JOURNAL OF CELLULAR PHYSIOLOGY 145:95-101 (1990)

Modulation of Growth and Differentiation in Normal Human Keratinocytes by Transforming Growth Factor+ KUNIO MATSUMOTO,* KOJIHASHIMOTO, MAKOTO HASHIRO, HIDENOBU YOSHIMASA, AND KUNlHlKO YOSHIKAWA Department of Dermatology, Osaka University School of Medicine, Fukushirna, Osaka 553, japan The effect of transforming growth factor-type f3 l(TGF-P) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TCF-P markedly inhibited the growth of keratinocytes at the concentrations >2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [ H1thym idine incorporation. The decrease of [3 HIthym idine incorporation was observed as early as 3 hr after addition of TGF-P. TGF-P also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-P. This rapid reduction of c-myc mRNA expression by TGF-P treatment i s possibly one of the main factors in the process of TGF-P-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-P under high Ca2 conditions (1.8 m M Ca2+, differentiation-promoting culture environment) was examined. TGF-P inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2 conditions, suggesting that it i s a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-P at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-P: TGF-P at 20 nglml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-0 on keratinocyte differentiation i s Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there i s a clear discrepancy between TGF-P effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TCF-P is direct and not induced by differentiation. +

+

TGF-p (TGF-p1) is a homodimeric peptide growth factor of 25,000 M,,originally defined as a factor stimulating the cell growth of normal rat kidney fibroblasts in soft agar (Roberts et al., 1981). Recent work demonstrated that it has multifunctional biological activities. It can either stimulate, or inhibit cell growth, and has numerous other effects, such as enhancement of the extracellular matrix formation and regulation of proteolytic activity (Sporn et al., 1987). It stimulates growth of cells of mesenchymal origin. By contrast, it is a potent growth inhibitor for cultured epithelial cells, such as bronchial epithelial cells (Masui et al., 19861, and endothelial cells (Takehara et al., 1987). TGF-P also has a profound inhibitory effect on the 0 1990 WILEY-LISS, INC

growth of cultured keratinocytes. Moses et a1 (1985) reported that TGF-P caused a marked reduction of [3H]thymidine labeling in cultured human prokeratinocytes. Shipley et al. (1986) showed reversible growth arrest of prokeratinocytes by TGF-P. Interestingly, these workers described the secretion of a TGF-p-like molecule by prokeratinocytes and the presence of a specific surface receptor for this molecule, suggesting the possibility that TGF-p is a self-regulatory factor

Received January 9, 1990; accepted June 5, 1990. *To whom reprint requestdcorrespondence should be addressed.

