The Journal of Dermatology Vol. 18: 252-257, 1991

Stimulation of Fibronectin Secretion in Cultured Human Keratinocytes by Transforming Growth Factor-f3 Not by Other Growth Inhibitory Substances Makoto Hashiro, Kunio Matsumoto, Koji Hashimoto and Kunihiko Yoshikawa Abstract

We investigated the effects of various growth inhibitory substances on fibronectin secretion in cultured normal human keratinocytes. Fibronectin secretion in keratinocytes was expressed by two methods: immunofluorescent staining of cells with anti-human fibronectin antibody, and sodium dodecyl-sulphate-polyacrylamide gel electrophoresis of [SSS]-labeled proteins secreted by the cells followed by autoradiography. With immunofluorescent staining, the extracellular fibronectin was observed as coarse fibrils only in some cells of the control culture, while cells treated with 20 ng/ml transforming growth factor-Sl (TGF-f3l) showed intense fluorescences in a radiating pattern around most of the cells, indicating that TGF-j31 markedly increased fibronectin secretion in keratinocytes. Analysis of [S5S]-methionine labeled proteins also revealed that TGF131 increased fibronectin secretion eight fold in culture medium. TGF-j32 has also induced weak increase of fibronectin secretion 1.8 fold in keratinocytes. In contrast, other growth inhibitory substances, such as tumor necrosis factor-a, 1,25dihydroxyvitamin D, and cyclosporin A, did not show any significant effects on fibronectin secretion. These results indicated that increase of fibronectin secretion is not associated with growth inhibitory effect, but rather with a specific effect of TGF-j3. Key words:

human keratinoeytes; fibronectin; TGF-I3; vitamin D s ; growth inhibitory substances

Introduction The interaction between cells is mediated by a variety of extracellular matrices such as fibronectin, collagen, laminin, and vitronectin. Fibronectin is a 220kd-glycoprotein produced by various types of cells, and is detected in serum, in cytoplasm, and on cell surfaces (l). Normal human keratinocytes produce fibronectin (2), which has various important roles for epidermal keratinocyte biology, such as cell adhesion and spreading (3, 4), cell motilization (5), cell differentiation, and wound healing (6). Thus the regulation of fibronectin secretion in epidermal keratinocytes seems to be important Received October 17, 1990; accepted for publication April 16, 1991. Department of Dermatology, Osaka University School of Medicine, Osaka, Japan. Reprint requests to: Makoto Hashiro, M.D., Department of Dermatology, Osaka University School of Medicine, 1-1-50, Fukushima, Fukushima-ku, Osaka 553,japan.

in the regulation of growth, differentiation, motilization, and wound healing. Transforming growth factor-13 (TGF-f3) is known to be a multifunctional peptide growth factor (see review, Ref. 7). Various types of cells, including normal human keratinocytes, produce TGF-/3 (7, 8). Blood platelets, in particular, contain large amounts of TGF-13 (9), which suggests that TGF-13 has an important role in wound healing processes. Recently, Wikner et al. reported that TGF-131 stimulates fibronectin secretion in normal human keratinocytes; it also functions as a potent growth inhibitor for human keratinocytes (10). These results led us to study whether the fibronectin secretion of human keratinocytes is affected by other growth inhibitory substances. TGF-132 is a family ofTGF-13 related polypeptides which inhibits proliferation of epithelial cells (8). Tumor necrosis factor (TNF)-a (11), 1,25-dihydroxyvitamin D 3 (l,25(OH)2 D 3) (12), and cyclosporin A (13) are

Fibronectin Secretion in Human Keratinocytes by TGF-f3 also known to inhibit the growth of human keratinocytes. In the present study, we investigated the effects of TGF-131, TGF-132, TNF-a, 1,25(OH)2Dg, and cyclosporin A on fibronectin secretion in normal human keratinocytes cultured under serum-free conditions. We found that TGF-131 and TGF-132 increased fibronectin secretion, but that the other three substances had no effect.

