Control of Growth Regulatory and Differentiation-specific Genes in Human Epidermal Keratinocytes by Interferon y ANTAGONISM BY RETINOIC ACID AND TRANSFORMING GROWTH FACTOR pl*

Interferon y (JFN-y) is a potent inducer of squamous differentiation in normal human epidermal keratinocytes. This induction is characterized by a 295% de- crease in the mRNA level of two growth regulatory genes, cdc2 and E2F-1, and a 7-16-fold increase in the expression of two squamous cell-specific genes, transglutaminase type I and cornifin. In contrast to the de- crease in cdc2 and E2F-1 expression, the increase in transglutaminase type I and cornifin mRNAs by IFN-y occurs after a lagtime of more than 12 h. These results are consistent with the hypothesis that in normal hu- man epidermal keratinocyte cells irreversible growth arrest precedes the expression of the squamous-differ- entiated phenotype. The action of IFN-y on the expression of squamous cell-specific genes is antagonized by retinoic acid and transforming growth factor pl. Both factors are potent suppressors of the induction of trans- glutaminase type I and cornifin; however, they do not prevent the commitment to irreversible growth arrest. Several squamous cell carcinoma cell lines do not show a detectable decrease in cdc2 or increase in transgluta- minase type I mRNA levels after IFN-y treatment and appear to be altered in their control of squamous differ- entiation. The epidermis is a stratified squamous epithelium that con-sists

Interferon y (JFN-y) is a potent inducer of squamous differentiation in normal human epidermal keratinocytes. This induction is characterized by a 295% decrease in the mRNA level of two growth regulatory genes, cdc2 and E2F-1, and a 7-16-fold increase in the expression of two squamous cell-specific genes, transglutaminase type I and cornifin. In contrast to the decrease in cdc2 and E2F-1 expression, the increase in transglutaminase type I and cornifin mRNAs b y IFN-y occurs after a lagtime of more than 12 h. These results are consistent with the hypothesis that in normal human epidermal keratinocyte cells irreversible growth arrest precedes the expression of the squamous-differentiated phenotype. The action of IFN-y on the expression of squamous cell-specific genes is antagonized by retinoic acid and transforming growth factor pl. Both factors are potent suppressors of the induction of transglutaminase type I and cornifin; however, they do not p r e v e n t the commitment to irreversible growth arrest. Several squamous cell carcinoma cell lines do not s h o w a detectable decrease in cdc2 or increase in transglutaminase type I mRNA levels after IFN-y treatment and appear to be altered in their control of squamous differentiation.
The epidermis is a stratified squamous epithelium that consists of four histologically distinct layers (1). In normal skin, cells in the basal layer comprise the proliferative compartment (2). These cells undergo irreversible growth arrest and start to express squamous cell-specific genes when they transit into the suprabasal (spinous) layer (1)(2)(3)(4)(5). Similar to other cell systems (6), irreversible growth arrest in NHEKl cells is closely associated with the expression of the differentiated phenotype (5).
The events that link the control of growth arrest with the induction of the squamous differentiated phenotype have yet to be elucidated. The expression of squamous differentiation-specific genes is induced at very specific stages during squamous differentiation and appears to be controlled at the transcriptional level (4, 7-9). A characteristic feature of squamous differentiation is the formation of the cross-linked envelope, a layer of cross-linked protein deposited just beneath the plasma membrane (10,11). Transglutaminase type I catalyzes the formation of e-(y-g1utamyl)lysine linkages between envelope precursor proteins such as involucrin, cornifin, and loricrin (9, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: NHEK, normal human epidermal keratinocytes; TGF, transforming growth factor; INF-y, interferon-y; kb, kilobase(s); GPDH, glyceraldehyde-3-dehydrogenase. [12][13][14][15][16][17][18]. A variety of factors including activators of protein kinase C, calcium ions, retinoids, and transforming growth factor P l (TGF-Pl) have been shown to influence epidermal differentiation (18)(19)(20)(21)(22).