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for keratinocyte growth. Recently, Akhust et al. phenylalanine, 0.045 mM tryptophan, 0.075 mM ty(1988) demonstrated the induction of TGF-P mRNA rosine) and antibiotics (100 IU/ml penicillin and 100 in mouse epidermis by 12-tetradecanoyl-phorbol-13- pg/ml streptomycin). Second-passage cells were used in acetate. These results indicate that TGF-P plays im- this experiment. Unless indicated otherwise, keratiportant physiological and pathological roles in human nocytes were plated in six-well trays (Falcon 3046, Becepidermis. However, the effect of TGF-P on differenti- ton Dickinson, Oxnard, CA). To induce differentiation, ation of epidermal keratinocytes has not been fully elu- keratinocytes were cultured in the medium containing cidated. The relationship between growth inhibition 1.8 mM Ca2+ (Hennings et al., 1980). Thus, keratiand differentiation is important for understanding the nocytes under undifferentiated and differentiated congrowth-inhibitory mechanism of TGF-P, since several ditions correspond to cells cultured in medium containrespectively. reagents inhibit cell growth by inducing differentia- ing 0.1 mM Ca2+ and 1.8 mM (=a2+, tion. Physiological differentiation of epidermal keratiMeasurement of cell growth area nocytes involves a series of changes in morphology and After the culture period, usually one week, the cells protein expression in the maturing cells. The differentiation of epidermal keratinocytes can be understood a t were fixed in 10% formalin in PBS and stained with the molecular level as alterations in expression of ker- 0.2% crystal violet in 0.1 M citric acid. The total atin intermediate filament (Fuchs and Green, 1978), stained area was measured by a computer-aided video the appearance of a major envelope precursor protein camera and image processor system (Hashimoto et al., involucrin (Rice and Green, 1979), and the formation of 1987). Briefly, stained cells were photographed with a the crosslinked cornified envelope (Sun and Green, three-tube-video-camera (Ikegami Tsushinki Co., To1976). One of the difficulties in evaluating differentia- kyo, model ITC-730);the image was then processed by tion is how to quantitate the differentiation marker. Nexus 6400 (Kashiwagi Research Corp., Tokyo) and For this purpose, we measured the number of cells im- the transformed information was sent to a personal munoreactive to involucrin using anti-involucrin anti- computer (NEC, Tokyo, model PC-9801-VM2), which body and a flow cytometer. We also quantitated the calculated the percentage of stained arealtotal area. cornified envelope by counting radioactivity after sol- We usually expressed cell growth as the percentage of ubilizing [35Sl-methionine-labeled cells with SDS-DTT stained area to the total area. In several experiments, solution. In the present study, we investigated the ef- cell growth was calculated by this method as well as fects of TGF-p on cell growth and differentiation induc- the conventional method of cell counting using a hetion of human keratinocytes. We report here that TGF- mocytometer, and results from both calculations were p inhibits the growth of human keratinocytes under in good agreement. Incorporation of I3H1thymidine both differentiated and undifferentiated conditions. By contrast, TGF-p inhibited the differentiation of human Keratinocytes were plated in 24-well trays (Falcon keratinocytes under low Ca2 conditions, but en- 3047, Oxnard, CA) at 5 x lo4 cells/well. After subconhanced it under high Ca2 conditions (differentiation- fluency, cells were treated with various concentrations promoting culture environment). of TGF-P for 40 hr, and pulse-labeled with 1 pCi of [meth~l-~HIthymidine (25 Cilmmol, CEA) for 3 hours. MATERIALS AND METHODS Next, cultures were washed three times with ice-cold PBS and twice with cold 10%TCA and solubilized with Transforming growth factor TGF-P1 prepared from human platelets was used in 1N NaOH. This solution was mixed with scintillation the present study. It was generously supplied by Mary- fluid and radioactivity was counted using a scintillaland Research Laboratories of Otsuka Phamaceutical tion counter. Averages of triplicate measurements were calculated. Co. LTD (Bethesda, MD). RNA purification and Northern Cell culture blot hybridization Human keratinocytes were cultured using a modifiTotal RNA was extracted from cultured human kecation of the technique of Wille et al. (1984). Briefly, ratinocytes by solubilization with guanidine thiocynormal human skin was obtained during plastic sur- anate followed by treatment with chloroform/phenol gery, cut into 3- to 5-mm pieces, and floated on dispase (Lazarus et al., 1987); 6 pg of total RNA was electrosolution (500 U/ml) overnight at 4°C (Kitano and phoresed on a 1.2% agarose gel, transferred onto a nyOkada, 1983). After separation of epidermis from der- lon membrane, then dried and baked according to Mamis by forceps the epidermal sheets were rinsed with niatis et al. (1982). The c-myc probe used in this study Ca2+ and Mg'+-free PBS and incubated in a 0.25% was a 2.2-kb fragment and was obtained from the Japtrypsin solution for 10 min a t 37"C, and the epidermis anese Cancer Research Resources Bank (Tokyo).Labelwas teased with forceps. Epidermal cells were sus- ing of the probe was performed using a Multiprime pended in optimized nutrient medium MCDB153 (Ky- DNA labeling kit (Amersham), according to manufacokuto Co., Tokyo, Japan) supplemented with EGF (10 turer's instructions. Hybridization was performed a t ng/ml), insulin (5 mg/ml),hydrocortisone (5 x M), 42°C for a minimum of 16 hr. The filters were then ethanolamine (0.1 mM), phosphoethanolamine (0.1 washed in 2 x SSC containing 0.1% SDS at 50°C.The mM), and bovine hypothalamic extract (150 pg/ml) (Pit- washing solution was changed every 15 min, at least telkow and Scott, 1986). The stock solution consisted of four times. Filters were covered with Saran wrap and MCDB153 supplemented with 0.1 mM Ca2+ plus ele- exposed for various periods to Kodak XAR film at vated concentrations of amino acids (0.75 mM isoleu- -70°C with a intensifier screen. Northern blots were cine, 0.24 mM histidine, 0.09 mM methionine, 0.09 mM quantitated by densitometric scanning using a dual+

