under a microscope. Western blot analysis Normal keratinocytes were seeded on 60 mm dishes at a density of 1 3 105 cells per 5 ml of culture medium in the presence of 0.09 mM or 1.8 mM calcium. Because TGase activity and cornified envelope formation were increased by activin A at the concentration of 1 nM or more and TGase activity in human keratinocytes is mostly due to TGase 1 (Kim et al, 1995a), 1 nM of activin A was used in this study to examine the effects of activin A on the synthesis of TGase 1, loricrin, keratin 1, and involucrin. Cells incubated with activin A for 24 h were washed twice with PBS, and harvested by PBS with 1 mM phenylmethylsulfonyl fluoride (Molecular Probes, Eugene, OR), 1 mM N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, Research Organics, Cleveland, OH), and 5 mM E64 (Enzyme System Products, Dublin, CA). We performed western blot analysis using monoclonal antibodies against human TGase 1 (Kim et al, 1995b), loricrin (Yoneda and Steinert, 1993), keratin 1 (34βB4, Enzo Diagnotics, Farmingdale, NY), and rabbit polyclonal antibody to involucrin (BT601, Biomedical Technologies, Stoughton, MA). Aliquots were mixed with sample buffer (10% 2-β-mercaptoethanol, 2% sodium dodecyl sulfate, 30% glycerol, 0.25 M Tris, final concentration), followed by heating at 98°C for 3 min. Thirty micrograms of protein was loaded per lane and reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 10% polyacrylamide gel for TGase 1, 20% gel for loricrin, and 8% gel for involucrin and keratin 1 by the method of Laemmli (1970). Protein standards for molecular weight (Bio Rad) were as follows: ovalbumin, 49 kDa; bovine serum albumin, 80 kDa; βgalactosidase, 116.5 kDa; and myosin, 205 kDa. Proteins were electroblotted onto Hybond-ECL (Amersham, Bucks, U.K.) using a semi-dry transfer apparatus (Bio Rad) for 30 min at 10 V. The membranes were preincubated with a blocking solution containing 5% skim milk and 0.2% Tween 20 in PBS for 1 h at room temperature. Membranes were then incubated overnight at 4°C with diluted primary antibodies. Following incubation with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (0.2 µl per ml) in the blocking solution for 2 h, membranes were washed and bound antibodies were detected by using ECL detection reagents (Amersham) and exposure to Hyperfilm. Northern blot analysis After keratinocytes were incubated without or with 1 nM of activin A for 12 h, total RNA was extracted from the cells with a buffered solution containing 4 M guanidium thiocyanate according
Figure 1. Suppression of keratinocyte proliferation by activin A. Dose-dependent effects of activin A on cell number (A, B) and [3H]thymidine incorporation (C, D) in low (A, C) or normal calcium (B, D) media are shown. After incubation with activin A at 1–100 nM for 24 h, the number of keratinocytes was markedly decreased compared with control under both low (A) and normal (B) calcium conditions. [3H]Thymidine incorporation was significantly reduced by exposure for 24 h to 1, 10, or 100 nM of activin A in both low (C) and normal (D) calcium conditions. Values are means of triplicate determinations from three separate experiments. Error bars: 1 SD. *p , 0.05, **p , 0.005, ***p , 0.001. to the method of Chomzynski and Sacchi (1987). The TGase 1 cDNA was labeled with α-32P-dCTP using a multiprime DNA labeling system, and hybridization was performed at 37°C for 15 h. After hybridization, the filters were washed and subjected to autoradiography. Data analysis Data are expressed as mean 6 1 SD, and statistical differences were evaluated using Student’s t test. p values less than 0.05 were taken as significant.
RESULTS Suppression of keratinocyte proliferation by activin A Cell numbers were counted to evaluate the effect of activin A on keratinocyte proliferation. Cell numbers as well as [3H]thymidine incorporation in the low calcium medium were higher than those in the normal calcium medium when activin A was not added. After incubation with activin A at 1–100 nM for 24 h, the number of keratinocytes was markedly decreased compared with control under both low and normal calcium conditions (Fig 1). Cell proliferation was decreased with time by 1 nM of activin A for up to 36 h (Fig 2). Further incubation in the medium without activin A (for 36 h) after 36 h incubation with 1 nM of activin A did not induce recovery of cell growth. In addition, [3H]thymidine incorporation was measured to examine the effect of activin A on DNA synthesis. DNA synthesis was significantly reduced by activin A at the concentrations of 1, 10, and 100 nM for 24 h incubation in either low or normal Ca21 concentration (Fig 1). Moreover, [3H]thymidine incorporation in cells incubated with 1 nM of activin A (9.83 6 0.93 3 10–4 cpm per 105 cells, n 5 3) for 24 h was restored to the basal level by pretreatment with 3 nM of
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Figure 2. Time-dependently reduced keratinocyte proliferation by activin A until 36 h. Time-dependent effects of activin A (1 nM) on cell number of human keratinocytes cultured in low calcium media are shown. Values are means of triplicate determinations from three separate experiments. Error bars: SD.