IFN-y, a cytokine produced by activated T-cells and natural killer cells, affects a vast array of different cellular processes, such as antiviral responses, cell growth and differentiation, and immunoregulatory functions (23,24). Cellular responses to IFN-y are mediated by a specific high-affinity receptor expressed at the cell surface (25)(26)(27). Binding of IFN-y to its receptor results in activation of a specific protein kinase and the subsequent phosphorylation and activation of a latent cytoplasmic protein, y-activated factor (28). After translocation to the nucleus, this factor interacts with a specific DNA element (y-activated site) in the promoter of target genes such as the guanylate-binding protein resulting in an altered rate of transcription (28)(29)(30). In addition, the activation of specific phosphatase(s) and kinase(s) by IFN-y may influence other signaling pathways such as protein kinase C (31) or sphingomyelin (32).
Evidence has been provided suggesting that IFN-y plays a role in the regulation of proliferation and differentiation in the epidermis (33). IFN-y has also been reported to inhibit growth and induce the production of TGF-a and intercellular adhesion molecule-1 in cultured epidermal keratinocytes (34,351. In the present study, we analyze in more detail the action of IFN-y on the expression of several growth regulatory and differentiationspecific genes in cultured NHEK cells and in several squamous cell carcinoma cell lines. In addition, we examine the interference of two established modulators of epidermal differentiation, retinoic acid, and TGF-P1, with the IFN-y-induced squamous differentiation. Both retinoic acid and TGF-P1 antagonize the induction of squamous cell-specific genes; however, these factors do not affect the induction of irreversible growth arrest by IFN-y. Our findings are consistent with the hypothesis that IFN-y is a n effective inducer of squamous cell differentiation in NHEK cells. saline and incubated in 1 ml of methanoVacetic acid (9:l) for 1 h at 4 "C. Cells were then treated with 0.5 ml of 0.2 M NaOH a t 37 "C for 30 min. After neutralization, aliquots were taken for the determination of protein and radioactivity. Colony forming efficiency was determined as described previously (22).
~ansglutaminase Assay-Cells grown in 60" dishes were washed in ice-cold phosphate-buffered saline containing 1 m~ EDTA and 1 m~ phenylmethylsulfonyl fluoride. Cells were disrupted by three freeze and thaw cycles. The homogenate was centrifuged at 105,000 x g, yielding the particulate and soluble fractions. Transglutaminase assays were performed on the total homogenate to determine total transglutaminase activity and on the particulate and soluble fractions to determine the activity of type I and type I1 transglutaminase, respectively. Transglutaminase activity was measured by determining the incorporation of t3H1putrescine (16.2 Cilmmol, Dupont) into casein hydrolysate (Sigma) as described previously (36), and was expressed as disintegrationdmin of [3Hlputrescine incorporated in 1 h/mg of total cellular protein.
cDNA Probes-Probes for E2F-1,2 human IFN-y receptor (using amplimers from Clontech) and cdc2 (37) were prepared by polymerase chain reaction amplification. The cDNA probe for c-myc was purchased from Oncogene Science (Uniondale, NY). The cDNA probes for Rb and p53 were obtained from Dr. Y. T. Fung (University of Southern California, Los Angeles, CA) and Dr. M. Oren (Weizmann Institute of Science, Rehovot, Israel), respectively. The recombinant cDNAclones pTG-7 and pTG3400 contain fragments of the coding region of transglutaminase type I and type 11, respectively (14,20). The cDNA probe for chicken glyceraldehyde-3-dehydrogenase consisted of a 1.12-kb PstI restriction fragment of the cDNA clone pGAD-28 (38). The recombinant cDNA clone SQ37 was digested with EcoRI yielding a 1.0-kb fragment encoding cornifin (18). Human collagen al(nr) cDNA was a 2.6-kb PstI restriction fragment of plasmid pHT-21 (39). All probes were gel purified and labeled with [a-32PldCTF' (3000 Ci/mmol; Amersham Corp.) via random priming using the kit and protocols supplied by Bethesda Research Laboratories.
