JOURNAL OF CELLULAR PHYSIOLOGY 146:277-289 (1991)

Growth Factor-Independent Proliferation of Normal Human Neonatal Keratinocytes: Production of Autocrine- and Paracrine-Acting Mitogenic Factors PAUL W. COOK, MARK R. PITTELKOW, AND GARY D. SHIPLEY* Department of Cell Biology and Anatomy, the Oregon Health Sciences University, Portland, OR 9720 1 (P. W.C., C.D.S.J, Department of Dermatology, Ma yo CliniclFounddtion, Rochester, MN 55905 (M.R. P.)

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10'lcells cm', the cells proliferated continuously and the addition of IGF-I, EGF, TGFa, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate,TGFP, or surarnin inhibited keratinocyte growth factor-independentproliferation. The addition of EGF, TCFa, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGFa peptide at a concentration of approximately 235 pgiml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derivedCM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes,keratinocytederived CM stimulated the incorporation of [ 'Hlthymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and ECF-receptorless mouse NR6 cells. CM-stimulated ['Hlthymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and 13H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-actingfactors produced by keratinocytes mediated their effect through the activation of both ECF receptordependent and EGF receptor-independentmitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin. The regulation of autocrine secretion of growth factors and the control of cellular proliferation have been incorporated into a hypothesis to account for the differences in growth regulation between normal and malignantly transformed cells. This hypothesis was originally developed from observations comparing the transformingigrowth-promotingproperties of proteins present in the conditioned medium of transformed and nontransformed mouse fibroblast cell lines (Sporn and Todaro, 1980). A corollary to the original hypothesis suggested that cells derived from normal tissue were dependent on exogenous mitogens for continued proliferation, while many tumorigenic cells often did not require exogenous growth factors. Exceptions to this generalization have become evident as growth factors, cytokines, or "transforming" growth factors have been implicated in normal regulation of cell proliferation during growth and development (Goustin et al., 1986; Deuel, 1987; Knochel and Tiedman, 1989; Derynck, 1988; Mercola and Stiles, 19881, dermal would repair and inflammation (Wahl et al., 1989; Clark and Henson. 1988), and liver regeneration (Braun et al., 1988; Kan et al., 1989; Mead and Fausto, 1989). Transforming growth factor type alpha (TGFa) (Derynck, 1988; 4:

1991 WILEY-LISS, INC.

Gottlieb et al., 1988; Madtes et al., 1988; Pittelkow et al., 1989; Coffey et al., 1987a; Mead and Fausto, 1989; Cook et al., 1990; M. R. Pittelkow, submitted), transforming growth factor type beta (TGFP) (Pittelkow et al., 1988), basic fibroblast growth factor (bFGF) (Sternfeld et al., 1988; Halaban et al., 1988; Cook et al., 19901, and platelet-derived growth factor (PDGF) (Deuel, 1987; Majesky et al., 1988) have also been shown to be expressed by normal human cells in vitro, suggesting that the regulation of growth in normal tissues and cell types can be affected by endogenously produced growth factors which act through autocrine and/or paracrine mechanisms. The human skin consists of a vascularized dermis that is separated by a basement membrane from the avascular epidermis. The epidermis is composed of several topologically organized compartments, including a proliferative basal layer and post-mitotic suprabasal layers which differentiate and form the keraReceived August 20, 1990; accepted October 26, 1990. "Towhom reprint requests/correspondence should be addressed at OHSU:L215 3181 SW Sam Jackson Park Road, Portland, OR 97201.

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tinized outer layer of the skin. It is generally regarded that proliferating keratinocytes within the basal layer of the epidermis must rely on the vascularized dermal layers for nutritive support. Several studies have suggested that neonatal human keratinocytes may produce autocrine- or paracrine-acting growth factors in vitro (i.e., Gilchrest et al., 1983) and identification of these factors and their potential mechanisms of action is ongoing. Moreover, little is known about the role these factors play in regulating keratinocyte proliferation in vivo. We, as well as others, have shown that human keratinocytes produce TGFa (Coffey et al., 1987; Pittelkow et al., 1989; Cook et al., 1990; M. R. Pittelkow, submitted), TGFp (Shipley et al., 1986; Pittelkow et al., 19881, bFGF (Halaban et al., 1988; Cook et al., 19901, and interleukins (Blanton et al., 1989; Kirnbauer et al., 1989). Although elevated expression of TGFa has been shown t o correlate with hyperproliferative psoriatic epidermis (Gottlieb et al., 1988;Elder et al., 19891, it is not known whether any of the undefined epidermal-derived factors are involved in autocrine and/or paracrine growth regulation of skin-derived cell types in vivo. TGFa and EGF exert their mitogenic effects through cell surface EGF receptors and have been demonstrated to be potent mitogens for human keratinocytes (Barrandon and Green, 1987; Shipley and Pittelkow, 1987a; Shipley et al., 1989; M. R. Pittelkow, submitted). TGFa mRNA and protein are expressed in rapidly growing keratinocytes and the levels of TGFa decrease as cells become confluent (M. R. Pittelkow et al., submitted). TGFa levels increase when keratinocytes in defined medium are treated with EGFITGFa, TPA, or serum (Coffey et al., 1987a; Pittelkow et al., 1989; Cook et al., 1990; M. R. Pittelkow, submitted). The effect of these agents is more pronounced when the cells are near or at confluency when TGFa expression is low. Although earlier studies suggested that human keratinocytes require EGF/TGFa or FGFs to form colonies at low density (Barrandon and Green, 1987; Shipley and Pittelkow, 1987; O’Keefe et al., 1988; Shipley et al., 1989), we have recently demonstrated that exogenous EGF/TGFu is only required for initiating clonal growth and that keratinocyte colonies become independent of exogenous EGFiTGFa or FGFs after a small colony has been established (M. R. Pittelkow, submitted). We have also shown that human keratinocytes plated at high density into medium which lacks EGF, TGFa, or FGFs continue to proliferate in the absence of exogenously added growth factors (Shipley et al., 1989). Furthermore, the high density, growth factor-independent proliferation of human keratinocytes was inhibited by treatment of these cultures with heparin sulfate (Shipley et al., 1989) or with a monoclonal antibody which acts as a competitive antagonist of the human EGF receptor (M. R. Pittelkow, submitted). Heparin sulfate is a sulfated glycosaminoglycan (GAG) with physiochemical properties that allow numerous molecular and cellular interactions (Hook et al., 1984). Heparin sulfate has been demonstrated to exert antiproliferative effects on several different cell types, in vitro and in vivo (Hook et al., 1984; Shipley et al., 1989; Clowes and Karnovsky, 1977; Imamura