follistatin, an activin binding protein, for 6 h under low calcium conditions (13.1 6 0.98 3 10–4 cpm per 105 cells, n 5 3, p , 0.001); however, low activin A concentration (0.1, 0.05, and 0.01 nM) did not affect either the cell numbers or the [3H]thymidine incorporation. Pretreatment with 0.3 nM of follistatin similarly did not affect cell number and [3H]thymidine incorporation at the low concentrations of activin A. Keratinocyte differentiation induced by activin A The effects of various concentrations of activin A on differentiation were examined by monitoring cornified envelope formation. Cornified envelope formation in 1, 10, or 100 nM of activin A-treated cells was significantly increased compared with control, but no change was observed in 0.1 nM of activin A (Fig 3). Because TGase 1 is a key enzyme for keratinocyte differentiation, we examined the effects of activin A on TGase activity, TGase 1 mRNA level, and TGase 1 synthesis. TGase activity was increased at 12 h, and reached a plateau at 36 h (Fig 4), in cells treated or not treated with activin A (1 nM). TGase activity was significantly increased by activin A at more than 1 nM compared with control at 24 h, but no increase was observed in low concentration of activin A (0.1 nM) (Fig 3). In addition, 1 nM of activin A-induced TGase activity at 24 h was significanty suppressed to the basal level by pretreatment with 3 nM of follistatin for 6 h in normal calcium medium (44.4 6 3.75 vs 21.9 6 3.60 3 10–3 cpm per µg prot. per 30 min, n 5 3, p , 0.005). The expression of TGase 1 mRNA and the synthesis of TGase, loricrin, keratin 1, and involucrin were enhanced in the normal calcium condition as compared with low calcium (Fig 5). Because the TGase activity and cornified envelope formation were increased by activin A at concentrations of 1 nM or more (Fig 3), 1 nM of activin A was employed to examine the effects of activin A on the expression of these differentiation markers. The synthesis of TGase 1, loricrin, keratin 1, and involucrin was enhanced by 1 nM of activin A under both normal and low calcium conditions (Fig 5A, C–E, respectively). The TGase 1 mRNA level examined by northern blot analysis was obviously increased by treatment with 1 nM of activin A for 12 h under both normal and low calcium conditions (Fig 5B).
Figure 3. Keratinocyte differentiation induced by 1–100 nM of activin A. Dose-dependent effects of activin A on cornified envelope formation (CE) (A, B) and transglutaminase (TGase) activity (C, D) in low (A, C) or normal calcium (B, D) media are shown. Cornified envelope formation and TGase activity were significantly increased by incubation with activin A at 1 nM or more for 24 h. Values are means of triplicate determinations from three separate experiments. Error bars: SD. *p , 0.05, **p , 0.001.
DISCUSSION In this study, two different calcium conditions, 0.09 mM (low concentration) and 1.8 mM (normal concentration), were employed for culture, because it is well known that a low concentration of calcium induces proliferation, and normal calcium induces differentiation in keratinocytes (Henning and Holbrook, 1983; Yuspa et al, 1989). As expected, the cell number and [3H]thymidine incorporation in the low calcium medium were significantly higher than those in normal calcium, and the expression of keratinocyte differentiation markers such as cornified envelope formation and TGase activity in the low calcium medium were lower than those in normal calcium, irrespective of the presence of activin A. The addition of activin A at different concentrations ranging from 1 to 100 nM inhibited the increase in the cell number and [3H]thymidine incorporation dose-dependently under both calcium conditions, indicating that activin A inhibits cell proliferation. Cornified envelope, which is formed beneath the plasma membrane and is resistant to detergents and reducing agents, is characteristic of the terminal differentiation of keratinocytes. TGase 1, a key enzyme for keratinocyte differentiation, is a Ca21-requiring enzyme that catalyses the cross-linking of membrane proteins to form cornified envelopes. The effect of various concentrations of activin A on the differentiation of cultured keratinocytes was examined by monitoring cornified envelope formation and TGase activity. Both markers were enhanced in a dose-dependent manner. This enhancement in TGase activity is mostly due to the increase in TGase 1 activity, because it is now known that the TGase activity
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in human keratinocytes is mostly due to TGase 1 (Kim et al, 1995a). Although Hohl et al (1991) showed that Ca21 concentrations above 0.1 mM were permissive for the expression of loricrin mRNA, our data clearly demonstrate that activin A even in low Ca21 contributes to the terminal differentiation of cultured human keratinocytes. In addition, the expression of TGase 1 mRNA, and the synthesis of TGase, loricrin, keratin 1, and involucrin were enhanced with even 1 nM of activin A. These findings suggest that activin A induces early and terminal differentiation of keratinocytes at concentrations of 1 nM or more.
Figure 4. Time-dependent increase in TGase activity by 1 nM of activin A. TGase activity was increased at 12 h, and reached plateau at 36 h in human keratinocytes cultured in normal calcium media. Values are means of triplicate determinations from three separate experiments. Error bars: 1 SD.
Figure 5. Enhanced expression of TGase 1, loricrin, keratin 1, and involcrin and increased TGase 1 mRNA level by 1 nM of activin A. The synthesis of TGase 1 (A), loricrin (C), keratin 1 (D), and involucrin (E) was enhanced by the incubation with 1 nM of activin A for 24 h in both low and normal calcium conditions. The TGase 1 mRNA expression (B) was also enhanced by the incubation with activin A (1 nM) for 12 h. Similar results were obtained in three independent experiments. Molecular weight markers (KD) are indicated in (A) (left). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as a control in northern blot analysis (B).