Preparation of RNA and Northern Blot Anulysis-Cultured NHEK cells were solubilized in guanidinium isothiocyanate and the extract centrifuged through a discontinuous CsCl gradient to pellet the total RNA (40). Poly(A)+ RNA was obtained by affinity chromatography through oligo(dT)-cellulose push columns (Stratagene, La Jolla, CA). RNA was electrophoresed through a 1.2% denaturing agarose-formaldehyde gel. Following electrophoresis, RNA was transferred to Nytran (Schleicher & Schuell) and then cross-linked by W irradiation. Northe m blots were prehybridized for 24 h a t 42 "C in a buffer containing 50% formamide, 5 x SSPE, 2 x Denhardt's, 1% SDS, and 250 pg/ml sheared salmon sperm DNA as described (40). Following addition of the labeled probe (3-15 nglml), hybridization was allowed to proceed overnight at 42 "C. Blots were washed a t a final stringency of 60 "C in 0.2 x SSC, 0.1% SDS. Blots were exposed to Kodak XAR5 film and quantitated by scanning laser densitometry using the GSXL2 software package (Pharmacia LKB Biotechnology Inc.).

RESULTS
To study the action of IFN-y on squamous cell differentiation in NHEK cells, we examined its effect on two stages of this differentiation process, irreversible growth arrest and expression of the squamous phenotype. IFN-y inhibited the proliferation of NHEK cells in a concentration-dependent manner (Fig.  LA). The concentration of half-maximum inhibition (ECS0) was 10 unitdml IFN-y. The inhibition of cell proliferation by IFN-y was an irreversible process since removal of IFN-y from the medium 3 h after addition failed to restore proliferation. This was confirmed by the observed reduction in the colony forming efficiency of NHEK cells from 43% to 50.1% after IFN-y treatment (Table I). These results indicate that IFN-y commits NHEK cells to a state of irreversible growth arrest. The growth inhibitory action was specific for IFN-y since the addition of 1000 unitdm1 IFN-a had little effect on the proliferation of NHEK cells (Fig. LA). The inhibition of the growth of NHEK cells by IFN-y was accompanied by changes in the expression of several growth control genes. As shown in Fig. 1   NHEK cells with IFN-y caused a 295% reduction in the level of p53, cdc2, and E2F-1 mRNA. In contrast, the level of Rb mRNA was not affected at all whereas IFN-y caused a reduction of about 50% in the level of c-myc mRNA. The responsiveness of c-myc expression to IFN-y varied with no change being observed in some experiments. NHEK cells treated with IFN-y acquired a squamous phenotype and became more adhesive to the substratum (not shown). These observations are consistent with an induction of squamous cell differentiation in IFN-y-treated NHEK cells. To investigate this further, we tested the effect of IFN-y on the expression of two established squamous cell-specific genes, transglutaminase type I and cornifin (13,18,35). Addition of IFN-y to cultured NHEK cells caused a dose-dependent increase in type I (particulate) transglutaminase activity and cornifin protein (Fig. 2, A and B ). The ECS0 for the induction of transglutaminase activity and cornifin was estimated to be 25 and 40 unitdm1 of IFN-y, respectively. The increase in transglutaminase was time-dependent ( Fig. 2C). After 3 days of treatment, IFN-y caused a 10-14-fold increase in transglutaminase type I activity above control levels. Previously it was shown that increasing Ca2+ concentration in the medium (1.8 instead of 0.15 mM) enhanced transglutaminase type I expression in confluent, differentiating cultures of NHEK cells whereas a high Ca2+ concentration had little effect in undifferentiated (exponential phase) cultures (34). These findings suggested that Ca2+ by itself does not trigger differentiation in NHEK cells but can enhance the expression of squamous cellspecific genes in cells already committed to differentiate (37). The observed effects of Ca2+ on IFN-y-treated and untreated cells are in agreement with this interpretation; high Ca2+ concentration in the medium increased the IFN-y-induced transglutaminase activity (Fig. 2, A and C) whereas high calcium by itself caused only a slight increase. Since cultures treated with high Ca2+ continue to grow and are at a higher cell density (but still at subconfluence) than IFN-y-treated cultures at the time cells are harvested, the small increase in squamous-specific genes by Ca2+ may be due to increased expression in a small fraction of cells already committed to squamous differentiation. The IFN-y-induced changes in transglutaminase type I activity and cornifin protein were shown to be related to an increase in the level of corresponding mRNA (Fig. 3). The presence of high Ca2+ concentration slightly increased the level of transglutaminase type I and cornifin mRNA whereas simultaneous treatment of NHEK cells with IFN-y and Ca2+ had a synergistic effect on transglutaminase type I and cornifin mRNA levels.