and Mitsui, 1987; Majesky et al., 1987; Robertson and Golstein, 1988; Wright et al., 1989).Heparan sulfate is closely related to heparin sulfate and has also been shown to exert growth inhibitory effects in some of the same cell systems. These GAGs are commonly found covalently linked to protein core molecules in the form of proteoglycans (PGs) that can be isolated from cells or cell culture medium (Hook et al., 1984; Rahemtulla et al., 1989) and are components of skin and basement membranes. Normal human keratinocytes have been shown to produce heparan, chondroitin and dermatan sulfate PGs which are localized in the medium or with the cell monolayer (Rahemtulla et al., 1989). Interestingly, this same study demonstrated that the cell associated PGs of differentiating keratinocyte cultures had higher levels of heparan sulfate than PGs isolated from proliferating cells. Collectively, these observations suggest that sulfated GAGs or PGs could act as regulators of growth and differentiation in vivo. In the current study, we have explored the proliferation of normal human keratinocytes cultured in the absence of exogenously added growth factors to determine if keratinocyte cultures produce factors which act as autocrine and/or paracrine stimulators of growth. The results of our current study demonstrate that normal human keratinocytes secrete autocrine/paracrine growth factors which stimulate various target cells, including dermal fibroblasts and epidermal keratinocytes. MATERIALS AND METHODS Media a n d supplements Medium MCDB 202a was prepared in our laboratory using previously described methods (Hammond et al., 1984; Mckeehan and Ham, 1977). The high amino acid (HAA) formulation of MCDB 153 was prepared by adding the appropriate amount of a lOOx concentrated amino acid stock to the basal medium at the final concentrations previously described (Shipley and Pittelkow, 19871, or obtained from Clonetics Corp. (San Diego, CA) as keratinocyte basal medium (KBM). MCDB 402 was prepared in our laboratory as previously described (Shipley and Ham, 1981). DME was purchased from Gibco Laboratories (Grand Island, NY). Highly purified human platelet derived TGFpl was a generous gift from B. E. Magun (OHSU, Portland, OR) and was stored lyophilized at -20°C and used as described in the text. Human recombinant EGF and IGF-I were purchased from Amgen Corp. (Thousand Oaks, CA). EGF was dissolved in Hepes buffered saline solution A (Shipley and Ham, 1981) and stored at -20°C. Human recombinant TGFa was a generous gift from R. Derynck (Genentech, South San Francisco, CA) and was stored at -80°C in 0.1% TFA until use. Bovine insulin (Sigma Chemical Co., St. Louis, MO) was dissolved in 10 mM HCI at 5 mg/ml, filter sterilized, and stored at -20°C. IGF-I was dissolved in 10 mM HCI at 10 mg/ml and stored at -20°C. Phosphoethanolamine, and ethanolamine (Sigma Chemical Co., St. Louis, MO), dissolved in solution A at 1 0 0 0 the ~ final concentration and stored at - 20°C. Hydrocortisone (Sigma Chemical Co., St. Louis, MO) was dissolved in 95% ethanol at 1 0 0 0 ~the final concentration and

KERATINOCYTE AUTOCRINE GROWTH

stored at -20°C. Suramin was purchased from Mobay Chemical Corporation. Heparin sulfate purified from porcine intestinal mucosa (Sigma Chemical Go., St. Louis, MO; or Hepar Industries Franklin, OH) was dissolved in solution A at 10 mgiml and stored a t - 20°C. Mouse anti-human EGF receptor monoclonal antibodv (monoclonal antibodv LA1. lot 10055) was purchaied from Upstate Bio{echnology, Inc., (Lake Placid, NY). Cells Primary human foreskin fibroblasts (normal human fibroblast, strain 5) were isolated by collagenase digestion of minced foreskin tissue and grown in MCDB 202a supplemented with 5% FBS as previously described (Shipley et al., 1989). Normal human fibroblasts were cryopreserved in the same medium containing 10% DMSO and 10% FBS. Cells used for the experiments reported here were between population doubling level 13 and 25. Primary cultures of human foreskin keratinocytes were isolated by the trypsin float technique as previously described (Wille et al., 1984). Stock cultures were maintained (Shipley et al., 1989; Shipley and Pittelkow, 1987) in an actively growing state in HAA medium MCDB 153 supplemented with 0.2% (v/v) bovine pituitary extract (BPE), culture grade EGF (10 ng/ml), insulin (5 pg/ml), hydrocortisone (5 x lo-’ M), ethanolamine (1 x l o p 4 M) and phosphoethanolamine (1 x M), gentamicin sulfate (10 ug/ml) or in KBM with the same supplements, hereafter referred t o as “complete” medium. Complete medium without BPE, EGF, and insulin is referred t o in the text as “standard” medium. Primary or secondary cultures of keratinocytes were used to innoculate growth and conditioned medium experiments. The AKR-2B mouse cell line was cultured as previously described (Shipley et al., 1984). The NR6 (EGF receptor minus variant) subclone of the mouse swiss 3T3 cell line (Pruss and Hershmann, 1979; Schneider et al., 1986) was routinely maintained at subconfluent densities in DME supplemented with 10% FBS and in 10 ugiml gentamicin sulfate. Keratinocyte clonal growth assays The clonal growth of keratinocytes was performed in 6-well tissue culture plates. Briefly, cells were removed from stock culture flasks using 0.025% trypsin/0.01% EDTA and resuspended in cold solution A containing 0.54 FBS. The cells were centrifuged at 180g and the pellet resuspended in complete medium. The cells were inoculated into the 6-well plates containing 2 mls of complete medium (600 cells/well, 9 cm‘) and incubated for 24 hours to allow the cells to attach to the surface. Cultures were then washed 2 times with 3 mls of solution A, and 2 mls of standard medium supplemented with insulin (5 pg/ml) was added to each well. Experimental factors were subsequently added as described in the legends to figures. The keratinocytes were grown without a medium change for 10 days. After the incubation, the colonies which developed were fixed in 10% formalin and stained with crystal violet for photography.

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High density keratinocyte proliferation assay Keratinocytes were plated in complete medium at a density of 1 x lo4 or 0.5 x lo4 cells/cm2 in 6-well (9 cm? tissue culture plates. After 24 hours of culture, the cells were washed 2 times with 3 ml of solution A, and 2 ml of standard medium was added to each well. Experimental factors were then added as described, and the cells were cultured 1-5 days, with 1 medium change at day 3. At the end of each incubation period, the cells were fixed in 4 mls of 70% EtOH and later assayed to determine total DNA contentiwell; 70% EtOH was added to several blank wells to serve as blank controls in the DNA assay (described below). Determination of total cellular DNA Determination of total DNA per well from the keratinocyte proliferation experiments was carried out utilizing a modification of previously described protocols (Hinegardner, 1971; Firestone et al., 1986).Briefly, the 70% EtOH fixative was removed from each well and the wells were air dried. Herring sperm DNA standards (0-10 pg) were added to some of the blank control wells; 0.9 mls of 10% DABA (3,5-diaminobenzoic acid dihydrochloride, Aldrich Chemical Go.) solution was added to each well. The lids of the multi-well plates were taped in place and the samples were subsequently incubated in a 60°C water bath for 1 hour. Four and one-half mls of 1N HC1 was then dispensed to each well, and after shaking each plate, 2.5 mls from each well was transferred to a 10 x 75 mm borosilicate glass test tube. Fluorescence of each sample was monitored at 465-540 nm when excited at 405 nM in a Turner model 112 fluorometer. The fluorometer was zeroed with DABA solution from blank control wells. The emission filter slit width was adjusted in the presence of DABA solution from wells containing 10 kg herring sperm DNA, such that 1 fluorescence unit equalled 1 X lo4 keratinocytes (as determined by an assay which compared predetermined numbers of cells to DNA standard fluorescent units). DNA synthesis assays Measurement of DNA synthesis in AKR-2B cells was performed as previously described (Shi ley et al., 1984). AKR-2B cells were plated at 5 x 10Bcells/cm2in 24 well dishes in McCoys 5A supplemented with 5 8 FBS and cultured for five days. The medium in each well was replaced with MCDB 402 without serum and the cells were incubated for 48 additional hours. After this culture period, the medium in each well was removed and replaced with 1 ml of MCDB 402 supplemented with insulin (0.5 ug/ml), penicillin (100 U/ml), and streptomycin (100 pgiml). Experimental factors were added to each well as described in the figure legends. Relative incorporation of I3H]thymidine was determined as previously described (Shipley et al., 1984). Normal human fibroblast stock flasks were trypsinized at 4°C with 0.05% trypsin, resuspended in solution A containing 0.54 FBS, centrifuged and resuspended in medium MCDB 202a supplemented with 1.0 ugiml insulin. The fibroblasts were plated at