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In other cells, activin A inhibits neural differentiation (Hashimoto et al, 1990) and stimulates glucose production in hepatocytes (Mine et al, 1989) at 1 nM, modulates osteoblast proliferation and differentiation at 10 nM (Hashimoto et al, 1992), inhibits the growth of mammary epithelial cells at 100 nM, and induces erythroid differentiation at 1 µM. On the other hand, low concentration (0.01 nM) of activin A stimulates the differentiation of human lung fibroblasts into myofibroblasts and proliferation of fibroblasts (Ohga et al, 1996). This different responsiveness to activin A might be due to differences in cell types, quantities, or binding activities of activin receptors or activin inhibitors. Activin receptors are transmembrane serine/threonine kinases, among them type I receptors (ARI and ARIB) and type II receptors (ARII and ARIIB) (Ca´rcamo et al, 1994; Lebrun and Vale, 1997). Follistatin is an activin binding protein exerting a neutralizing effect on activin activity (Nakamura et al, 1990). Mad proteins function downstream of the activin receptor pathway (Chen et al, 1996). The endogenous activin A activity in cultured DJM-1 cells, a squamous cell carcinoma line, corresponded to the activity generated by 0.32 nM of recombinant activin A. No activin was detected in normal human keratinocytes (less than 0.02 nM) (Seishima et al, 1996). Activins are reported to be secreted in an autocrine or paracrine manner (Shiozaki et al, 1992). Therefore, the effect of activin A was compared with control, which lacked exogenous activin A. We have previously demonstrated that activin A mRNA (βA) is detected at an early stage of keratinocyte culture and that activin A is observed at the wound edge in an in vitro tissue injury model, where cell migration and proliferation are activated without terminal differentiation (Seishima et al, 1996). The induction of activin A expression was also found in wounded skin, especially granulation tissue below the eschar, within 7 d of skin injury, suggesting that activin A plays an important role in tissue repair (Hu¨bner et al, 1996). These observations appear to be in disagreement with our results. Namely, our previous results suggest that activin A acts as a potent inducer of proliferation, but this study indicates that activin A is an inhibitor of proliferation and a stimulator of differentiation. This apparent discrepancy can be explained as follows: activin A may upregulate proliferation and stimulate migration at the wound edge or early stage of cultivation, but it may downregulate
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proliferation and stimulate differentiation at higher concentrations given exogenously. In other words, activin A might be an initial modulator of differentiation in keratinocytes. In conclusion, activin A appears to play a role in the proliferation and differentiation of cultured human keratinocytes. Further in vivo studies on the precise regulation mechanisms for proliferation and differentiation by activin A, including the signaling pathway and nuclear responses, are needed to elucidate the roles of activin A in epidermis. REFERENCES Ca´rcamo J, Weis FMB, Ventura F, Wieser R, Wrana JL, Attisano L, Massague´ J: Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor β and activin. Mol Cell Biol 14:3810–3821, 1994 Chen Y, Lebrun J-J, Vale W: Regulation of transforming growth factor β- and activin- induced transcription by mammalian Mad proteins. Proc Natl Acad Sci USA 93:12992–12997, 1996 Chomzynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159, 1987 Eto Y, Tsuji T, Takezawa M, Takano S, Yokogawa T, Shibai H: Purification and characterization of erythroid differentiation factor isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095–1103, 1987 Gonzalez-Manchon C, Vale W: Activin-A, inhibin and transforming growth factorβ modulate growth of two gonadal cell lines. Endocrinol 125:1666–1672, 1989 Hashimoto M, Kondo S, Sakurai T, Etoh Y, Shibai H, Muramatsu M: Activin/EDF as an inhibitor of neural differentiation. Biochem Biophys Res Commun 173:193– 200, 1990 Hashimoto M, Shoda A, Inoue S, et al: Functional regulation of osteoblastic cells by the interaction of activin A with follistatin. J Biol Chem 267:4999–5004, 1992 Hennings H, Holbrook KA: Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. Exp Cell Res 143:127–142, 1983 Hohl D, Lichti U, Breitkreutz D, Steinert PM, Roop DR: Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid. J Invest Dermatol 96:414–418, 1991 Hu¨bner G, Werner S: Serum growth factors and proinflammatory cytokines are potent inducers of activin expression in cultured fibroblasts and keratinocytes. Exp Cell Res 228:106–113, 1996 Hu¨bner G, Hu Q, Smola H, Werner S: Strong induction of activin expression after injury suggests an important role of activin in wound repair. Dev Biol 173:490– 498, 1996 Kim S-Y, Chung S-I, Steinert PM: Highly active soluble processed forms of the transglutaminase 1 enzyme in epidermal keratinocytes. J Biol Chem 270:18026– 18035, 1995a Kim S-Y, Chung S-I, Yoneda K, Steinert PM: Expression of transglutaminase 1 in human epidermis. J Invest Dermatol 104:211–217, 1995b Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685, 1970 Lebrun JJ, Vale WW: Activin and inhibin have antagonistic effects on ligand-