Previous reports indicated that the controls of growth and differentiation are interrelated (2,5). To study the link between growth arrest and the induction of the squamous-differentiated phenotype further, we examined the action of IFN-y on several growth regulatory and squamous cell-specific genes as a function of the duration of IFN-treatment. As shown in Fig. 4A treatment of NHEK cells with IFN-y caused a rapid decline in the level of cdc2 and E2F-1 mRNAs. This decrease coincided with the inhibition of the incorporation of [3H]-thymidine (Fig.  4A). In contrast, the changes in the level of transglutaminase type I and cornifin mRNA were observed after a lag time of more than 12 h (Fig. 4B ). These findings indicate that changes in the expression of growth regulatory genes occur more rapidly than that of squamous cell-specific genes and suggest that growth arrest precedes the induction of differentiation-specific genes. These observations are consistent with the hypothesis that in normal cells the control of growth arrest and differentiation are closely linked (5). Several studies have reported that retinoic acid inhibits squamous differentiation in phorbol ester-treated and confluent cultures of NHEK cells (5, 38). It has been demonstrated that retinoids do not block irreversible growth arrest but abol- ish the expression of squamous-specific genes (5). Therefore, we examined the effect of retinoic acid on IFN-"induced squamous differentiation. Simultaneous treatment of NHEK cells with retinoic acid and IFN-y had no effect on the growth inhibitory effect of IFN-y (Fig. 1). Moreover, retinoic acid did not prevent the reduction in colony forming efficiency (Table I) by IFN-y indicating that retinoic acid did not block IFN-y-induced irreversible growth arrest. However, retinoic acid was able to suppress effectively the induction of several squamous cellspecific genes. Retinoic acid inhibited the IFN-y-induced transglutaminase type I activity in a concentration-dependent man- ner (Fig. 5). The ECS0 of this inhibition was about 0.08 m. Retinoic acid also blocked the increase in cornifin protein (Fig.  6). These observations are in agreement with the hypothesis that retinoic acid prevents the induction of the squamous-differentiated phenotype.
In contrast to IFN-y, TGF-P1 induces reversible growth arrest in NHEK cells and does not induce squamous differentiation (20,41). The results with TGF-P1 described in Figs. 6-8 are in agreement with these observations. Interestingly, study of the effect of TGF-p1 on IFN-y-induced squamous differentiation in NHEK cells demonstrated an antagonistic action between these two cytokines. Although TGF-pl did not prevent the IFN-y-induced irreversible growth arrest as determined by colony forming efficiency (Table I), it suppressed the induction of the squamous-specific genes, cornifin (Fig. 6) and transglutaminase type I (Fig. 7). Additionally, while suppressing the IFN-y-induced transglutaminase type I activity increasing concentrations of TGF-01 enhanced type I1 transglutaminase activity (Fig. 7). The latter is consistent with previous observations (20) showing induction of transglutaminase type I1 by TGF-P1 in NHEK cells and indicates that the action of TGF-p1 on the expression of transglutaminases prevails over that of The antagonism between IFN-y and retinoic acid-TGF-Pl on differentiation was reflected in changes in the mRNA levels of the differentiation-specific genes (Fig. 8). The increase in transglutaminase type I and cornifin mRNA by IFN-y was abrogated by both retinoic acid and TGF-P1. The action of TGF-P1 on gene expression was dominant over the effect of IFN-y as demonstrated by the effects on transglutaminase type I1 and collagen IV. TGF-p1 either in the presence or absence of IFN-y increased the level of type I1 transglutaminase mRNA. Similarly, the expression of collagen al(IV), another gene induced by TGF-p1 in NHEK cells (20,42), was also enhanced by TGF-P1 in the presence of IFN-y.