+

TGF-P EFFECTS ON HUMAN KERATINOCYTES

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wavelength flying-spot scanner (CS-9000, Shimadzu, Kyoto). Quantitative evaluation of keratinocyte differentiation Keratinocyte differentiation was evaluated by measuring cornified envelope formation and the expression of involucrin. The formation of the cornified envelope was quantitated by the method of King et al. (1986). Briefly, subconfluent cells3pown in six-well plates were labeled with 1 pCi [ Slmethionine (1,272 Ci/ mmol, Amersham) for 96 hr under appropriate conditions. The cells were removed by trypsinEDTA treatment and suspended in MCDB153 medium containing 1.8m M Ca2' and calcium ionophore X537A (HoffmanLaRoche, Nutley, NJ). After incubation for 2 hr, collected cells were dissolved in 2% SDS solution containing 20 mM dithiothreitol. Cornified envelopes were quantitated using a liquid scintillation counter aRer collecting on 10-mm pore size filters (LCWPO2500,Nihon Millipore Kogyo K.K., Yonezawa, Japan). Immunofluorescent staining of involucrin was carried out as described by Wilke et al. (1988) with some modifications. Keratinocytes dissociated by trypsid EDTA treatment were fed with FCS and centrifuged at 1,000 rpm for 5 min. Precipitated cells were fixed with 3.7% formaldehyde in PBS, cold ethanol, and then cold acetone. Immunochemical staining was performed using a commercially obtained staining kit (Biomedical Technologies Inc., Cambridge, MA). FITC-conjugated goat antirabbit IgG (Miles Biochemicals, Elkhart, IN) was used as a second antibody. Tris-buffered saline (10 mM Tris, 0.15 M NaCl, pH 7.6) containing 0.2% (w/v) Tween 20 was used as washing buffer and dilution buffer. The addition of 0.2% Tween 20 prevented the aggregation of cells without any change in the staining pattern. A total of lo4 stained cells were analyzed in each experiment by flow cytometry (FACScan, Becton Dickinson, Oxnard, CAI.

TGF- p ( n g l ml) Fig. 1. Effect of TGF-p on the growth of normal human keratinocytes cultured under low Ca2+ conditions (0.1 mM Ca2'). Cells were plated on a six-well plate at a density of 1.5 x lo4 cells/well. Next day, the medium was changed, and TGF-f3 was added at the various concentrations. Cultures were refed at day 4 with the same medium and culture was stopped afier 7 days. Cells were stained with crystal violet and the cell growth was calculated using a Nexus 6400 image processor system. Substantially similar results were obtained in two other experiments.

r

RESULTS Inhibition of human keratinocyte growth by TGF-0 The growth of normal human keratinocytes cultured under low Ca2 conditions (nondifferentiation-inducing environment) was inhibited by addition of TGF-P at 0.25 to 10 ng/ml (Fig. 1).Striking growth inhibition occurred a t 2 ng/ml. TGF-p at 2 ng/ml reduced cell growth by >20%. Significant inhibition was observed even at 0.5 ng/ml (data not shown). This dose dependency was almost the same among human keratinocytes from six donors of different ages (data not shown).

lrrl 0.5

1

5

10

+

Effect of TGF-f3on incorporation of [3Hlthpidine The effect of TGF-p on [3Hlthymidine incorporation was examined to determine whether TGF-f3 inhibits DNA synthesis in human keratinocytes. TGF-p at 0.5 ng/ml decreased r3H1thymidine uptake by 87.5%, as shown in Fig. 2. The data indicate that TGF-P suppressed DNA synthesis in human adult skin keratinocytes.