dependent heteromerization of the type I and type II activin receptors and human erythroid differentiation. Mol Cell Biol 17:1682–1691, 1997 Li L, Tennenbaum T, Yuspa SH: Suspension-induced murine keratinocyte differentiation is mediated by calcium. J Invest Dermatol 106:254–260, 1996 Ling N, Ying S-Y, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R: Pituitary FSH is released by a heterodimer of the β-subunits from the two forms of inhibin. Nature 321:779–782, 1986 Matzuk MM, Kumar TR, Vassalli A, Bickenbach JR, Roop DR, Jaenisch R, Bradley A: Functional analysis of activins during mammalian development. Nature 374:354–356, 1995a Matzuk MM, Lu N, Vogel H, Sellheyer K, Roop DR, Bradley A: Multiple defects and perinatal death in mice deficient in follistatin. Nature 374:360–363, 1995b McCarthy SA, Bicknell R: Inhibition of vascular endothelial cell growth by activinA. J Biol Chem 268:23066–23071, 1993 Mine T, Kojima I, Ogata E: Stimulation of glucose production by activin-A in isolated rat hepatocytes. Endocrinol 125:586–591, 1989 Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H: Activin-binding protein from rat ovary is follistatin. Nature 247:836–838, 1990 Ogawa Y, Schmidt DK, Nathan RM, et al: Bovine bone activin enhances bone morphogenetic protein- induced ectopic bone formation. J Biol Chem 267:14233–14237, 1992 Ohga E, Matsuse T, Teramoto S, Katayama H, Nagase T, Fukuchi Y, Ouchi Y: Effects of activin A on proliferation and differentiation of human lung fibroblasts. Biochem Biophys Res Commun 228:391–396, 1996 Roberts VJ, Sawchenko PE, Vale W: Expression of inhibin/activin subunit messenger ribonucleic acids during rat embryogenesis. Endocrinol 128:3122–3129, 1991 Seishima M, Takagi H, Okano Y, Mori S, Nozawa Y: Ganglioside-induced terminal differentiation of human keratinocytes: early biochemical events in signal transduction. Arch Dermatol Res 285:397–401, 1993 Seishima M, Nojiri M, Akiyama T, Seishima M, Noma A, Etoh Y, Kitajima Y: Expression of activin A in human keratinocytes at early stage of cultivation. FEBS Lett 398:120–124, 1996 Shiozaki M, Sakai R, Tabuchi M, Nakamura T, Sugino K, Sugino H, Eto Y: Evidence for the participation of endogenous activin A/erythroid differentiation factor in the regulation of erythropoiesis. Proc Natl Acad Sci USA 89:1553– 1556, 1992 Sugino H, Nakamura T, Hasegawa Y, et al: Erythroid differentiation factor can modulate follicular granulosa cell functions. Biochem Biophys Res Commun 153:281–288, 1988 Sun TT, Green H: Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope. Cell 9:511–521, 1976 Vale W, Rivier J, Vaughan J, et al: Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321:776–779, 1986 Yasuda H, Mine T, Shibata H, et al: Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. J Clin Invest 92:1491–1496, 1993 Yoneda K, Steinert PM: Overexpression of human loricrin in transgenic mice produces a normal phenotype. Proc Natl Acad Sci USA 90:10754–10758, 1993 Yuspa SH, Ben T, Hennings H, Lichti U: Phorbol ester tumor promoters induce epidermal transglutaminase activity. Biochem Biophys Res Commun 97:700– 708, 1980 Yuspa SH, Kilkenny AE, Steinert PM, Roop DR: Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 109:1207–1217, 1989
Activin A, a member of the TGFβ-superfamily, is well known to play important roles in the growth and differentiation of various target cells. We have previously demonstrated that activin A is produced at an early stage of cultivation at both the protein and the mRNA levels in cultured human keratinocytes. In this study, the effects of activin A on differentiation and proliferation of human keratinocytes were examined. Activin A (^1 nM) induced cornified envelope formation and the synthesis of loricrin, keratin 1, involucrin, and transglutaminase 1. In addition, transglutaminase
activity and mRNA level of transglutaminase 1 were increased by activin A. [3H]Thymidine incorporation and cell number were reduced by activin A (^1 nM) compared with control, suggesting an inhibitory effect of activin A on cell proliferation. On the basis of these findings, it is likely that activin A contributes to differentiation and suppression of proliferation in human keratinocytes. Key words: loricrin/proliferation/ transforming growth factor β-superfamily/transglutaminase 1. J Invest Dermatol 112:432–436, 1999
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development and hyperkeratotic skin (Matzuk et al, 1995b). These findings suggest that activin A plays an important role in keratinocyte functions; however, the role of activins in keratinocytes has not been fully determined. The aim of this study was to investigate the effects of activin A on proliferation and differentiation in cultured human keratinocytes.