The level of cdc2 mRNA in NHEK cells treated under the various conditions correlated closely with the extent of I3H1thymidine incorporation (Fig. 8). The down-regulation of cdc2 mRNA by IFN-y was not reversed by retinoic acid and supports the conclusion that retinoic acid does not prevent growth arrest (Table I) rest in NHEK cells, only the growth arrest by TGF-P1 was accompanied by a 295% reduction in c-myc mRNA levels (not shown) whereas cdc2 mRNA was down-regulated by both agents. When NHEK cells were treated with IFN-y and TGF-P1 simultaneously the action of TGF-P1 on c-myc expression prevailed (not shown). These data support the hypothesis that retinoic acid and TGF-P1 suppress the IFN-y-induced expression of squamous-specific genes but do not prevent IFNymediated irreversible growth arrest. Squamous carcinoma cells exhibit defects in the mechanisms that control growth and differentiation. To determine whether squamous carcinoma cells and transformed epidermal cells are altered in their response to IFN-y, we examined the effect of IFN-y on the incorporation of [3H]thymidine and compared this with the effects on cdc2 and transglutaminase type I mRNA expression. IFN-y had little effect on the incorporation of [3H]thymidine and on the level of cdc2 mRNA in the carcinoma cell lines K14, SCC13, and SQCCrYl (Fig. 9). In addition, IFN-y did not induce expression of the squamous cell-specific gene, transglutaminase type I. These results suggest that these cells are resistant to IFN-y-induced squamous differentiation. "his resistance appears not to be due to the inability to express IFN-y receptors since mRNA for these receptors was detected in all these cells (Fig. 9).

DISCUSSION
In this study, we show that treatment of NHEK cells with IFN-y induces two stages characteristic of squamous differentiation, irreversible growth arrest and expression of squamous cell-specific markers. These findings indicate that IFN-y is a potent inducer of squamous cell differentiation and are consistent with the ultrastructural changes reported previously in IFN-y-treated NHEK cells (33). The irreversible growth arrest of NHEK cells induced by IFN-y was accompanied by a decrease in the levels of cdc2, p53, and E2F-1 mRNA whereas the levels of c-myc and Rb mRNA were not greatly altered. The decrease in the expression of cdc2 and E2F-1 mRNAs appears to precede the increase in the expression of two differentiationspecific genes, transglutaminase type I and cornifin. Our results are consistent with the hypothesis that the control of growth arrest and expression of squamous cell-specific genes are separate but interdependent processes. A similar coupling has been demonstrated previously for squamous differentiation in phorbol ester-treated NHEK cells and in confluent NHEK cultures (5,22). In several other cell systems that undergo terminal differentiation, a link between proliferation and differentiation has been established (6). In muscle cell and adipocyte differentiation, specific "master genes," like myoD and CEBP, respectively, have been shown to induce growth arrest as well as the expression of certain differentiation-specific genes and appear to be involved in coupling growth arrest and expression of the differentiated phenotype (43,44). Although the existence of such putative master genes in squamous differentiation has been postulated, such genes have yet to be identified (5).