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5 x lo4 cells/cm2in Corning 24 multi-well plates and incubated for 48 hours at 37°C. After this culture period, varied concentrations of experimental factors were added to the individual wells, as described in the figure legends. Mouse anti-human EGF receptor monoclonal antibody (mAb LAl), which has been reported to competitively block EGF binding and subsequent activation of the human EGF receptor, was also added to some of the experimental wells just prior to the addition of the growth factors as described in the text. After 20 hours incubation, [3H]thymidine (50-80 Ci/mmole, NEN) was added t o each well at a final concentration of 1 uCi/ml and the cells were incubated for an additional 4 hours. Incorporation of 13Hlthymidine was determined as described above for AKR-2B cells. NR6 cells were plated at 2 x lo4 cells/cm’ in 24 well plates in MCDB 402 supplemented with 5% FBS and cultured for 3 days. After this culture period, the medium from each well was removed and replaced with MCDB 402 containing 0.5% FBS and the cells were cultured for an additional 48 hours. The medium was then removed from each well and replaced with 1ml of the same medium containing insulin (5 ug/ml). Experimental factors and L3H]thymidine (1 uCi/ml) were added to each individual well as described in the Figure legends. After 24 hours incubation, incorporation of [3Hlthymidine was determined as described above for AKR-2B cells. Preparation of concentrated conditioned medium Keratinocytes were plated in complete medium at a density of 2.5 x lo3 cells/cm2in 100 mm tissue culture dishes. After 48 hours of incubation, the cells were fed fresh complete medium and 24 hours later the cells were washed 3 times with solution A and the medium replaced with standard medium. After 24 hours of additional incubation, the medium was discarded and replaced with fresh standard medium. Standard medium conditioned by human keratinocytes was collected every 24 hours for 4-5 days until the cells reached 90-95% confluency. Keratinocyte conditioned medium (CM) was collected and frozen at -80°C. CM was later, thawed, pooled and concentrated 43 fold (at 4°C) using a Millipore Minitan concentrator utilizing 10,000 molecular weight cut-off filters. TGFa radioimmune assay (RIA) Concentrated CM was assayed for the presence of immunoreactive TGFa using a TGFa RIA kit (Biotope Inc., Seattle, WA). The manufacturer’s protocol was utilized for these experiments. RESULTS We have previously demonstrated that keratinocytes plated at densities above 1 x lo3 cellsicm’ will proliferate in the absence of exogenous growth factors such as EGF, TGFa, or FGFs and that proliferation under these conditions is inhibited by the presence of heparin sulfate or a monoclonal antibody which competitively antagonizes EGF binding to the human EGF receptor (Shipley et al., 1989; M.R. Pittelkow, submitted). In order to explore this phenomenon further, we performed experiments to test the effect of both growth

stimulators and inhibitors on the EGF-independent proliferation of high density keratinocyte cultures. Exogenously applied growth factors exert only minor effects o n the EGF-independent proliferation of human keratinocytes in standard medium Traditionally the growth of human keratinocytes has been accomplished with medium containing EGF/ TGFa (Barrandon and Green, 1987; Shipley et al., 1989; Shipley and Pittelkow, 1987). In light of our recent discovery that human keratinocytes proliferate at high cell densities without the addition of exogenous EGF/TGFa or FGFs (Shipley et al., 1989; M. R.Pittelkow, submitted), we decided to further characterize this growth state. We compared the proliferation of keratinocytes in the presence or absence of different growth factors (bFGF, aFGF, TGFa), with or without the addition of IGF-1 (Fig. la). Keratinocytes cultured in the absence of any polypeptide growth factors (standard medium) underwent approximately 2.5 doublings in 5 days and the addition of IGF-I resulted in only a slight enhancement of this rate of proliferation. Addition of TGFa, bFGF, or aFGF in the presence or absence of IGF-I produced essentially the same result. Because the addition of IGF-I or insulin resulted in consistently good growth with all keratinocyte strains, we included either IGF-I or insulin in the medium for all other keratinocyte proliferation experiments reported here. For clarity of presentation, we refer to growth in standard medium with IGF-I or insulin as “EGFindependent”. Collectively, the results shown in Figure 1A indicated that normal human keratinocytes proliferated in the absence of exogenously added growth factor and that the addition of growth factors to keratinocyte cultures had little effect on growth rate. Heparin sulfate and TGFP inhibit the EGF-independent proliferation of human keratinocytes We previously reported that heparin inhibits the EGF-independent proliferation of human keratinocytes (Shipley et al., 1989). This observation led us to determine the optimal dose at which heparin mediates the inhibition of EGF-independent keratinocyte rowth. Heparin inhibited the EGF-independent proli eration of keratinocytes in a dose-dependent manner with 112 maximal inhibition occurring at approximately 0.1 pgiml heparin, while maximal inhibition occurred between 3-30 pgiml (Fig. lb). We have previously shown that TGFP inhibits EGF-stimulated growth of human keratinocytes (Shipley et al., 1986). Thus, we determined whether TGFP would act as an inhibitor of EGF-independent keratinocyte growth. Treatment of human keratinocyte cultures with various doses of human platelet-derived TGFpl caused inhibition of proliferation in the absence of EGF (Fig. lc). The growth inhibitory effect exerted by TGFp was 112 maximal at approximately 1 ng/ml. Heparin-induced inhibition of EGF-independent growth is reversed by EGF, TGFa, aFGF, and bFGF The inhibitory effect of heparin sulfate on the EGFindependent growth of human keratinocytes (Fig. 2a)

B

KERATINOCYTE AUTOCRINE GROWTH

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Fig. 1A: The effect of growth factors on the proliferation of high density keratinocytes cultured in standard medium. Keratinocytes were plated at 1 x lo4 cells/cm2 in complete medium, cultured for 24 hours, and washed, and standard medium was added to each well as described in Methods. IGF-I (10 ngiml) was added to some of the wells as indicated. bFGF (3 ngiml), aFGF (3ngiml), or TGFu (10 ngirnl) was added to wells treated with and without IGF-I. One set of wells containing keratinocytes was fixed a t the time of washing and the experimental cultures were fixed 5 days later. DNA content and cell numbedwell was determined a s described in Methods. Arrow indicates mean number of cells present immediately after the cells were washed. “Control” indicates cells cultured in standard medium alone. Each point represents the mean of separate determinations on 3 different wells, ? the SEM. B: The effect of heparin sulfate on the EGF-independent growth of high density human keratinocytes. Keratinocytes were plated at 5 x lo3cellsicm2 and cultured as described in Figure la, except that the standard medium was supplemented with 5 kgiml insulin a t the indicated concentrations of heparin sulfate. After 5 days of culture, the cell numberiwell was determined as described in Figure l a and in Methods. Each point represents the mean of separate determinations on 3 different wells, t- the SEM. C: The effect of TGFp on the EGF-independent growth of high density human keratinocytes. Keratinocytes were plated and cultured as described in Figure 1B in the presence of the indicated concentrations of TGFP. After 5 days of culture the cell numberiwell was determined as described in Figure l a and in Methods. Each point represents the mean of separate determinations on 3 different wells, 2 the SEM.