Materials and Methods Growth inhibitory substances: TGF-f31, recombinant TNF-a, 1,25(OH)2D" and cyclosporin A were kindly provided by Dr. DeLarco (14), Dainippon Pharma. Ltd. (Osaka), Teijin Ltd. (Tokyo), and Sandoz Pharma Ltd. (Basel, Switzerland), respectively. Porcine TGF-f32 was obtained from R&D Systems (Minneapolis, MN). Keratinocyte culture: Normal human skin samples were obtained during plastic surgery, and keratinocytes were obtained and cultured as previously described (12). Briefly, epidermal keratinoeytes were cultured in nutrient medium MCDB 153 (Kyokuto Co., Tokyo) supplemented with insulin (5 j.lg/ml), hydrocortisone (5 x 10-7 M), ethanolamine (O.lmM), phosphoethanolamine (O.lmM), and bovine hypothalamic extract (150 j.lg protein/ml). The stock solution consisted of MCDB 153 supplemented with 0.1mM Ca 2+ and elevated concentrations of amino acids (0.75mM isoleucine, 0.24mM histidine, 0.09mM methionine, 0.09mM phenylalanine, 0.045mM tryptophan, 0.075mM tyrosine) and kanamycin (60 mg/I). Second passaged cells were used in this experiment. Immunofluorescent staining: Keratinocytes (8 x 104 per well) were cultured on a chamber slide glass (Nunc Inc., Naperville, Ill). TGF-f31 (20 ng/ml), TGF-f32 (20 ng/ml), TNF-a (100 ng/ml), I,25(OH)2D, (10-6 M), or cyclosporin A (5 x 10-6 M) Was added and incubated for 72 hours. After the medium was discarded, cells were washed with phosphate buffered saline (PBS). After blocking with 1% (w/v) bovine serum albumin (BSA) in PBS, rabbit anti-human fibronectin serum (Organon Teknika Corp. West Chester, PA), diluted 1:200 in PBS containing 1% BSA, was added and shaken for 30 min at room temperature. After a rinse with PBS, FITC-Iabeled goat anti-rabbit IgG (£-Y Laboratories Inc., San Mateo. CA), diluted I :200 with PBS con-

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taining 1% BSA, was added and shaken for 30 min at room temperature. After further washing, the slide glass was mounted and observed under a fluorescent microscope. Analysis ofjibronedin in conditioned medium: Keratinocytes (8 x 105) cultured on a 30-mm dish (Becton Dickinson, Oxnard, CA) were incubated in the presence of TGF-,81 (2 ng/ml, 20 ng/ml), TGF-,82 (2 ng/ml, 20 ng(ml), TNF-a (25 ng/ml, 100 ng/ml), 1,25(OH)2D, (10- 7 M, 10-6 M) or cyclosporin A (1 x 1{)-6 M, 5 X 10-6 M) for 8 hours. Cells were labeled with 1.11 MBq/ml of [35S]_ methionine (Amersham) in low methionine (10-6 M) MCDB 153 medium containing these reagents at the same concentration for 16 hours at 37°C in 5% CO 2, The conditioned medium was dialyzed against 5mM ammonium acetate containing ImM phenylmethylsulphonyl fluoride (Nacarai Tesque, Kyoto), 10 j.lg/ml pepstatin A (Sigma) and 5 j.lg/ml £64 (Peptide Institute Inc., Osaka) at 4°C. The dialyzed medium was lyophilized and solubilized in IOmM Tris-HCI (pH 7.4) buffer containing 0.I5M NaCI and 0.02% (w/v) NaN,. Labeled materials were analyzed by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with 5-20% gradient gel followed by autoradiography. Immunodepletion of fibronectin in conditioned medium was performed as follows. Labeled materials in lOmM Tris-HCI (pH 7.4) buffer containing 0.15M NaCI and 0.02% NaN, were incubated with 1/15 volume of rabbit anti-human fibronectin serum for 2 hours at 4°C. Protein A sepharose CL-4B (Pharmacia) suspension (20% v/v) in the Tris buffer with the same volume as labeled materials was added and incubated for 1 hour at 4°C. Protein A sepharose was precipitated by centrifugation, and the supernatant was analyzed in the same way.

