transforming growth factor-β1 inhibits human keratinocyte proliferation by upregulation of a...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 228, 807–812 (1996)ARTICLE NO. 1736

Transforming Growth Factor-b1 Inhibits Human KeratinocyteProliferation by Upregulation of a Receptor-Type Tyrosine

Phosphatase R-PTP-k Gene Expression

Young Yang,*,† Minchan Gil,† Si Myung Byun,* Inpyo Choi,†Kwang Ho Pyun,† and Hyunjung Ha‡,1

*Department of Biological Sciences, KAIST, Research Center for New Bio-Materials in Agriculture, Seoul NationalUniversity, Suwon; †Immune Cell Signal Transduction RU, Korea Research Institute of Bioscience and

Biotechnology, KIST, Taejon 305-600; and ‡Division of Life Sciences,Chungbuk National University, Cheongju 360-763, Republic of Korea

Received October 7, 1996

We studied regulation of a receptor-type tyrosine phosphatase R-PTP-k gene expression in a humankeratinocyte cell line, HaCaT. Addition of TGF-b1 to the HaCaT cells markedly induced the expressionof R-PTP-k mRNA in a time- and dose-dependent manner. The induction of R-PTP-k mRNA expressionwas observed at a dose as low as 0.02 ng/ml TGF-b1 and reached a peak at 2 ng/ml TGF-b1 after 6 htreatment. The TGF-b1-induced R-PTP-k mRNA expression was suppressed by sodium orthovanadate, atyrosine phosphatase inhibitor, and H7, a serine/threonine kinase inhibitor, but not by genistein, a tyrosinekinase inhibitor. In addition, the inducing effect is not dependent on de novo protein synthesis. Takentogether, these results suggest that TGF-b1 inhibits the human keratinocyte proliferation in vitro, possiblythrough induction of R-PTP-k gene expression. q 1996 Academic Press, Inc.

Transforming growth factor-b (TGF-b) family of hormonal polypeptides is critical for avariety of physiologic activities including cellular proliferation and differentiation, morphogen-esis, wound healing, tissue repair, and other functions in many cell types (1, 2). TGF-b1 wasshown to stimulate as well as inhibit cell proliferation, depending on the cell types and theculture conditions (3, 4). In fibroblasts and epithelial cells, TGF-b1 displays a reversiblegrowth arrest, but many mesenchymal cells exhibit a proliferative effect and an increasedexpression of several extracellular matrix components and protease inhibitors (5-7).

The mechanism by which TGF-b inhibits cell proliferation following binding to its cellularreceptor has not yet been elucidated. We have investigated the mechanism of TGF-b1 growthinhibition in a human keratinocyte cell line, HaCaT. The keratinocytes required epidermalgrowth factor and transforming growth factor-a (EGF/TGF-a) for proliferation and were foundto be reversibly inhibited in their growth by TGF-b1 (8, 9). It has been demonstrated thattreatment of the keratinocytes with TGF-b1 markedly results in the decrease of c-myc geneexpression through inhibition of transcriptional initiation in 5* cis-regulatory element, sug-gesting that the c-myc may play a potentially important role in the keratinocyte proliferation(10). Recently, evidence has demonstrated showing that TGF-b1 inhibits cell growth by uncou-pling of ras p21 to the phosphodiesterase-mediated hydrolysis of phosphatidylcholine in mousekeratinocytes (11). In addition, previous studies showed that TGF-b1 inhibited proliferationof hamster lung fibroblasts, but did not alter activation of protein kinase C and polyphosphoino-sitide breakdown (12). Moreover, in human bone marrow fibroblasts, PDGF-receptor autophos-phorylation by PDGF receptor tyrosine kinase activity was decreased in the presence of TGF-

1 To whom correspondence should be addressed. Fax: 82-431-64-9600.

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b1, and was restored by okadaic acid treatment, suggesting the possible involvement of proteinphosphatases. However, the mechanism of signal transduction by which TGF-b inhibits cellgrowth is still conflicting and unclear depending on the cell types, growth conditions, and thebiological responses observed.

In an attempt to decipher an important role of protein phosphatases for the antiproliferativeeffects of TGF-b1 in human keratinocytes, in the present study, we have investigated theregulation of the R-PTP-k gene expression triggered by TGF-b1 and the effects of the severalsignal transduction inhibitors on the induction of the R-PTP-k mRNA in the HaCaT humankeratinocyte cell line.