initial lag-phase of 1 day, the cultures underwent one doubling between 1-3 days, followed by 2 doublings from days 3-5. Addition of TGFp or heparin sulfate to these cultures at T=O caused marked inhibition of this EGF-inde endent proliferation, limiting the keratinocyte cu tures to less than 1 doubling in 5 days. Suramin inhibits the proliferation of human keratinocytes cultured in the absence of EGF Heparin (pg/rni) Our results suggested that normal human keratinocytes were capable of autocrine proliferation. To examine this property further, we determined whether suramin inhibited the EGF-independent proliferation of human keratinocytes. Suramin is a polyanionic compound which has been utilized as an anti-trypanosoma1 therapeutic agent. More recently, suramin has been demonstrated to interrupt ligand (growth factor) interactions with cell surface receptors and may disrupt the action of autocrine acting growth factors in tumor cells (Coffey et al., 198713;Betsholtz et al., 1986). Suramin inhibited the high density, EGF-independent proliferation of the keratinocyte cultures (Fig. 2c). One-half maximal growth inhibition occurred between 0.003 and 0.01 mM suramin. These results suggested that autocrine acting growth factors were produced by keratinocytes. 1 O04, 0.1 1 .o 10 Conditioned medium from human keratinocytes cultured at high cell density in the absence of TGFB (no/ml) was partially reversed by the addition of bFGF to the growth factors stimulates the growth of human keratinocytes at clonal cell density culture medium and was completely overridden by the To more thoroughly test the hypothesis that keratiaddition of EGF, TGFa, or aFGF at the concentrations tested. Thus, heparin inhibition of EGF-independent nocytes produce autocrine acting mitogenic factors, keratinocyte growth at high cell densities can be over- conditioned medium (CM) from moderate to high density keratinocyte cultures grown in the absence of come by the addition of exogenous growth factors. growth factors (standard medium), was concentrated Proliferation kinetics of human keratinocytes and tested for its ability to stimulate the proliferation cultured in the absence of EGF of keratinocvtes cultured at clonal cell densitv. As is Figure 2b displays the proliferation kinetics of the shown in Fkure 3, concentrated keratinocyte-derived EGF-independent growth of keratinocytes. After an CM stimulated the clonal growth of keratinocytes when

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Fig. 2.A:The ability of exogenous growth factors to overcome heparininduced inhibition of high density keratinocyte proliferation. Keratinocytes were plated and washed as described in Figure 1B. After the cells were washed, standard medium supplemented with IGF-I (10 ngiml) was added to each well. Heparin sulfate (15 pgiml), bFGF, aFGF, EGF or TGFn (1nM) was added to some of the wells as indicated. The cells were fixed and cell number was determined as described in Figure 1A and in Methods. Control indicates cells cultured in standard medium plus IGF-I alone. Each point represents the mean of separate determinations on 3 different wells, i the SEM. B:The effect of heparin sulfate and TGFp on the proliferation kinetics of high density keratinocytes cultured in standard medium supplemented with IGF-I. Keratinocytes were plated and washed as described in Figure 1B and were placed in standard medium plus IGF-I (10 ngiml). Heparin sulfate (15 pgiml) or TGFp (5 ngiml) were added to corresponding wells. Cells were fixed a t the indicated time points and cell numberiwell was determined as described in Figure 1A and Methods. Each point represents the mean of separate determinations on 3 different wells, 2 the SEM. C: The effect of suramin concentration on the EGF-independent growth of high density keratinocytes. Keratinocytes were plated a t and cultured as described in Figure 1B in the presence of the indicated concentrations of suramin. After 5 days of culture the cell numberiwell was determined as previously described in Figure 1A and Methods. Each point represents the mean of separate determinations on 3 different wells, t the SEM.

Plur

Ulnur Haparin

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TGFP), or possibly supraphysiological levels of mitogenic growth factors, present in the CM result in the inhibition (or absence) of keratinocyte proliferation. Collectively, these results show that high density cultures of keratinocytes produced extracellular factors which stimulated, and under some circumstances were non-permissive for, proliferation of keratinocytes at clonal cell densities. 0 1 0

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Monoclonal antibody (mAb LAl), a competitive antagonist to the human EGF receptor, blocks EGF/TGFa and keratinocyte-derived CM stimulation of human keratinocyte clonal growth We have previously shown that mAb LA1 specifically blocks the EGF-independent proliferation of human keratinocytes at high density (M. R. Pittelkow, submitted). In addition, mAb LA1 specifically blocks the mitogenic action of EGFITGFa, but not bFGF on quiescent human fibroblast cultures, and irrelevant monoclonal antibodies (anti-human growth hormone) do not affect the clonal growth of human keratinocytes (M. R. Pittelkow, submitted). These observations led us to determine whether mAb LA1 could block the mitogenic effect of EGFiTGFa or keratinocyte-derived CM on the clonal growth of keratinocytes. Either EGF or TGFa (0.33 nm) stimulated colony formation by keratinocytes, and this mitogenic effect was blocked by the addition of mAb LA1 (33 nM) to the culture medium (Fig. 4).However, the clonal cultures were still capable of responding to the mitogens in the presence of the mAb, as raising the level of EGFITGF-a to 33 nM resulted in significant clonal growth. In a separate experiment (Fig. 5), mAB LA1 (33 nM) completely blocked CM- and TGFa-induced clonal growth of human keratinocytes. We have also shown that a monoclonal antibody which neutralizes the mitogenic effect of mature human TGFa (monoclonal antibody TGFal) on keratinocytes does not neutralize the mitogenic activity of keratinocyte-derived CM on clonal density human keratinocytes (data not shown) and does not

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added at concentrations less than or approximating the original conditioned medium concentration (0.3-1 .O x ) in the presence of insulin. Interestly, concentrated conditioned medium above the original medium concentration (>1X ) failed tc stimulate clonal growth. This latter result indicates that inhibitors (such as

KERATINOCYTE AUTOCRINE GROWTH

Con.Med.

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Fig. 3. Effect of keratinocyte-derived CM on the clonal growth of keratinocytes. Keratinocytes were plated, washed, and cultured in standard medium plus insulin (control) as described in Methods. Keratinocyte-derived conditioned medium (Con. Med.) (0.W X ) or TGFu (0.3nM) was added to indicated wells and the cells were subsequently cultured for a period of 10 days before fixation and staining. “Control” represents cells cultured in standard medium plus insulin. Each experimental point was performed in triplicate. The symbol “X” refers to fold volume concentration.

Keratinocyte-derivedconditioned medium stimulates DNA synthesis in normal human fibroblasts and fibroblast-like cell lines through both EGF receptor-independentand -dependent pathways We postulated that potential paracrine stimulators of cell growth could also be present in keratinocytederived CM. In order to test this hypothesis, we tested Heparin sulfate partially blocks the mitogenic concentrated keratinocyte-derived CM for its ability to effect of keratinocyte-derived conditioned stimulate DNA synthesis in normal human dermal medium on the clonal growth of fibroblasts and 2 different mouse fibroblast-like cell human keratinocytes lines. Keratinocyte-derived CM stimulated DNA synBecause heparin sulfate inhibits the EGF-indepen- thesis in quiescent normal human fibroblasts, producdent growth of human keratinocytes at high cell den- ing a 112 maximal response at approximately 1.5 fold sity, we asked whether this compound would effect volume concentration (Fig. 6a). The maximal response CM-induced keratinocyte clonal growth. TGFa or ke- produced by the CM was equal in magnitude to that ratinocyte-derived CM were added to keratinocyte produced by optimal doses of bFGF, EGF or TGF-a clonal cultures in the presence or absence of heparin (data not shown). The quiescent fibroblast cultures sulfate. As can be seen in Figure 5, the addition of appeared less sensitive to the mitogenic effects of heparin sulfate to TGFa treated cultures had a slight keratinocyte-derived CM than the keratinocyte clonal inhibitory effect on clonal proliferation. Heparin had a cultures as the 1/2 maximal responses were < 0.3 more significant effect on the CM-induced clonal x and 1.5 x for keratinocytes and fibroblast.. respecgrowth, causing an approximately 75% reduction of tively. Supramaximal levels (> 10 x )L “ keratinocytecolony size when compared to the CM alone. Thus, derived CM also caused less stimulation of DNA synheparin sulfate inhibits the ability of keratinocyte- thesis. Keratinocyte-derived CM also was capable of derived CM factorb) to stimulate the clonal growth of stimulating DNA synthesis in quiescent AKR-2B cells (Fig. 6b), but with less potency (112 maximal stimulahuman keratinocytes. inhibit the EGF-independent proliferation of these cells cultured at high cell densities (M. R. Pittelkow, submitted). These results indicate that CM stimulation of keratinocyte clonal growth is dependent upon freely functional EGF receptors, while the exact identities of the CM-derived factors which stimulate keratinocyte clonal growth are not clear.