ctivin A, a member of the TGFβ-superfamily that was originally purified from ovarian fluids, stimulates secretion of follicle-stimulating hormone from the anterior pituitary gland (Ling et al, 1986; Vale et al, 1986). Erythroid differentiation factor was independently isolated from human monocytic leukemia cells (Eto et al, 1987). Molecular cloning of cDNA has shown that activin A and erythroid differentiation factor are identical. Activins have been identified as homo- or heterodimers of the βA and βB chain and have been classified into three types: activin A (βAβA), activin B (βBβB), and activin AB (βAβB). Activins induce differentiation of hematopoietic cells (Eto et al, 1987), osteoblasts (Ogawa et al, 1992), and endocrine cells (Sugino et al, 1988), and inhibit proliferation of gonadal cell lines (Gonzalez-Manchon and Vale, 1989), hepatocytes (Yasuda et al, 1993), and endothelial cells (McCarthy and Bicknell, 1993). We have previously shown that mRNA of activin A (βA), but not βB, is detected in normal human epidermis and in keratinocytes at an early culture stage (Seishima et al, 1996). In contrast to our results, Roberts et al showed that activin βA is expressed in the developing hair bulb and also in the dermal layer of embryonic skin, but not in adult skin (Roberts et al, 1991). It has been reported that activin A expression is enhanced in keratinocytes by cutaneous injury (Hu¨bner et al, 1996) and by the addition of interleukin 1β, tumor necrosis factor α, or serum (Hu¨bner and Werner, 1996). On the other hand, mice lacking the activin βA-chain in whiskers and whisker follicles are abnormal (Matzuk et al, 1995a). Furthermore, mice deficient in follistatin, an activin binding protein, show disturbed whisker
MATERIALS AND METHODS Cell culture and cell number count Normal human keratinocytes from foreskin (Kurabo, Osaka, Japan) were grown in Eagle’s minimum essential medium (with 0.09 mM Ca2) supplemented with antibiotics (100 units penicillin per ml, 100 mg streptomycin per ml) and 10% fetal calf serum (HyClone Laboratories, Logan, UT) on 60 mm dishes (Corning Plastics) at 37°C with 5% CO2/95% air in a humidified incubator. To adjust the Ca21 level in the medium to 0.09 mM, fetal calf serum treated with 8% Chelex 100 (Bio-Rad Laboratories, Richmond, CA) and 0.09 mM Ca2 were added to Ca21 and Mg21 free-minimum essential medium (Whittaker Bioproducts, Walkerville, MD) (Li et al, 1996). Cells were used after one passage and the medium was changed every 2–3 d. After reaching confluence, the cells were treated with Hank’s balanced salt solution containing 0.02% trypsin and 0.02% ethylenediamine tetraacetic acid for 15 min at 37°C, and were then resuspended in the culture medium. The cells were seeded onto 35 mm dishes at a density of 13105 per ml of culture medium in the presence of 0.09 mM (low) or 1.8 mM (normal concentration) of calcium. After incubation with activin A at different concentrations (0, 0.1, 1, 10, or 100 nM) for 24 h, the cells were washed with phosphate-buffered saline (PBS) and were dissociated by treatment with 0.25% trypsin and 0.1% ethylenediamine tetraacetic acid solution at 37°C for 10 min. After centrifugation at 1000 3 g for 10 min, the cells were resuspended in 10 mM Tris-HCl buffer, pH 7.5 and then the cell number was counted under a microscope. Subconfluent and monolayer culture was observed after 36 h incubation without activin A.
Manuscript received November 19, 1998; revised December 18, 1998; accepted for publication December 22, 1998. Reprint requests to: Dr. Mariko Seishima, Department of Dermatology, Ogaki Municipal Hospital, Minaminokawa-cho 4-86, Ogaki City, Japan 503-8502.
Measurement of DNA synthesis DNA synthesis was assessed by measuring the incorporation of [3H]thymidine. After keratinocytes were cultured with activin A at different concentrations for 24 h, the cells were labeled with 1.0 µCi [3H]thymidine per ml for 4 h. Cultures were washed
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three times with PBS and then lyzed with 2N NaOH at 37°C for 20 min and neutralized with 2N HCl. Acid-insoluble material was precipitated by addition of four volumes of ice-cold trichloroacetic acid and was collected on GF/A glass-fiber filters (Whatman, Ann Arbor, MI). After washing three times with 10% trichloroacetic acid and once with ethanol, the filters were dried at room temperature for measurement of radioactivity with a liquid scintillation counter (Beckman LS 7500). Transglutaminase activity assay Tranglutaminase (TGase) activity was assayed by measuring the formation of ε-amino-γ-glutamyl bonds between [3H]putrescine and casein (Yuspa et al, 1980; Seishima et al, 1993). After incubation with 0.1, 1, 10, or 100 nM of activin A for 24 h, cells were scraped off and sonicated in 10 mM Tris HCl buffer (pH 7.4) containing 10 mM dithreitol, 0.5 mM ethylenediamine tetraacetic acid, and 0.1% Triton X-100. The homogenates were centrifuged at 105,000 3 g for 60 min and the resultant supernatants were used for the TGase activity assay. One hundred microliters of cell lysate was incubated with 100 liters of 50 mM Tris-HCl buffer (pH 7.4) containing casein (20 g per ml) and [3H]putrescine (5 mM). After incubation for 30 min at 37°C, the reactions were terminated by adding the same volume of ice-cold 10% trichloroacetic acid and the mixture was kept on ice for 30 min. The acid-insoluble material was collected on GF/A glass-fiber filters. After three washes with 5 ml of 5% ice-cold trichloroacetic acid containing 1% putrescine, the filters were dried at room temperature. The dried filters were mixed with scintillation fluid and the radioactivity was determined with a liquid scintillation counter. Scoring of cornified envelopes The scoring of cornified envelope formation was performed essentially as