MATERIALS AND METHODS

Cell culture and reagents. The HaCaT cells, a spontaneously immortalized aneuploid human keratinocyte cell line,were a gift from Dr. N.E. Fusenig (German Cancer Research Center, Heidelberg, Germany). The cells were culturedin Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO, Grand Island,NY), 2mM glutamine, 100 units of penicillin, and 50 mg/ml streptomycin, and maintained and subcultured in ahumidified 5% CO2 atmosphere at 37 7C. Transforming growth factor-a (TGF-a) and transforming growth factor-b1(TGF-b1) were purchased from R&D Systems (Minneapolis, MN), and protein kinase inhibitors, H7 and genistein,were from GIBCO (Grand Island, NY). Sodium orthovanadate and cycloheximide were purchased from SigmaChemical Company (St. Louis, MO). The human R-PTP-k, a receptor-type protein tyrosine phosphatase, cDNA probe(5.7 kb, full-length) was prepared from normal human keratinocyte cDNA library (manuscript in preparation).

Northern blot analysis. The HaCaT cells grown in DMEM were plated on 10-cm dishes at a concentration of 1 1106 cells per dish and cultured for 24 h. The cells were treated with 2ng/ml TGF-b1 and washed with cold phosphate-buffered saline at the indicated time intervals. Northern blot analysis and extraction of total cellular RNA wereperformed as described previously (13). The total cellular RNA was prepared with RNAzolTM B (Biotecx LaboratoriesInc., Houston, TX) under conditions recommended by the manufacturer.

Keratinocyte proliferation assay. Proliferative growth was assessed by [3H]thymidine incorporation. The HaCaTcells grown in DMEM containing 10% FBS were plated in 24-well flat-bottomed microplates (Costar, Cambridge,MA) at a concentration of 3 1 104 cells per well in a final volume of 1 ml, and cultured for 24 h. To evaluate theeffect of TGF-b1 on the keratinocyte growth, TGF-b1 was added at the indicated concentrations, and the cells wereincubated for 24 h with [3H]thymidine (0.5 mCi/well) added for the last 6 h. After harvesting the cells with trypsin,the amount of [3H]thymidine uptake was counted by liquid scintillation spectrometry (Beckman LS6000A). Resultswere calculated as the means{ SEM of triplicate cultures.

Statistical analysis. Statistical analysis was performed on [3H]thymidine incorporation using the Student t test, andall values are represented as the mean { SEM.

RESULTS

TGF-b1 inhibits proliferation of keratinocytes in vitro. The response of the HaCaT cells toTGF-b1 was assessed by measuring [3H]thymidine incorporation. We treated cells with increas-ing concentrations of TGF-b1 (0, 0.1, and 1 ng/ml) for 30 h in DMEM. The results in Fig. 1show that TGF-b1 significantly decreased [3H]thymidine incorporated by the HaCaT cells ina concentration-dependent manner. To evaluate the proliferative effect of IL-4 and TGF-a onHaCaT cells as a positive control, the cells were treated with IL-4 and TGF-a in the presenceof various concentrations ranging from 0 to 10 ng/ml. The growth-stimulatory effects of IL-4 and TGF-a were observed similar to those obtained from normal human keratinocytes (datanot shown).

TGF-b1 modulates R-PTP-k gene expression. We investigated the possibility that the activa-tion of protein tyrosine phosphatase may represent an additional signal pathway in growtharrest induced by TGF-b1. To test this possibility, we employed Northern blot analysis tomeasure the modulation of R-PTP-k mRNA expression by TGF-b1. The expression of R-PTP-k and c-myc were examined in HaCaT cells exposed to 1 ng/ml TGF-b1 for the indicatedtimes (0, 3, 6, or 24 h). As shown in Fig. 2A, a decline in c-myc mRNA expression was observedafter TGF-b1 treatment, whereas there was a slight increase in c-myc mRNA expression after24 h, indicating that the reduction of c-myc mRNA by TGF-b1 was transient and the expression

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FIG. 1. Effects of TGF-b1 on growth of a human keratinocyte cell line, HaCaT. HaCaT cells were plated at aconcentration of 3 1 104 cells/well in 24-well flat-bottomed microplates and cultured for 24 h. Cells were treatedwith the indicated concentration of TGF-b1 for 24 h and then incubated with [3H]thymidine for 6 h. The amount of[3H]thymidine uptake was assessed by liquid scintillation counting. Data represent the means of two separate experi-ments { SEM carried out in triplicate. *, p õ 0.05 versus control.