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EGF

TGFa Fig. 4. Effect of anti-EGF receptor mAb on EGF and TGFa-induced stimulation of keratinocyte clonal growth. Keratinocytes were plated, treated, fixed, and stained as described for Figure 3 and in Methods, Cells were cultured in the indicated concentrations of EGF or TGFa in the absence or presence of 33 nM anti-EGF receptor mAb LA1. “Control” represents cells cultured in standard medium plus insulin. Each experimental point was performed in triplicate.

tion approximately equal to 1x ) than that observed for clonal density keratinocytes. These results show that concentrated keratinocyte-derived CM contained factors which could act as paracrine mediators of growth on neighboring dermal fibroblasts. TGFa mRNA and protein are produced by cultured keratinocytes proliferating in the absence of exogenously added EGF or TGFa (M. R. Pittelkow, submitted). Using a RIA with mature TGFa as a standard, we determined that the concentrated (43x ) keratinocytederived CM contained TGFa immunoreactivity corresponding to 10.1 ng/ml of the mature polypeptide and indicated that the original unconcentrated (1X ) CM contained immunoreactive TGFa at a concentration of approximately 235 pg/ml. This immunoreactive material most likely represents higher molecular weight forms of TGFa, as the original conditioned medium was concentrated with a filter that removes polypeptides with molecular weights less than 10,000 daltons. To determine whether growth factors other than those that act through the EGF receptor were present in the keratinocyte-derived CM, we tested the CM for its ability to stimulate DNA synthesis in the EGF-receptorless NR6 cell line, which has been shown to be growth responsive to bFGF but not EGF/TGFa (Pruss

and Herschman, 1979; Schneider et al., 1986). Interestingly, keratinocyte-derived CM stimulated DNA synthesis is quiescent NR6 cells (Fig. 6c) but with significantly less potency (1/2 maximal stimulation approximately equal to 5 ~ than ) that observed for clonal keratinocytes, normal human fibroblasts, or AKR-2B cells. Our results with the NR6 cells indicated that keratinocyte-derived CM must contain growth factors which mediate some of their mitogenic effects through EGF receptor-independent pathways. We utilize mAb LA1 to test the hypothesis that keratinocyte-derived CM contained mitogens which utilized EGF receptor-independent pathways. We have previously demonstrated that this antibody specifically blocks the activity of EGF and TGFa on normal human fibroblasts but has no effect on the mitogenic activity of bFGF (M. R. Pittelkow, submitted). We used mAb LA1 to quantitate the contribution of CM-derived, EGF receptor-independent, mitogenic activity on quiescent normal human fibroblast cultures. As shown in Figure 6a, addition of 33 nM mAb LA1 reduced the maximal CM-induced stimulation of quiescent normal human fibroblasts by approximately 70% and caused the 112 maximal stimulation concentration to shift to the right (3 x 1. These experiments demonstrated that kerati-

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Fig. 5. Effect of heparin sulfate and anti-EGF receptor mAb on keratinocyte-derived CM- and TGFa-stimulation of keratinocyte clonal growth. Keratinocytes were plated, treated, fixed, and stained as described for Figure 3 and in Methods. Cells were exposed to TGFu (0.3 nM) or keratinocyte-derived CM (0.3 X ) in the absence or presence of 33 nM anti-EGF receptor mAb LA1 or 30 kg/ml heparin sulfate as indicated. “Control” represents cells cultured in standard medium plus insulin. Each experimental point was performed in triplicate.

nocyte-derived CM is capable of stimulating normal human fibroblasts and suggests that fibroblast growth stimulation is mediated by mitogens which exert their action through both EGF receptor-dependent and EGF receptor-independent pathways.

Heparin sulfate partially blocks the mitogenic activity of keratinocyte-derived CM on AKR-2B cells Keratinocyte-derived CM stimulated the growth of several cell types including mouse AKR-2B cells (Fig. 6b).We tested heparin sulfate for its ability to diminish the mitogenic effect of CM on this mouse cell line. TGFa and keratinocyte-derived CM stimulated DNA synthesis in quiescent mouse AKR-2B cells in a dosedependent fashion (Fig. 7a). In the experiment shown in Figure 7b, addition of heparin sulfate to AKR-2B cultures potentiated the activity of TGFu (1nM), while the addition of heparin caused a 50% inhibition of the mitogenic stimulation induced by 3~ CM. In other experiments we have demonstrated that heparin sulfate does not inhibit the mitogenic activity of EGF, TGFa, aFGF, or bFGF in this cell type (data not shown). As was the case for human keratinocytes (Fig.

51, heparin sulfate inhibited the mitogenic activity of keratinocyte-derived CM on AKR-2B cells.

DISCUSSION Autocrine growth control occurs when a cell produces and secretes a growth factorb) which in turn stimulateshhibits the proliferation of the same cell. Paracrine growth control describes the stimulation/ inhibition of cells by a growth factor that is produced by a neighboring cell. The autocrine hypothesis was initially applied to explain aberrant growth regulation observed in chemically and virally transformed mouse fibroblast cell lines (Sporn and Todaro, 1980). More recently, loss of growth regulation due to continuous autocrine stimulation of cell proliferation is thought to occur in some tumor cells (Goustin et al., 1986; Bates et al., 1988;Smith et al., 1987).Some growth factors are produced by normal cells during growth and development and during non-neoplastic pathological states (Goustin et al., 1986; Deuel, 1987; Knochel and Tiedman, 1989; Derynck, 1988; Mercola and Stiles, 1988; Wahl et al., 1989; Clark and Henson, 1988; Braun et al., 1988; Kan et al., 1989; Mead and Fausto, 1989). For example, normal human keratinocytes have been

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Fig. 6. Effect of keratinocyte-derived CM on the incorporation of i3Hlthymidine into DNA in quiescent normal human fibroblasts and mouse fibroblast-like cell lines. A: Quiescent normal human fibroblasts were exposed to the indicated concentrations (0-30 XIof keratinocyte-derived CM in the absence or presence of 33 nM antiEGF receptor mAb LA1, as described in Methods. After 20 hours of incubation, relative incorporation of [3Hlthymidine was determined as described in Methods. Each point represents the mean incorporation of separate determinations on 3 different wells, 2 the SEM. B: Effect of keratinocyte-derived CM on the incorporation of ['Hlthymidine into DNA in quiescent AKR-2B cells. Quiescent AKR2B cells were exposed to the indicated concentrations (0-3 X ) of keratinocyte-derived CM. After 22 hours of incubation, relative incorporation of L3HJthymidinewas determined as described in Methods. Arrows indicate mean incorporation in cultures exposed to bFGF (0.1 nM) or TGFa (1.0 nM). Each point represents the mean incorporation of separate determinations on 3 different wells, the SEM. C: Effect of keratinocyte-derived CM on the incorporation of VHIthymidine into DNA in quiescent NR6 cells. Quiescent NR6 cells were exposed to the indicated concentrations (0-30 x of keratinocytederived CM and incubated for 24 hours with L3H1thymidine. Relative incorporation of L3H1thymidine was determined as described in Methods. Arrows indicate mean incorporation of cultures exposed to bFGF (0.1 nM) or TGFa (1.0 nM). Each point represents the mean incorporation of separate determinations on 3 different wells, 2 the SEM.