TGF- p ( n g l ml) Fig. 2. Inhibition of L3H]thymidine incorporation by TGF-B. Cells were plated in 24-well plates at 5 x lo4 cellsiwell and cultured under low Caz+ conditions (0.1 mM Ca2+).After subconfluency, cells were treated with TGF-p at various concentrations for 40 hr, and pulselabeled with 1 pCi of [methyL3H]thymidinefor 3 hr. The averages of triplicate measurements (2SD)are shown.

c-myc mRNA expression and its relationship to DNA sjmthesis Expression of the c-myc gene has been reported to be closely linked to cellular proliferation (Kelly et al., 1983; Kaczemarek et al., 1985). We investigated the regulation of the c-myc gene by TGF-P in human.keratinocytes. The level of c-myc mRNA declined in cells exposed to 5 ng/ml of TGF-p for 30 min and for 1 hr, although it increased to the control level once again

MATSUMOTO ET AL.

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TGF-0 1-treated

Untreated Time (h)

0.5

1

3

0.5

3

1

Fig. 3. Northern blot analysis of c-myc expression in human keratinocytes treated and untreated with TGF-p. Confluent cells were left untreated or were treated with 5 ng/ml TGF-p for 30 rnin, 1 hr, and 3 hr, and total RNA was extracted; 6 kg total RNA was added to each lane.

after 3 hr (Fig. 3), showing that the decrease of c-myc mRNA was transient. Scanning densitometry of the bands revealed an 83% reduction in c-myc expression a t 30 min. To examine the relationship between the decrease of c-myc and DNA synthesis, we measured 13H]thymidineincorporation after 1 , 3 , 6 24,and 48 hr exposure to 5 ng/ml of TGF-p (Fig. 4).['HIThymidine incorporation had not decreased 1 hr after addition of TGF-P. However, a significant decrease of 28.6%was observed at 3 hr, and inhibition was almost complete after 24 hr. By contrast, the striking decrease in the c-myc mRNA level was observed as early as 30 min after the addition of TGF-P, when l3H1thymidineincorporation showed no significant decline. The decrease of c-myc mRNA clearly preceded the decrease of l3H]thyrnidine incorporation. This suggests that reduction of the c-myc mRNA level precedes the growth arrest of human keratinocytes. Effect on keratinocyte differentiation Since growth inhibition and induction of differentiation are closely related to each other in keratinocytes, the above results led us to examine the relationship between TGF-P and induction of differentiation. Therefore, we investigated the effect of TGF-p on the differentiation of human adult skin keratinocytes under low Ca2+ conditions (0.1 mM Ca") and high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment). First, the inhibitory effect of TGF-p on keratinocytes under high Ca2 conditions was examined. As shown in Figure 5, cell growth was inhibited by TGF-P, and the dose dependency was almost identical to that of keratinocytes under low Ca2+conditions (see Fig. 1). This demonstrates that TGF-p inhibited human keratinocyte growth in a similar manner regardless of cellular conditions, i.e., low or high Ca2+. Next, keratinocytes under both low and high Ca2+ conditions were cultured in the presence of TGF-p a t 1 and 20 ng/ml or in its absence for 96 hr, and involucrinpositive cells were detected using a flow cytometer (Fig. 6). With keratinocytes cultured under low Ca2' +

I

I

24

-0

48

Incubation period ( h )

Fig. 4. Time course of inhibition of PHIthymidine incorporation by 5 ng/ml TGF-p. Cells were plated on 24-well plates at 5 x lo4 cells/ well and cultured under low Ca2+ conditions (0.1 mM Ca''). Next day, cells were treated with 5 ng/ml TGF-P for 1, 3, 6, 24, and 48 hr and pulse-labeled with 1 FCi [methyl-3Hlthymidinefor 1 hr. The averages of triplicate measurements (?SD)are shown.

conditions, 7.0% of cells were positive for involucrin. The shift to high Ca2+ conditions increased involucrinpositive cells to 9.3%. In high Ca2+ conditions, TGF-P enhanced involucrin expression in a dose-dependent manner. The percentages of involucrin-positive cells increased from 9.3%to 14.4%and to 18.3%by addition of TGF-p a t 1 ng/ml and 20 ng/ml, respectively. By contrast, TGF-P had the opposite effect on involucrin expression under low Ca2+conditions. TGF-P inhibited involucrin expression from 7.0% to 5.5%and to 3.3%at 1 ng/ml and 20 ng/ml, respectively. This bidirectional effect of TGF-P was also observed in cornified envelope formation (Fig. 7). The shift from low Ca2+ to high Ca2+ increased cornified envelope formation by 78%. TGF-p enhanced cornified envelope formation under high Ca2 conditions in a dose-dependent manner, i.e., by 18.8%and 30.7% in the presence +