Results Immunofluorescent staining: Normal human keratinocytes were cultured in the absence and presence of various growth inhibitory substances and submitted to immunofluorescent staining using anti-human fibronectin antibody. In control culture cellular fibronectin was observed mainly as p~rinuc1ear staining in most cells; the extracellular fibronectin was observed as coarse fibrils around some large cells (Fig. 1, a).

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Hashiro et al

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 1. Immunofluorescent staining of fibronectin in human keratinocytes, Cells were cultured (a) or treated with 20 ng/rnl TGF-,81 (b), 20 ng/ml TGF-,82 (c), 100 ng/mllNF-a (d), 10--6 M 1,25(OH)2Ds (e) or 5 x 10-6 M cyclosporin A (f). Cells were stained using rabbit anti-human fibronectin serum and FITClabelled anti-rabbit antibody.

Cells treated with 20 ng/ml TGF-,81 showed more intense fluorescence in a radiating pattern, particularly around the TGF-,81-treated cells, than in control cells (Fig. 1, b). TGF-,82 also caused increased immunofluorescence due to fibronectin (Fig. 1, c). In contrast, TNF-a (100 ng/rnl), 1,25(OH)2Ds (10-6 M), and cyclosporin A (5 x 10-6 M) had

almost no effects on immunofluorescent staining, either cellular or extracellular (Fig. 1, d, e and f). Fibronectin secretion in conditioned medium: In order to see whether extracellular fibronectin secreted by human keratinocytes was affected by growth inhibitory substances, we analyzed [S5S]-labeled proteins secreted into conditioned medium. TGF-,81 treatment increased the secretion of various proteins

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Fibronectin Secretion in Human Keratinocytes by TGF-fJ

MW (k)

MW

0

2

0

2

20 ng/ml

25 100 ng/m-'l

20ng/ml

200-

(k) 200 200-

11692.5-92.5 66.2-

11692.566.2 -

11692.566.2-

-92.5 -66.2 45.0-

45.0-

-45.0

- 66.2 -

. 45.0-

-21.3 -14.4

- 21.5 - 14.4 (b)

(a)

MW (k)

10-7 10-6 M

(k)

-45.0

45.0

- 31.0

-21.5 -14.4

0

-92.5 -66.2

-31.0

-31.0

MW

0

MW (k)

0

(c)

10-6 5 X 10-6 M

200-

Fig. 2.

Autoradiographs of proteins secreted by human keratinocytes. Cells were treated with TGF-tn (a), TGF-fJ2 (b), TNF-a (c), I,25(OH)2D~ (d) or cyclosporin A (e). The positions and molecular weights of standard marker proteins are indicated. [~SS]-labelled

11692.5-

20011692.566.2-

-92.5 -66.2

45.0-

-45.0

66.2-

-92.5 -66.2

45.0-

-45.0 -31.0

-31.0 -21.3 -14.4

-21.5 -14.4 (d)

into culture medium. To identify the 220kd protein as fibronectin, secreted proteins from TGF-fJI treated cells were mixed with anti-human fibronectin antibody, the immune complex was precipitated with protein A-sepharose, and the resultant supernatant was analyzed by autoradiography (Fig. 3). Since the 220kd protein was depleted by anti-human fibronectin antibody, it was identified as fibronectin. Relative intensities of the 220kd band on autoradiographs were measured by dual-wavelength fiying-spot scanner CS-9000 (Shimadzu, Kyoto). In panicular, TGF-fJI at 2 ng/ml induced an 8-fold increase of 220kd-sized protein (Fig. 2, a). Condi-

(e) tioned medium with TGF-fJ2-treated keratinocytes labeled with pSS]-methionine also showed a l.8-fold increase ofthe same 220kd protein band (Fig. 2, b). Autoradiographs of celllysates treated with TGF-m and TGF-fJ2 did not show any increased intensity of the 220kd protein band (not shown). We then examined the effects of other growth inhibitory substances on fibronectin secretion in culture medium. TNF-a and I,25(OH)2D~ had no effects on fibronectin secretion (Fig. 2, c and d), while cyclosporin A seemed to decrease secretion of all proteins, inclUding fibronectin (Fig. 2, e).