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shown to produce TGFa in vitro (Pittelkow et al., 1989; Coffey et al., 1987a; Cook et al., 1990; M. R. Pittelkow, submitted) while psoriatic epidermis has been shown to express elevated levels of TGFa in vivo (Gottlieb et al., 1988; Elder et al., 1989). Our recent discovery that keratinocytes display heparin-sensitive, EGF-independent proliferation (Shipley et al., 19891,led us to more completely characterize this phenomenon and to investigate the effect of vari-

ous growth modulators in this system. Our current results extend our previous observations and demonstrate that addition of EGF, TGFa, bFGF, or aFGF with or without IGF-1 to standard medium resulted in only minor increases in the proliferation rate of keratinocytes cultured at high cell density. In agreement with our previous study (Shipley et al., 19891, heparin inhibition of EGF-independent growth was completely reversed by the addition of EGFPTGFa, or aFGF to the culture medium, while the addition of bFGF only partially reversed the inhibitory effects of heparin. These results clearly demonstrated that heparin did not non-specifically block the ability of our keratinocyte cultures to proliferate and that these growth factors (EGFITGFa, bFGF, aFGF) were probably not sensitive to the growth inhibitory effects of heparin. In addition, we show here that both TGFp and suramin act as potent inhibitors of keratinocyte growth in the absence of exogenously added polypeptide growth factors. The inhibitory effect of suramin suggested that, under these culture conditions, keratinocyte proliferation may have resulted from the production of autocrine stimulators of growth. Moreover, we do not believe that the EGF-independent growth observed resulted from "carry-over" of residual EGF derived from the plating medium, as clonal-density keratinocytes which were plated, washed, and cultured using an identical protocol failed to proliferate in the absence of EGF or CM. In order to demonstrate that autocrine acting growth factors are involved in the EGF-independent proliferation of keratinocytes, we examined the mitogenic properties of medium conditioned by high density keratinocytes. Results from this study clearly demonstrated that keratinocyte-derived CM can replace EGFi TGFa in stimulating the clonal growth of human keratinocytes. Our previous studies have demonstrated that TGFa mRNA and mature TGFa peptide are expressed in rapidly growing subconfluent keratinocytes cultured in standard medium supplemented with insulin (M. R. Pittelkow, submitted). In the current study, we have shown via the use of a TGFa RIA, which detects multiple forms of TGFa, that kerati-

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Fig. 7. Dose response of quiescent AKR-2B cells to TGFa and keratinocyte-derived CM. A: Quiescent AKR-2B cells were treated with the indicated concentrations of TGFa or keratinocyte-derived CM and relative incorporation of [3H]thymidine was determined as described for Figure 6b and in Methods. FVC indicates fold volume concentration. For keratinocyte-derived CM, each point represents the mean incorporation of separate determinations on 3 different wells, ? the SEM. For TGFa, each point represents the mean value of separate determinations on 2 different wells. B: Effect of heparin sulfate (10 Fgiml) on TGFa or keratinocyte-derived CM-induced stimulation of ['Hlthymidine incorporation in AKR-PB cells. Quiescent AKR-2B cells were treated with TGFa (1.0 nM) or keratinocyte-derived CM (3.0 FVC) and specific incorporation of [3Hlthymidine was determined as described for Figure 4b and in Methods. Control indicates results from cells not exposed to CM or growth factor (? heparin sulfate). For keratinocyte-derived CM, the data represents mean incorporation of separate determinations on 3 different wells, 2 the SEM. For TGFa, the data represents the mean value of separate determinations on 2 different wells.

nocyte-derived CM contains TGFa immunoreactive material at concentrations which would be sufficient to stimulate keratinocyte clonal growth. However, our results with human fibroblasts treated with the LA1 mAb indicated that keratinocyte-derived CM contained both EGF receptor and non-EGF receptor interacting growth factors. These observations are substantiated by the fact that the EGF-receptorless cell line, NR6, was mitogenically sensitive to factors present in keratinocyte-derived CM. We do not believe that these non-EGF receptor interacting mitogenic factors represent acidic or basic FGF as we fail to detect these growth factors in the keratinocyte-derived CM (data not shown). We have observed that PDGF AA peptide

cultured in standard medium (data not shown). Other factors produced by human keratinocytes could mediate autocrine growth stimulation. For example, Interleukin-6 has been shown to be produced by human keratinocytes (Kirnbauer et al., 19891, and may be mitogenic for these cells in vitro (Grossman et al., 1989). Thus, the EGF receptor-independent mitogenic components of keratinocyte-derived CM could be PDGF AA, other known cytokines, or other as yet uncharacterized factors. For concentration of the CM we used filters with a molecular weight cut-off of 10,000 daltons. This procedure may have eliminated factors with molecular weights below this cut-off. Therefore, any mitogenic factors retained in the concentrated conditioned medium were probably not due to mature TGFa or other low molecular weight factors. Our current study demonstrates that some of the mitogenic activity in keratinocyte-derived CM was inhibited by addition of heparin sulfate to the culture medium. The inhibitory effect was observed when the mitogenic activity was tested in keratinocyte clonal growth assays or DNA synthesis assays in quiescent AKR-2B cells. Since we have shown that heparin does not inhibit the mitogenic effect of EGF, TGFa, or aFGF on keratinocytes or AKR-2B cells (data not shown), novel heparin-sensitive growth factors may have been produced by autonomously growing keratinocytes. In this regard, several other groups have reported potentially novel growth factors which display similar properties (Halper and Carter, 1989; Masuda et al., 1987). Heparin and heparan have been shown to exert antiproliferative effects on several cell types in vitro and in vivo (Hook et al., 1984; Shipley et al., 1989; Clowes and Karnovsky, 1977; Imamura and Mitsui, 1987; Majesky et al., 1987; Robertson and Goldstein, 1988; Wright et al., 1989). Other investigators have demonstrated that cellular production of sulfated GAGs may be involved in mediating density-dependent growth inhibition and differentiation observed in normal cells (Hook et al., 1984; Rahemtulla et al., 1989). Our current observations suggest that sulfated GAGs elaborated by keratinocytes (Rahemtulla et al., 1989) or other cell types could play important roles regulating keratinocyte growth and differentiation. Interestingly, keratinocyte-derived CM was not stimulatory for the clonal growth of keratinocytes a t concentrations greater than the original (1X ) CM concentration. The reason for this result is not clear; however, it is possible that keratinocyte CM may have contained growth inhibitors which exerted their effects when concentrations were elevated above those present in CM (1x ). TGFp inhibits the rowth of keratinocytes and is known also to be pro uced by these cells, largely in an inactive latent form (Shipley et al., 1986; Pittelkow et al., 1988). Latent TGFp present in the CM could have been activated by freeze-thawing or during other manipulations of the CM. In support of the notion that the keratinocyte-derived CM contained some active TGFP, we have observed that elevated levels of keratinocytederived CM abrogated the growth stirnulatory effect of EGF on keratinocyte clonal cultures (data not shown). Supraphysiological concentrations of growth stimula-