TGF-I3 EFFECTS ON HUMAN KERATINOCYTES

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0 : control : 1 nglrnl TGF-n

TGF-

p (ng/ml>

Fig. 5. Effect of TGF-P on the growth of normal human keratinocytes cultured under high Ca2+ conditions (1.8 mM CaZ+).Cells were plated on a six-well plate at a density of 1.5 x lo4 cells/well. Next day, the medium was changed and TGF-P was added at the various concentrations. Cultures were refed at day 5 with the same medium and culture was stopped after 9 days. Cells were stained with crystal violet, and cell growth was calculated using a Nexus 6400 image processor system. Substantially the same results were obtained in two other experiments.

ICa2+conc.l I 1

0.1 mM

1 I

1.8 mM

I I

Fig. 7. Effeet of TGF-6 on the cornified envelope formation of human keratinocytes. After subconfluenc , cultures were refed with fresh medium supplemented with 1pCi ["Slmethionine, and cultured under low and high CaZ+conditions in the presence or absence of TGF-P for 96 hr. Then, cells were dissociated by EDTNtrypsin treatment and submitted to quantitation of the cornified envelope. The averages of triplicate measurements (? SD) are shown.

nocytes. Our results are in agreement with previous reports (Moses et al., 1985; Shipley et al., 1986; k i s s and Sartorelli., 1987). In an attempt to understand the I 0 :control I mechanism of growth inhibition by TGF-p in normal human keratinocytes, its effects on DNA synthesis, cmyc expression and markers of differentiation, i.e., involucrin and cornified envelope, were examined. We showed that inhibition of DNA synthesis was observed only 3 hr after the addition of TGF-p, indicating that this inhibition is one of the early phenomenon in TGF-p growth inhibition of human keratinocytes. The remarkable decline in the c-myc mRNA level preceded the inhibition of DNA synthesis and it was observed as early as 30 min after exposure to TGF-p. This decrease of c-myc expression was intensively studied in an epidermal growth factor (EGFbdependent cell line derived from mouse epidermal keratinocytes (Coffey et al., 1988a). After 1 hr, TGF-p caused a remarkable decrease in c-myc expression in both EGF-restimulated and rapidly growing Balb/MK cells. c-myc expression ICa*+conc.l 0.1 m M I 1.8 m M I returned to the control level in EGF-restimulated cells after 3 hr, as was observed in our experiments. A reFig. 6. Effect of TGF-b on involucrin expression in normal human keratinocytes. After subconfluency, cultures were refed with fresh duction in c-myc expression by TGF-p was also remedium and cultured under low and high Ca2' conditions in the ported in human endothelial cells (Takehara et al., presence or absence of TGF-P for 96 hr. Then, cells were dissociated by EDTNtrypsin treatment and submitted to immunochemical staining 1987) and a human breast cancer cell line (Fernandezand subsequent analysis by flow cytometry. The averages of triplicate Pol et al., 1987). These findings suggest that reduction measurements (?SD) are shown. of the c-myc mRNA level is a common occurrence in response to TGF-P. The c-myc gene plays a n important role in the regulation of cellular processes such as proof TGF-j3 a t 1 ng/ml and 20 ng/ml, respectively. How- liferation and differentiation (Cole, 1986). Thus, the ever, under low Ca2+ conditions, it inhibited cornified reduction of c-myc expression is possibly one of the envelope formation by 20.6% and 53% a t 1ng/ml and main factors in the growth inhibition of human keratinocytes by TGF-P. 20 ng/ml, respectively. The regulation of proliferation is closely related to DISCUSSION cell differentiation in various cells. To understand the We have demonstrated in this report that TGF-P is a mechanism of growth inhibition of human keratipotent growth inhibitor of normal human kerati- nocytes by TGF-P, it is important to clarify the rela-