described by Sun and Green (1976). The cells incubated with activin A at various concentrations for 24 h were washed with PBS, and were dissociated by treatment with 0.25% trypsin and 0.1% ethylenediamine tetraacetic acid solution at 37°C for 10 min. After centrifugation at 1000 3 g for 10 min, the cells were resuspended in 10 mM Tris-HCl buffer, pH 7.5, containing 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol at a density of 1–2 3 106 cells per ml. After the cell suspensions had been kept at room temperature for 10 min, sodium dodecyl sulfate- and mercaptoethanol-resistant cornified envelopes were counted under a microscope. Western blot analysis Normal keratinocytes were seeded on 60 mm dishes at a density of 1 3 105 cells per 5 ml of culture medium in the presence of 0.09 mM or 1.8 mM calcium. Because TGase activity and cornified envelope formation were increased by activin A at the concentration of 1 nM or more and TGase activity in human keratinocytes is mostly due to TGase 1 (Kim et al, 1995a), 1 nM of activin A was used in this study to examine the effects of activin A on the synthesis of TGase 1, loricrin, keratin 1, and involucrin. Cells incubated with activin A for 24 h were washed twice with PBS, and harvested by PBS with 1 mM phenylmethylsulfonyl fluoride (Molecular Probes, Eugene, OR), 1 mM N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, Research Organics, Cleveland, OH), and 5 mM E64 (Enzyme System Products, Dublin, CA). We performed western blot analysis using monoclonal antibodies against human TGase 1 (Kim et al, 1995b), loricrin (Yoneda and Steinert, 1993), keratin 1 (34βB4, Enzo Diagnotics, Farmingdale, NY), and rabbit polyclonal antibody to involucrin (BT601, Biomedical Technologies, Stoughton, MA). Aliquots were mixed with sample buffer (10% 2-β-mercaptoethanol, 2% sodium dodecyl sulfate, 30% glycerol, 0.25 M Tris, final concentration), followed by heating at 98°C for 3 min. Thirty micrograms of protein was loaded per lane and reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 10% polyacrylamide gel for TGase 1, 20% gel for loricrin, and 8% gel for involucrin and keratin 1 by the method of Laemmli (1970). Protein standards for molecular weight (Bio Rad) were as follows: ovalbumin, 49 kDa; bovine serum albumin, 80 kDa; βgalactosidase, 116.5 kDa; and myosin, 205 kDa. Proteins were electroblotted onto Hybond-ECL (Amersham, Bucks, U.K.) using a semi-dry transfer apparatus (Bio Rad) for 30 min at 10 V. The membranes were preincubated with a blocking solution containing 5% skim milk and 0.2% Tween 20 in PBS for 1 h at room temperature. Membranes were then incubated overnight at 4°C with diluted primary antibodies. Following incubation with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (0.2 µl per ml) in the blocking solution for 2 h, membranes were washed and bound antibodies were detected by using ECL detection reagents (Amersham) and exposure to Hyperfilm. Northern blot analysis After keratinocytes were incubated without or with 1 nM of activin A for 12 h, total RNA was extracted from the cells with a buffered solution containing 4 M guanidium thiocyanate according
Figure 1. Suppression of keratinocyte proliferation by activin A. Dose-dependent effects of activin A on cell number (A, B) and [3H]thymidine incorporation (C, D) in low (A, C) or normal calcium (B, D) media are shown. After incubation with activin A at 1–100 nM for 24 h, the number of keratinocytes was markedly decreased compared with control under both low (A) and normal (B) calcium conditions. [3H]Thymidine incorporation was significantly reduced by exposure for 24 h to 1, 10, or 100 nM of activin A in both low (C) and normal (D) calcium conditions. Values are means of triplicate determinations from three separate experiments. Error bars: 1 SD. *p , 0.05, **p , 0.005, ***p , 0.001. to the method of Chomzynski and Sacchi (1987). The TGase 1 cDNA was labeled with α-32P-dCTP using a multiprime DNA labeling system, and hybridization was performed at 37°C for 15 h. After hybridization, the filters were washed and subjected to autoradiography. Data analysis Data are expressed as mean 6 1 SD, and statistical differences were evaluated using Student’s t test. p values less than 0.05 were taken as significant.
RESULTS Suppression of keratinocyte proliferation by activin A Cell numbers were counted to evaluate the effect of activin A on keratinocyte proliferation. Cell numbers as well as [3H]thymidine incorporation in the low calcium medium were higher than those in the normal calcium medium when activin A was not added. After incubation with activin A at 1–100 nM for 24 h, the number of keratinocytes was markedly decreased compared with control under both low and normal calcium conditions (Fig 1). Cell proliferation was decreased with time by 1 nM of activin A for up to 36 h (Fig 2). Further incubation in the medium without activin A (for 36 h) after 36 h incubation with 1 nM of activin A did not induce recovery of cell growth. In addition, [3H]thymidine incorporation was measured to examine the effect of activin A on DNA synthesis. DNA synthesis was significantly reduced by activin A at the concentrations of 1, 10, and 100 nM for 24 h incubation in either low or normal Ca21 concentration (Fig 1). Moreover, [3H]thymidine incorporation in cells incubated with 1 nM of activin A (9.83 6 0.93 3 10–4 cpm per 105 cells, n 5 3) for 24 h was restored to the basal level by pretreatment with 3 nM of
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Figure 2. Time-dependently reduced keratinocyte proliferation by activin A until 36 h. Time-dependent effects of activin A (1 nM) on cell number of human keratinocytes cultured in low calcium media are shown. Values are means of triplicate determinations from three separate experiments. Error bars: SD.