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tory factors present in the CM may also have contributed to the growth inhibition. Future investigations designed to separate and purify growth inhibitory agents from keratinocyte-derived CM will be required to more clearly delineate the factors that mediate this effect. Keratinocytes cultured in the absence of exogenous growth factors may secrete paracrine acting factors. Our results clearly support this concept, as quiescent cultures of human fibroblasts were stimulated by keratinocyte-derived CM. Keratinocyte-derived CM was also mitogenic for the fibroblast-like mouse AKR-2B cell line and the EGF-receptorless, fibroblast-like NR6 mouse cell line. These observations suggest that growth factors expressed by human keratinocytes in vivo could act in a paracrine fashion on neighboring dermal fibroblasts. Two factors which are produced by human keratinocytes, TGFa (Coffey et al., 1987a; Pittelkow et al., 1989; Cook et al., 1990; M. R. Pittelkow, submitted) and interleukin-1 (Blanton et al., 19891, have also been shown to act as growth factors for human dermal fibroblasts (M. R. Pittelkow, submitted; Schmidt et al., 1982).It has been recently demonstrated that proteins synthesized and secreted by keratinocytes in vivo may traverse the basement membrane into the dermis where the protein is ultimately detected systemically (Fenjves et al., 1989). Thus, we speculate that mitogenic factors produced and secreted by human keratinocytes may mediate growth responses of dermal fibroblasts or act as distant sites in vivo. This study represents the first definitive evidence that the proliferation of human keratinocyte cultures in the absence of polypeptide growth factors is mediated by the production of keratinocyte-derived autocrine factors. Production of various forms of TGFa (Coffey et al., 1987a; Pittelkow et al., 1989; Gottlieb et al., 1988; Cook et al., 1990; M. R. Pittelkow, submitted), including transmembrane TGFa, (Derynck, 1988; Bringman et al., 1987; Brachmann et al., 1989)by these cells may mediate keratinocyte autocrine growth. However, our results suggest that keratinocyte cultures also produced heparin-sensitive and/or EGF receptorindependent factors which may be involved in the autocrine stimulation of keratinocyte growth or paracrine stimulation of neighboring cell types. Culturing human keratinocytes at subconfluent densities may simulate the wounded or pathological states in vivo. Isolation and characterization of the factors present in medium conditioned by human keratinocytes under these conditions may reveal the presence of both previously characterized and novel growth factors. Identification of these factors and the mechanisms that regulate their expression and activity will be important in understanding the processes of wound healing, development of psoriasis, and other cutaneous disorders such as skin neoplasia and aging. ACKNOWLEDGMENTS The authors wish t o thank Winifred W. Keeble and Norma Swanson for their technical assistance on this project. We also thank Dr. Rodney L. Sparks for his critical review of this manuscript. This work was supported by NIH grant CA42409 (GDS);a grant from Clonetics Corp. (GDS); the Mayo Foundation (MRP);

and an American Cancer Society postdoctoral training fellowship to P.W.C. LITERATURE CITED Barrandon, Y., and Green, H. (1987) Cell migration is essential for sustained growth of keratinocyte colonies: the role of transforming growth factor-a and epidermal growth factor. Cell, 50:1131-1137. Bates, S.E., Davidson, N.E., Valverius, E.M., Freter, C.E., Dickson, R.B., Tam, J.P., Kudlow, J.E., Lippman, M.E., and Salomon, D.S. (1988) Expression of transforming growth factor a and its messenger ribonucleic acid in human breast cancer: Its regulation by estrogen and its possible functional significance. Mol. Endo., 2543555. Betsholtz, C., Johnsson, A,, Heldin, C.-H.,and Westermark, B. (1986) Efficient reversion of simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin. Proc. Natl. Acad. Sci. U.S.A., 83:6440-6444. Blanton, R.A., Kupper, T.S., McDougall, J.K., and Dower, S. (1989) Regulation of interleukin 1 and its receptor in human keratinocytes. Proc. Natl. Acad. Sci. U.S.A., 86:1273-1277. Brachmann, R., Lindquist, P.B., Nagashima, M., Kohr, W., Lipari, T., Napier, M., and Derynck, R. (1989) Transmembrane TGF-(Yprecursors activate EGFITGF-a receptors. Cell, 56691-700. Braun, L., Mead, J.E., Panzica, M., Mikumo, R., Bell, G.I., and Fausto, N. (1988) Transforming growth factor p mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation. Proc. Natl. Acad. Sci. U.S.A., 85153%1543. Bringman, T.S., Lindquist, P.B., and Derynck, R. (1987) Different transforming growth factor-a species are derived from a glycosylated and palmitoylated transmembrane precursor. Cell, 48:429440. Clark, R.A.F., and Henson, P.M., eds. (1988) The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York, pp. 1-597. Clowes, A.W., and Karnovsky, M.J. (1977) Suppression by heparin of smooth muscle proliferation in injured arteries. Nature, 265625626. Coffey, R.J. Jr., Derynck, R., Wilcox, J.N., Bringman, T.S., Goustin, A S . , Moses, H.L., and Pittelkow, M.R. (1987a) Production and autoinduction of transforming growth factor-a in human keratinocytes. Nature, 328817-820. Coffey, R.J. Jr., Leof, E.B., Shipley, G.D., and Moses, H.L. (1987b) Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B Cells. J. Cell. Physiol., 132:143-148. Cook, P.W., Magun, B.E., Pittelkow, M.R., Coffey, R.J. Jr., and Shipley, G.D. (1990) Expression and regulation of mRNA coding for heparin binding growth factors and transforming growth factor type-alpha in human skin-derived cells. Molec. Endo., in press. Derynck, R. (1988) Transforming growth factor (Y. Cell, 54:593-595. Deuel, T.F. Peptide growth factors: roles in normal and abnormal cell growth. (1987) Annu. Rev. Cell Biol., 3:43-92. Elder, J.T., Fisher, G.J., Lindquist, P.B., Bennett, G.L., Pittelkow, M.R., Coffey, R.J. Jr., Ellingsworth, L., Derynck, R., and Voorhees, J.J. (1989) Overexpression of transforming growth factor CI in psoriatic epidermis. Science, 2432311-814. Fenjves, E.S., Gordan, D.A., Pershing, L.K., Williams, D.L., and Taichman, L.B. (1989) Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications epidermal function and gene therapy. Proc. Natl. Acad. Sci. U.S.A., 86:8803-8807. Firestone, G.L., John, N.J., and Yamamoto, K.R. (1986)Glucocorticoid regulated glycoprotein maturation in wild type and mutant rat cell lines. J. Cell Biol., 103:2323-2331. Gilchrest, B.A., Karassik, R.L., Wilkens, L.M., Vrabel, M.A., and Maciag, T. (1983) Autocrine and paracrine growth stimulation of cells derived from human skin. J. Cell Physiol., 117235-240. Gottlieb, A.B., Chang, C.K., Posnett, D.N., Fanelli, B., and Tam, J.P. (1988) Detection of transforming growth factor (Y in normal, malignant, and hyperproliferative human keratinocytes. J. Exp. Med., 167670-675. Goustin, A.S., Leof, E.B., Shipley, G.D., and Moses, H.L. (1986) Growth factors and cancer. Cancer Res., 46:1015-1029. Grossman, R.M., Krueger, J., Yourish, D., Garnelli-Piperno, A,, Murphy, D.P., May, L.T., Kupper, T.S., and Sehgal, P.B. (1989) Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proc. Natl. Acad. Sci. U.S.A., 86:6367-6371. Halaban, R., Langdon, R., Birchall, N., Cuono, C., Baird, A., Scott, G.,