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tionship between TGF-p-induced growth inhibition and terminal differentiation. In epidermal keratinocytes in vitro, Ca2+ is important in inducing terminal differentiation (Fuchs et al., 1988). A shift of Ca2+ concentration from low to high in the medium induces terminal differentiation and decreases DNA synthesis in keratinocytes (Hennings et al., 1980). The present study shows that TGF-p inhibited human keratinocyte growth in a dose-dependent manner, regardless of the Ca2+ concentration of the medium. This suggests that TGF-P inhibits human keratinocyte growth, regardless of differentiation. However, it is of interest t o determine whether TGF-(3 can modulate differentiation of human keratinocytes. Several groups reported on the relationship of TGF-P and differentiation in epithelial cells, including human keratinocytes. TGF-p induced terminal differentiation in normal human bronchial epithelial cells (Masui et al., 1986), while it had no effect on the expression of epithelial differentiation markers in a murine keratinocyte cell line, Balb/MK (Coffey et al., 1988b). TGF-p stimulation of differentiation of human keratinocytes was reported in a medium containing 1 mM Ca2+ (Reiss and Sartorelli, 1987). The same group also reported that the membrane-associated form of transglutaminase, a marker of differentiation, is not affected by TGF-p treatment of established murine skin keratinocytes (Reiss and Zhou, 1989). There is a clear discrepancy among these reports, which our results may clarify to some extent, although variation in the response of different cell types to TGF-p has also been demonstrated (Moses et al., 1985). We found that TGF-p has bidirectional effects on the differentiation of human keratinocytes, depending on the calcium concentration in the medium: TGF-p inhibited differentiation under low Ca2+ conditions while it enhanced the differentiation under high Ca2+conditions. In previous reports, the Ca2+ concentration differed and this might be the source of the conflicting results. The biological role of TGF-i3 bidirectional regulation of keratinocyte differentiation is not clear. However, physical analysis of tissue sections from mouse and human epidermis has demonstrated the existence of a Ca2+ gradient in which the Ca2+ content in the basal layer is extraordinarily low, while the Ca" content of the granular layer is extremely high (Menon et al., 1985; Malm uist et al., 1984). Induction of differentiation by results in an increase of TGF-p2 mRNA and peptide but produces a decrease in the expression of TGF-PI in mouse keratinocytes (Glick et al., 1990). These data indicate that TGF-p1 has more significant biological roles in regulating growth and differentiation of cells in basal layer than in the upper layer. It is possible to speculate that the major role of TGF-pl in vivo could be to induce growth arrest and prevent terminal differentiation in basal cells. Basal cells'are supposed to retain pruliferative potential without entering terminal differentiation while they are growth-arrested reversibly (Shipley et al., 1986). In this study, we demonstrated.that TGF-p can uncouple the inhibition of proliferation from the induction of terminal differentiation in human keratinocytes. This finding is consistent with previbus reports on mouse keratinocytes (Coffey et al., 1988b; Reiss and Zhou, 1989). Taken together, it seems likely

C3+

that growth inhibition of human keratinocytes by TGF-P is not the result of induction of differentiation. TGF-P may have an important role in the delicate regulation of growth and differentiation of epidermal keratinocytes in cooperation with Ca2 +