follistatin, an activin binding protein, for 6 h under low calcium conditions (13.1 6 0.98 3 10–4 cpm per 105 cells, n 5 3, p , 0.001); however, low activin A concentration (0.1, 0.05, and 0.01 nM) did not affect either the cell numbers or the [3H]thymidine incorporation. Pretreatment with 0.3 nM of follistatin similarly did not affect cell number and [3H]thymidine incorporation at the low concentrations of activin A. Keratinocyte differentiation induced by activin A The effects of various concentrations of activin A on differentiation were examined by monitoring cornified envelope formation. Cornified envelope formation in 1, 10, or 100 nM of activin A-treated cells was significantly increased compared with control, but no change was observed in 0.1 nM of activin A (Fig 3). Because TGase 1 is a key enzyme for keratinocyte differentiation, we examined the effects of activin A on TGase activity, TGase 1 mRNA level, and TGase 1 synthesis. TGase activity was increased at 12 h, and reached a plateau at 36 h (Fig 4), in cells treated or not treated with activin A (1 nM). TGase activity was significantly increased by activin A at more than 1 nM compared with control at 24 h, but no increase was observed in low concentration of activin A (0.1 nM) (Fig 3). In addition, 1 nM of activin A-induced TGase activity at 24 h was significanty suppressed to the basal level by pretreatment with 3 nM of follistatin for 6 h in normal calcium medium (44.4 6 3.75 vs 21.9 6 3.60 3 10–3 cpm per µg prot. per 30 min, n 5 3, p , 0.005). The expression of TGase 1 mRNA and the synthesis of TGase, loricrin, keratin 1, and involucrin were enhanced in the normal calcium condition as compared with low calcium (Fig 5). Because the TGase activity and cornified envelope formation were increased by activin A at concentrations of 1 nM or more (Fig 3), 1 nM of activin A was employed to examine the effects of activin A on the expression of these differentiation markers. The synthesis of TGase 1, loricrin, keratin 1, and involucrin was enhanced by 1 nM of activin A under both normal and low calcium conditions (Fig 5A, C–E, respectively). The TGase 1 mRNA level examined by northern blot analysis was obviously increased by treatment with 1 nM of activin A for 12 h under both normal and low calcium conditions (Fig 5B).
Figure 3. Keratinocyte differentiation induced by 1–100 nM of activin A. Dose-dependent effects of activin A on cornified envelope formation (CE) (A, B) and transglutaminase (TGase) activity (C, D) in low (A, C) or normal calcium (B, D) media are shown. Cornified envelope formation and TGase activity were significantly increased by incubation with activin A at 1 nM or more for 24 h. Values are means of triplicate determinations from three separate experiments. Error bars: SD. *p , 0.05, **p , 0.001.
DISCUSSION In this study, two different calcium conditions, 0.09 mM (low concentration) and 1.8 mM (normal concentration), were employed for culture, because it is well known that a low concentration of calcium induces proliferation, and normal calcium induces differentiation in keratinocytes (Henning and Holbrook, 1983; Yuspa et al, 1989). As expected, the cell number and [3H]thymidine incorporation in the low calcium medium were significantly higher than those in normal calcium, and the expression of keratinocyte differentiation markers such as cornified envelope formation and TGase activity in the low calcium medium were lower than those in normal calcium, irrespective of the presence of activin A. The addition of activin A at different concentrations ranging from 1 to 100 nM inhibited the increase in the cell number and [3H]thymidine incorporation dose-dependently under both calcium conditions, indicating that activin A inhibits cell proliferation. Cornified envelope, which is formed beneath the plasma membrane and is resistant to detergents and reducing agents, is characteristic of the terminal differentiation of keratinocytes. TGase 1, a key enzyme for keratinocyte differentiation, is a Ca21-requiring enzyme that catalyses the cross-linking of membrane proteins to form cornified envelopes. The effect of various concentrations of activin A on the differentiation of cultured keratinocytes was examined by monitoring cornified envelope formation and TGase activity. Both markers were enhanced in a dose-dependent manner. This enhancement in TGase activity is mostly due to the increase in TGase 1 activity, because it is now known that the TGase activity
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in human keratinocytes is mostly due to TGase 1 (Kim et al, 1995a). Although Hohl et al (1991) showed that Ca21 concentrations above 0.1 mM were permissive for the expression of loricrin mRNA, our data clearly demonstrate that activin A even in low Ca21 contributes to the terminal differentiation of cultured human keratinocytes. In addition, the expression of TGase 1 mRNA, and the synthesis of TGase, loricrin, keratin 1, and involucrin were enhanced with even 1 nM of activin A. These findings suggest that activin A induces early and terminal differentiation of keratinocytes at concentrations of 1 nM or more.
Figure 4. Time-dependent increase in TGase activity by 1 nM of activin A. TGase activity was increased at 12 h, and reached plateau at 36 h in human keratinocytes cultured in normal calcium media. Values are means of triplicate determinations from three separate experiments. Error bars: 1 SD.