Hashiro et al

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MW (k)

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b

c

20011692.566.2-

-92.5 -66.2

45.0-

-45.0 -31.0 -21.3 -14.4

Fig. 3. Autoradiograph of immunodepletion using anti-human fibronectin serum. Immunodepletion of fibronectin in conditioned medium was performed as follows. Labelled materials were incubated with PBS (lane a), normal rabbit serum (lane b), or rabbit antihuman fibronectin serum (lane c) for 2 hours at 4°C. Immune complexes were depleted with protein A sepharose CL-4B and subsequent centrifugation. The resultant supernatant was analyzed by SDS-PAGE and subsequent autoradiography.

Discussion TGF-Jjs are known to inhibit the growth of epithelial cells, including normal human keratinocytes (8, 15, 16). Another biological effect of TGF-Jjl is to simulate the expression of fibronectin and collagen (17). Recently, Wikner et al. showed that TGF-Jjl increased fibronectin secretion in normal human keratinocytes (10). We therefore hypothesized that increased secretion of fibronectin might be related to the growth inhibitory effect of TGF-Jjl. Since TNF-a, 1,25(OH)2D3' and cyclosporin A are also known to inhibit keratinocyte growth (11-13), we examined the effects of these growth inhibitory substances on the secretion of fibronectin in normal human keratinocytes. The growth of human keratinocytes is inhibited by TGF-Jjl (16), TNF-a (11), 1,25(OH)2D3 (12), and cyclosporin A (13) at the concentrations of 2 ng/ml, 5 ng/ml, 10-7 M, 8.3 X 10-7 M respectively. Our results showed that, unlike TGF-{31 and TGF-{32, these substances have no

ability to induce fibronectin secretion, suggesting that such secretion is not a common feature of growth inhibitory substances. TGF-Jj2 is a family of TGF-{3 related proteins which has approximately 70% amino acid sequence homology with TGF-Jjl. It inhibits epithelial cell proliferation in a manner similar to that of TGF-Pl (8). TGF-Jj2 arrests growth of human keratinocytes at 2 ng/ml, although TGF-Jjl is effective at 0.5 ng/ml (unpublished data). We therefore examined the effect of TGF-p2 on fibronectin secretion and observed that TGFJj2 also stimulates this secretion. Our results showed that TGF-Pl was much stronger than TGF-Jj2 in terms of induction of fibronectin secretion. There are several possibilities to explain this finding. When the growth inhibitory effect on human keratinocytes was examined, TGF-Jjl was more potent than TGFJj2. This indicates that the specific activity of TGF-Jj2 could be lower than that of TGF-Jjl. Another explanation is a possible difference of affinity to receptor between human TGF-Pl and porcine TGF-Jj2. It should be noted that production of fibronectin in cell lysates was not increased by addition of TGF-Jjl and TGF-Jj2, although immunofluorescent staining showed that cells treated with TGF-Jjl and TGF-Jj2 showed more intense fluorescent staining. The reasons for this discrepancy are not clear. TGF-Jjl and TGF-Jj2 may modulate localization of fibronectin in human keratinocytes, resulting in the increase of intensity of immunofluorescent staining. Recently we reported that the effect ofTGFJjl on keratinocyte differentiation is dependent on the Ca2+ concentration in the culture medium (16). It enhances differentiation on human keratinocytes under high Ca 2+ conditions, but inhibits differentiation under low Ca 2+ conditions. It is not thought that TGF-Jjl may induce differentiation of keratinocytes under any conditions in vivo. In wound healing processes, epidermal keratinocytes first undergo migration on a provisional matrix formed at the site of the wound without cell proliferation or terminal differentiation. Since human platelets contain TGF-,8s in large quantities, it is