KERATINOCYTE AUTOCRINE GROWTH Moellmann, G., and McGuire, J . (1988) Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J . Cell Biol., 107:1611-1619. Halper, J., and Carter, B.J. (1989) Modulation of growth of human carcinoma SW-13 cells bv heuarin and "growth factors. J. Cell. Physiol., 141:16-23. Hammond, S.L., Ham, R.G., and Stampfer, M.R. (1984) Serum-free erowth of human mammarv eDithelia1 cells: h a i d clonal growth in aefined medium and extendedserial passage whh pituitary extract. Proc. Natl. Acad. Sci. U.S.A., 81:5435-5439. Hinegardner, R.T. (1971) An improved fluorometric assay for DNA. Anal. Biochem., 39:197-201. Hook, M., Kjellen, L., and Johansson, S. (1984)Cell surface glycosaminoglycans. Annu. Rev. Biochem., 53:847-869. Imamura, T., and Mitsui, Y. (1987) Heparan sulfate and heparin as a potentiator or a suppressor of growth of normal and transformed vascular endothelial cells. Exp. Cell Res., 17292-100. Kan, M., Huang, J., Mansson, P.-K., Yasurnitsu, H., Carr, B., and McKeehan, W.L. (1989) Heparin-binding growth factor type 1 (acidic fibroblast growth factor): A potential biphasic autocrine and paracrine regulator of hepatocyte regeneration. Proc. Natl. Acad. Sci. U.S.A., 86:7432-7436. Kirnbauer, R., Kock, A,, Schwarz, T., Urbanski, A., Krutmann, J . , Borth, W., Damm, D., Shipley, G., Ansel, J.C., and Luger, T.A. (1989) IFN-P2, B cell differentiation factor, or hybridoma growth factor (IL-6)is expressed and released by human epidermal cells and epidermoid carcinoma cell lines. J . Immunol., 142:1922-1928. Knochel, W., and Tiedmann, H. (1989) Embryonic inducers, growth factors, transcription factors and oncogenes. Cell Diff. and Devel., 26:163-17 1. Madtes, D.K., Raines, E.W., Sakariassen, K.S., Assoian, R.K., Sporn, M.B., Bell, G.I., and Ross, R. (1988) Induction of transforming growth factor-a in activated human alveolar macrophages. Cell, 53:285-293. Majesky, M.W., Benditt, E.P., and Schwartz, S.M. (1988) Expression and developmental control of platelet-derived growth factor A-chain and R-chaidsis .~~~~~~~ eenes in rat aortic smooth muscle cells. Proc. Natl. Acad. Sci. U.S:K, 8531524-1528. Majesky, M.W., Schwartz, S.M., Clowes, M.M., and Clowes, A.W. 11987) Heuarin reeulates smooth muscle S phase entry in the injured rai carotid irtery. Circ. Res., 61:296-300. Masuda, Y., Yoshitake, Y., and Nishikawa, K. (1987) Secretion of DNA synthesis factor (DSF) by A431 cells that can grow in protein-free medium. Cell Biol. Int. Rep., I1:359-366. McKeehan, W.L., and Ham, R.G. (1977) Methods for reducing the serum requirement for growth in vitro of nontransformed diploid fibroblasts. Dev. Biol. Stand., 3797-108. Mead, J.E., and Fausto, N. Transforming growth factor a may be a physiological regulator of liver regeneration by means of a n autocrine mechanism. (1989) Proc. Natl. Acad. Sci. U.S.A., 86:15581562. Mercola, M., and Stiles, C. 11988) Growth factor superfamilies and mammalian embryogenesis. Development, 102:451460. O'Keefe. E.J.. Chiu. M.L.. and Pavne, R.E. (1988) Stimulation of growth of keratinocytes by basic fibroblast growth factor. J. Invest. Dermatol., 90:767-769. Pittelkow. M.R.. Coffev, R.J., and Moses, H.L. (1988) Keratinocytes produce' and are regulated by transforming growth factors. In: Endocrine, Metabolic and Immunologic Functions of Keratinocytes. L.M. Milestone and R.L. Edelson, eds. Ann. N.Y. Acad. Sci., New York, Vol. 548, pp. 211-224. "

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Pittelkow, M.R., Lindquist, P.B., Abraham, R.T., Graves-Deal, R., Derynck, R., and Coffey, R.J. J r . (1989) Induction of transforming growth factor-a expression in human keratinocytes by phorbol esters. J . Biol. Chem., 264:5164-5171. Pruss, R.M., and Herschman, H.R. (1979) Mitogenic effects of murine serum and fibroblast growth factor on EGF nonproliferative variants of 3T3. J. Supra. Struc., 12:467470. Rahemtulla, F., Moorer, C., and Wille, J.J. J r . (1989) Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium. J . Cell. Physiol., 140:98-106. Robertson, P.L., and Goldstein, G.W. (1988) Heparin inhibits the growth of astrocytes in vitro. Brain Res., 447341-345. Schmidt, J.A.,Mizel, S.B., Cohen, D., andGreen, I. 11982)Interleukin1, a potential regulator of fibroblast proliferation. J . Immunol., 128:2177-2182. Schneider, C.A., Lim, R.W., Tenvilliger, E., and Herschman, H.R. (19861 EDidermal a o w t h factor-nonresuonsive 3T3 variants do not contain 'epidermd growth factor receptor-related antigens or mRNA. Proc. Natl. Acad. Sci. U.S.A., 83:333-336. Shiplev, G.D.. Childs, C.B., Volkenant. M.E., and Moses. H.L. (1984) Diffifential effects of epidermal growth factor, transforming growth factor, and insulin on DNA and protein synthesis and morphology in serum-free cultures of AKR-2B cells. Cancer Res., 44:710-716. Shipley, G.D., and Ham, R.G. (1981) Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of swiss 3T3 cells. In Vitro, 17656670. Shipley, G.D., Keeble, W.W., Hendrickson, J.E., Coffey, R.J. Jr., and Pittelkow, M.R. (1989) Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor. J. Cell. Physiol., 138511-518. Shipley, G.D., and Pittelkow, M.R. (1987) Control of growth and differentiation in vitro of human prokeratinocytes cultured in serum-free medium. Arch. Dermatol., 123:1541a-l544a. Shipley, G.D., Pittelkow, M.R., Wille, J.J., Jr., Scott, R.E., and Moses, H.L. (1986)Reversible inhibition of normal human prokeratinocyte proliferation by type p transforming growth factor-growth inhibitor in serum-free medium. Cancer Res., 462068-2071. Smith, J.J., Derynck, R., and Korc, M. (1987) Production oftransforming growth factor (Y in human pancreatic cancer cells: evidence for a superagonist autocrine cycle. Proc. Natl. Acad. Sci. U.S.A., 84:7567-7570. Sporn, M.B., and Todaro, G.J. (1980) Autocrine secretion and malignant transformation of cells. N. Engl. J. Med., 303:878-880. Sternfeld, M.D., Hendrickson, J.E., Keeble, W.W., Rosenbaum, J.T., Robertson, J.E., Pittelkow, M.R., and Shipley, G.D. 11988) Differential expression of mRNA coding for heparin-binding growth factor type-2 in human cells. J. Cell. Physiol., 136297304. Wahl, S.M., Wong, H., and McCartney-Francis, N. (1989) Role of growth factors in inflammation and repair. J. Cell. Bioch., 40:19% 199. Wille, J.J. Jr., Pittelkow, M.R., Shipley, G.D., and Scott, R.E. (1984) Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium clonal analysis, growth kinetics, and cell cycle studies. J. Cell. Physiol., 121:31-44. Wright, T.C. Jr., Castellot, J.J. Jr., Petitou, M., Lormeau, J.-C., Choay, J., and Karnovsky, M.J. (1989) Structural determinants of heparins growth inhibitory activity. J. Biol. Chem., 264:1534-1542.