ACKNOWLEDGMENTS This work was supported by a grant to K. Yoshikawa from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED Akhust, R.J., Fee, F., and Balmain, A. (1988) Localized production of TGF-P mRNA in tumor promotor-stimulated mouse epidermis. Nature (Lond.), 331t363-365. Coffey, R.J., Jr., Bascom, C.C., Sipes, N.J., Graves-Deal, R., Weissman, B.E., and Moses, H.L. (1988a) Selective inhibition of growthrelated gene expression in murine keratinocytes by transforming growth factor p. Mol. Cell. Biol., 8t3088-3093. Coffey, R.J. Jr., Sipes, N.J., Bascom, C.C., Graves-Deal, R., Pennington, C.Y., Weissman, B.E., and Moses, H.L. (1988b) Growth modulation of mouse keratinocytes by transforming growth factors. Cancer Res., 48:1596-1602. Cole, M.D. (1986) The c-myc oncogene: Its role in transformation and differentiation. Annu. Rev. Genet., 20:361-384. Fernandez-Pol, J.A., Talkad, V.D., Klos, D.J., and Hamilton, D.J. (1987) Suppression of the EGF-dependent induction c-myc protooncogene expression by transforming growth factor p in a human breast carcinoma cell line. Biochem. Biophys. Res. Commun., 144: 1197-1205. Fuchs, E., and Green, H. (1978) The expression of keratin genes in epidermis and cultured epidermal cells. Cell, 15:887-897. Fuchs, E., Albers, K., and Kopan, R. (1988) Terminal differentiation in cultured human epidermal cells. Adv. Cell Cult., 6:l-33. Glick, A.B., Danielpour, D., Morgan, D., Sporn, M.B., and Yuspa, S.H. (1990) Induction and autocrine receptor binding of transforming growth factor-p2 during terminal differentiation of primary mouse keratinocytes. Mol. Endocrinol., 4t46-52. Hashimoto, M., Hata, R., Isomoto, A,, Tyuma, I., and Fukuda. M. (1987) Color analysis method for estimating the oxygen saturation of hemoglobin using a n image-input and processing system. Anal. Biochem., 162t178-184. Hennings, H., Michaels, D., Cheng, C., Steinert, P., Holbrook, K., and Yuspa, S.H.(1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell, Z9:245-254. Kaczemarek, L., Hyland, J.K., Watt, R., Rosenberg, M., and Baserga, R. (1985) Microinjected c-myc as a competent fador. Science, 228: 1313-1314. Kelly, K., Cochran, B.H., Stiles, C.D., and Leder, P. (1983) Cell-specific regulation of the c-myc gene by lymphocyte mitogens and plateletderived growth factor. Cell, 35~603-610. King, I., Sharon, L., Mella, and Sartorelli, A.C. (1986) A sensitive method to quantify the terminal differentiation of cultured epidermal cells. Exp. Cell Res., 167:252-256. Kitano, Y., and Okada N. (1983) Separation of the epidermis sheet by dispase. Br. J. Dermatol. 108:555-560. Lazarus, G., Hashimoto, K., Vassali, J.D., Gubler, P., Jensen, P., Baird, J., and Belin, D. (1987) mRNA for c-fos and a d i n is present in epidermis and epidermal cell cultures. J. Invest. Dermatol., 88: 502 (abst). Malmquist, K.C., Carlson, L.E., Forslind, B., Roomans, G.M., and Akselsson, D.R. (1984) Proton and electron microprobe analysis of human skin. Nucl. Instrum. Methods Phys. Res., 3:611-617. Maniatis, T., Fritsh, E.F., and Sambrook, J . (1982) Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Masui, T., Wakefield, L.M., Lechner, J.F., LaVeck, M.A., Sporn, M.B., and Harris, C.C. (1986) Tvue beta transformine factor is the urimary differentiation-induci& serum factor for nGrmal human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA, 83:2438-2442. Menon, G . B , Grayson, S., and Elias, P.M. (1985) Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytometry. J. Invest. Dermatol., 74.508-512. Moses, H.L., Tucker, R.F., Leof, E .B., Coffey , R.J., Jr., Halper, J., and Shipley, G.D. (1985) Type beta transforming growth factor is a growth stimulator and a growth inhibitor. In: Cancer Cells. J . Feramisco, B. Ozanrfe, and C. Stiles, eds. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, Vol. 3, pp. 65-71.

TGF-B EFFECTS ON HUMAN KERATINOCYTES Pittelkow, M.R., and Scott, R.E. (1986) New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin. Proc., 61:771-777. Reiss, M., and Sartorelli, A.C. (1987) Regulation of growth and differentiation of human keratinocytes by type p transforming growth factor and epidermal growth factor. Cancer Res., 47:6705-6709. Reiss, M., and Zhou, Z-L. (1989) Uncoupling of the calcium-induced terminal differentiation and the activation of membrane-associated transglutaminase in murine keratinocytes by type-beta transforming growth fador. Exp. Cell Res., 183:lOl-111. Rice, R.H., and Green, H. (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linkage envelope: Activation of the cross-linking by calcium ions. Cell, 18:681-694. Roberts, A.B., Anzano, M.A., Lamb, L.C. Smith, J.M., and Sporn, M.B.(1981) New class of transforming growth factors potentiated by epidermal growth factor: Isolation from non-neoplastic tissues. Proc. Natl. Acad. Sci. USA, 78.5339-5343. Shipley, G.D., Pittelkow, M.R.,Wille, J.J., Scott, R.E., and Moses, H.L. (1986) Reversible inhibition of normal human prokeratinocyte

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