Fibronectin Secretion in Human Keratinocytes by TGF-fi

possible to speculate that TGF-l3s, secreted from platelets in injured skin, increase the amount of fibronectin secretion by keratinocytes and that this fibronectin constructs the network for keratinocyte attachment and migration. Thus the human keratinocytes' growth arrest and concomitant stimulation of fibronectin secretion may be a rather specific event caused by TGF-13 as situations like wound healing, and thus not a feature common to other growth substances inhibitory to human keratinocytes. References 1) Mosher DF: Fibronectin, ProgHaemostasis Thromb, 5: 111-151,1983. 2) O'Keefe E], Woodley DT, Falk R], Gammon WR, Briggaman RA: Production of fibronectin by epithelium in a skin equivalent,] Invest Dermatol, 88: 634-639, 1987. 3) Takashima A, Grinnell F: Human keratinocyte adhesion and phagocytosis promoted by fibronectin,] Invest Dermatol, 83: 352~358, 1984. 4) Underwood PA, Bennett FA: A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin,] Cell Sci, 93: 641-649, 1989. 5) NickoloffB], Mitra RS, Riser BL, Dixit VM, VaraniJ: Modulation of keratinocyte motility: correlation with production of extracellular matrix molecules in response to growth promoting and anti proliferative factors, Am] Pathol, 132: 543-551,1988. 6) Denefte ]P, Zhu QL, Lechaire ]P: Anti-fibronectin serum inhibits the disorganization of the dermalepidermal junction in cultured wounded skin, Bioi of the Cell, 67: 91-95, 1989. 7) Rizzino A: Review: Transforming growth factor {3: Multiple effects on cell differentiation and extracellular matrices, Develop Bioi, 130: 411-422, 1988.

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8) Cheifetz S, Weatherbee ]A, Tsang ML-S, et al: The transforming growth factor-{3 system, a complex pattern of cross-reactive ligands and receptors, Cell, 48: 409-415,1987. 9) Assoian RK, Komoriya A, Meyer CA, Miller DM, Sporn MB: Transforming growth factor-{3 in human platelets,] Bioi Chem, 258: 7155-7~60, 1983: 10) Wikner NE, Persichitte KA, Baskin ]B, Nielsen LD, Clark RAF: Transforming growth factor-{3 stimulates the expression of fibronectin by human keratinocytes,] Invest Dermatol, 91: 207-212, 1988. 11) Symington FW: Lymphotoxin, tumor necrosis factor, and gamma interferon are cytostatic for normal human keratinocytes,] Invest Dermatol, 92: 798-805, 1989. 12) Matsumoto K, Hashimoto K, Nishida Y, Hashiro M, Yoshikawa K: Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal human keratinocytes cultured in serum-free medium, Biochem Biophys Res Commun, 166: 916-923, 1990. 13) Furue M, Gaspari AA, Katz SI: The effect of cyclosporin A on epidermal cells. II. Cyclosporin A inhibits proliferation of normal and transformed keratinocytes,] Invest Dermatol, 90: 796-800, 1988. 14) Cone ]L, Brown DR, DeLarco ]E: An improved method of purification of transforming growth factor, type {3 from platelets, Analytic Biochem, 168: 71-74, 1988. 15) Shipley GD, Pittelkow MR, WiIle.ll]r, ScottRE, Moses HL: Reversible inhibition of normal human prokeratinocytes proliferation by type beta transforming growth factor-growth inhibitor in serum-free medium, Cancer Res, 46: 2068-2071, 1986. 16) Matsumoto K, Hashimoto K, Hashiro M, Yoshikawa K: Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-{3,] Cell Physiol, 145: 95-101, 1990. 17) Ignotz RA, Massagu]: Transforming growth factor-S stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix, ] Bioi Chem, 261: 4337-4345, 1986.