The induction of transglutaminase type I and cornifin was delayed compared to IFN-y-induced growth arrest. Therefore, the induction of squamous cell-specific genes by IFN-y may be rather distal from the initial activation of its receptor and may not involve y-activated site elements in the promoter of these genes. Instead, the IFN-y-induced increase in squamous-specific genes may be a consequence of an alteration in the rate of transcription of genes (possibly the master genes mentioned above) that initiate events at an earlier stage in the differentiation process. The fact that no y-activated site elements are present in a 3-kb promoter region of the transglutaminase type I gene (7) could support this hypothesis. Alternatively, the action of IFN-y in NHEK cells may involve other signaling pathways such as the activation of protein kinase C (31) or increased turnover of sphingomyelin (32). Since phorbol estermediated activation of protein kinase C has been shown to induce squamous differentiation in NHEK cells (5,22), the induction of squamous differentiation by IFN-y could be mediated by the activation of protein kinase c. However, such a mechanism is not consistent with the observation that bryostatin, which blocks phorbol ester-induced squamous differentiation, has no effect on IFN-y-induced growth arrest Retinoic acid was unable to prevent the IFN-y-induced irreversible growth arrest but effectively antagonized the induction of the squamous cell-specific genes, transglutaminase type I and cornifin. Similarly, retinoic acid has been shown previously to inhibit the induction of squamous cell-specific genes in phorbol ester-treated NHEK cells and in confluent cultures without preventing irreversible growth arrest ( 5 , 20). These results suggest that retinoic acid affects specific stages during squamous differentiation. Retinoic acid may inhibit the induction of differentiation by phorbol esters and IFN-y a t a step that is common to both signal transduction pathways. Recently, we reported that phorbol esters and retinoic acid regulate transglutaminase type I expression at the transcriptional level (7). We are in the process of investigating what specific ele- ments in the transglutaminase type I promoter are involved in mediating the response to either the phorbol ester or IFN-y. It is likely that the nuclear retinoic acid receptors (RARs/RXRs) present in NHEK cells (45) play a role in the retinoid-mediated suppression of squamous-specific genes.
In agreement with previous reports (20,41), TGF-P1 by itself causes reversible growth arrest but does not induce squamous cell differentiation in NHEK cells. In this study we show that although TGF-01 does not prevent the IFN-y-induced irreversible growth arrest, it functions as a potent antagonist for the IFN-y-dependent induction of transglutaminase type I and cornifin. In addition, the induction of transglutaminase type I1 and collagen d ( W ) by TGF-/3 still Occur in the presence of IFN-y. In several cell types, including epidermal keratinocytes (46), retinoic acid has been shown to stimulate the production of TGF-P. Since both retinoic acid and TGF-P1 antagonize the induction of squamous-specific genes by IFN-y, the effect of retinoic acid could be mediated by TGF-P. However, this appears to be an unlikely explanation here since the expression of transglutaminase type I1 and collagen al(lV) which are upregulated by TGF-P are not increased by retinoic acid (Fig. 8). Also, antibodies against TGF-P1, 2, and 3 did not reduce the action of retinoic acid. 3 Malignant and transformed keratinocytes have been shown to respond differently to factors that regulate growth and differentiation. For example, squamous carcinoma cells have been shown to be resistant to phorbol ester-induced squamous differentiation (22). In this study, we show that several squamous cell carcinoma cell lines are not growth arrested and do not exhibit a reduced level of cdc2 mRNA after IFN-y treatment. In addition, no increase in the level of transglutaminase type I mRNA was observed. These results indicate that IFN-y does not induce squamous differentiation in these cells. This inability to induce differentiation is not due to a lack of IFN-y receptor expression since IFN-y receptor mRNA was detected in the carcinoma cells. In addition, these cell lines do not have an intrinsic defect in the IFN-y signaling pathway since the induction of guanylate-binding protein by IFN-y was not impaired in these cells.2 Since these cells also do not undergo differentiation a t confluence or when treated with phorbol esters (221, the defect in the control of squamous differentiation may be at a point that is common to the different signaling pathways.
IFN-y receptors have been reported to be uniformly expressed in the basal and suprabasal cell layers of the normal epidermis and are expressed in cultured keratinocytes (47,481 (Fig. 9). In keratinocytes of psoriatic skin lesions, a hyperproliferative skin disorder, the attenuated growth inhibitory response to IFN-y has been attributed a t least in part to the lack of IFN-receptors (47,48). Our study shows that IFN-y is a potent inducer of differentiation of epidermal keratinocytes in culture and provides evidence for complex interactions between several signaling pathways. Our observations are consistent with the concept that IFN-y is important in the control of cellular proliferation and differentiation in the epidermis (35,481.