Figure 5. Enhanced expression of TGase 1, loricrin, keratin 1, and involcrin and increased TGase 1 mRNA level by 1 nM of activin A. The synthesis of TGase 1 (A), loricrin (C), keratin 1 (D), and involucrin (E) was enhanced by the incubation with 1 nM of activin A for 24 h in both low and normal calcium conditions. The TGase 1 mRNA expression (B) was also enhanced by the incubation with activin A (1 nM) for 12 h. Similar results were obtained in three independent experiments. Molecular weight markers (KD) are indicated in (A) (left). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as a control in northern blot analysis (B).
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In other cells, activin A inhibits neural differentiation (Hashimoto et al, 1990) and stimulates glucose production in hepatocytes (Mine et al, 1989) at 1 nM, modulates osteoblast proliferation and differentiation at 10 nM (Hashimoto et al, 1992), inhibits the growth of mammary epithelial cells at 100 nM, and induces erythroid differentiation at 1 µM. On the other hand, low concentration (0.01 nM) of activin A stimulates the differentiation of human lung fibroblasts into myofibroblasts and proliferation of fibroblasts (Ohga et al, 1996). This different responsiveness to activin A might be due to differences in cell types, quantities, or binding activities of activin receptors or activin inhibitors. Activin receptors are transmembrane serine/threonine kinases, among them type I receptors (ARI and ARIB) and type II receptors (ARII and ARIIB) (Ca´rcamo et al, 1994; Lebrun and Vale, 1997). Follistatin is an activin binding protein exerting a neutralizing effect on activin activity (Nakamura et al, 1990). Mad proteins function downstream of the activin receptor pathway (Chen et al, 1996). The endogenous activin A activity in cultured DJM-1 cells, a squamous cell carcinoma line, corresponded to the activity generated by 0.32 nM of recombinant activin A. No activin was detected in normal human keratinocytes (less than 0.02 nM) (Seishima et al, 1996). Activins are reported to be secreted in an autocrine or paracrine manner (Shiozaki et al, 1992). Therefore, the effect of activin A was compared with control, which lacked exogenous activin A. We have previously demonstrated that activin A mRNA (βA) is detected at an early stage of keratinocyte culture and that activin A is observed at the wound edge in an in vitro tissue injury model, where cell migration and proliferation are activated without terminal differentiation (Seishima et al, 1996). The induction of activin A expression was also found in wounded skin, especially granulation tissue below the eschar, within 7 d of skin injury, suggesting that activin A plays an important role in tissue repair (Hu¨bner et al, 1996). These observations appear to be in disagreement with our results. Namely, our previous results suggest that activin A acts as a potent inducer of proliferation, but this study indicates that activin A is an inhibitor of proliferation and a stimulator of differentiation. This apparent discrepancy can be explained as follows: activin A may upregulate proliferation and stimulate migration at the wound edge or early stage of cultivation, but it may downregulate
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proliferation and stimulate differentiation at higher concentrations given exogenously. In other words, activin A might be an initial modulator of differentiation in keratinocytes. In conclusion, activin A appears to play a role in the proliferation and differentiation of cultured human keratinocytes. Further in vivo studies on the precise regulation mechanisms for proliferation and differentiation by activin A, including the signaling pathway and nuclear responses, are needed to elucidate the roles of activin A in epidermis. REFERENCES Ca´rcamo J, Weis FMB, Ventura F, Wieser R, Wrana JL, Attisano L, Massague´ J: Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor β and activin. Mol Cell Biol 14:3810–3821, 1994 Chen Y, Lebrun J-J, Vale W: Regulation of transforming growth factor β- and activin- induced transcription by mammalian Mad proteins. Proc Natl Acad Sci USA 93:12992–12997, 1996 Chomzynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159, 1987 Eto Y, Tsuji T, Takezawa M, Takano S, Yokogawa T, Shibai H: Purification and characterization of erythroid differentiation factor isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095–1103, 1987 Gonzalez-Manchon C, Vale W: Activin-A, inhibin and transforming growth factorβ modulate growth of two gonadal cell lines. Endocrinol 125:1666–1672, 1989 Hashimoto M, Kondo S, Sakurai T, Etoh Y, Shibai H, Muramatsu M: Activin/EDF as an inhibitor of neural differentiation. Biochem Biophys Res Commun 173:193– 200, 1990 Hashimoto M, Shoda A, Inoue S, et al: Functional regulation of osteoblastic cells by the interaction of activin A with follistatin. J Biol Chem 267:4999–5004, 1992 Hennings H, Holbrook KA: Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. Exp Cell Res 143:127–142, 1983 Hohl D, Lichti U, Breitkreutz D, Steinert PM, Roop DR: Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid. J Invest Dermatol 96:414–418, 1991 Hu¨bner G, Werner S: Serum growth factors and proinflammatory cytokines are potent inducers of activin expression in cultured fibroblasts and keratinocytes. Exp Cell Res 228:106–113, 1996 Hu¨bner G, Hu Q, Smola H, Werner S: Strong induction of activin expression after injury suggests an important role of activin in wound repair. Dev Biol 173:490– 498, 1996 Kim S-Y, Chung S-I, Steinert PM: Highly active soluble processed forms of the transglutaminase 1 enzyme in epidermal keratinocytes. J Biol Chem 270:18026– 18035, 1995a Kim S-Y, Chung S-I, Yoneda K, Steinert PM: Expression of transglutaminase 1 in human epidermis. J Invest Dermatol 104:211–217, 1995b Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685, 1970 Lebrun JJ, Vale WW: Activin and inhibin have antagonistic effects on ligand-
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