of c-myc was necessary for keratinocyte growth. In addition, expression of R-PTP-k signifi-cantly increased in the treated cells in a time-dependent manner after treatment with 1 ng/mlTGF-b1. To further determine if the increase of R-PTP-k mRNA expression was accompaniedby changes in the incubation time of treated TGF-b1, the R-PTP-k mRNA expression wasanalyzed by Northern blot analysis at various time points after the addition of 1 ng/ml TGF-b1 in DMEM. As shown in Fig. 2B, expression of R-PTP-k increased in a time-dependentmanner after treatment with 1 ng/ml TGF-b1, as early as 3 h after stimulation (Fig. 2A), andreached a peak at the approximately 24 h, and then we observed a plateau throughout TGF-b1 treatment for an additional 48 h. The treatment of the HaCaT cells for 6 h with increasingconcentrations of TGF-b1 (0 to 10 ng/ml) resulted in a dose-dependent increase of R-PTP-kmRNA expression up to 2 ng/ml, and a slight decline in R-PTP-k mRNA expression wasobserved after 10 ng/ml TGF-b1 treatment (Fig. 2B). To assess how the induction of R-PTP-k mRNA expression was regulated, HaCaT cells were treated either with TGF-b1 alone orwith TGF-b1 together with cycloheximide. As shown in Fig. 2C, the treatment with cyclohexi-mide (20 mg/ml) markedly increased the R-PTP-k mRNA expression compared with controlcultures. In addition, when the cells were treated with both TGF-b1 (2 ng/ml) and cyclohexi-mide, TGF-b1-induced upregulation of R-PTP-k mRNA expression was significantly in-creased, indicating that the direct stimulating effect of TGF-b1 may be not dependent on denovo protein synthesis. Taken together, these data suggest that TGF-b1 is sufficient to induceR-PTP-k mRNA expression in a dose-and time-dependent manner in the human keratinocytecell line HaCaT.

Sodium orthovanadate inhibits the induction of R-PTP-k mRNA by TGF-b1. It was previouslyshown that the TGF-b signal transduction pathway involves serine/threonine phosphorylation ofone of its receptors and activation of protein phosphatases in human keratinocytes (14). In orderto define and confirm the specific subsets of protein phosphatases, we used a variety of kinaseand phosphatase inhibitors to investigate whether or not the TGF-b1-induced upregulation of R-PTP-k mRNA expression was altered. As shown in Fig. 3, after treatment of HaCaT cells withTGF-b1 (2 ng/ml) and sodium orthovanadate (100 mM), a tyrosine phosphatase inhibitor, weobserved a significant reduction in the expression of R-PTP-k induced by TGF-b1. In contrast,

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FIG. 2. Effect of TGF-b1 on the regulation of R-PTP-k mRNA expression. (A) Effect of TGF-b1 on R-PTP-kand c-myc mRNA expression. Cells were treated with 1 ng/ml TGF-b1 in the presence of the indicated times (0, 3,6, or 24 h). Approximately 30 mg of total RNA was analyzed by Northern blot analysis for hybridization to R-PTP-k (Ç7.0 kb), c-myc (2.2 kb), and glyceraldehyde-3-phosphate dehydrogenase (1.3 kb) cDNA probes as describedunder Materials and Methods. (B) A time- and dose-dependency of R-PTP-k mRNA expression by TGF-b1. Cellscultured in DMEM supplemented with 10% FBS were incubated for 6 h in the presence of TGF-b1 (1 ng/ml) forthe indicated times. 1 1 106 cells were cultured for 24 h in DMEM containing 10% FBS prior to stimulation withTGF-b1 for 6 h at various concentrations ranging from 0 to 10 ng/ml. Total RNA (Ç30 mg/lane) from each treatmentwas assayed by Northern blot analysis. (C) Effect of cycloheximide on the TGF-b1 induction of R-PTP-k mRNAexpression. Cells were untreated or treated with 2 ng/ml TGF-b1 in the absence or presence of 20 mg/ml cycloheximidefor 24 h. Northern analysis of Ç30 mg total RNA from each treatment was described under Materials and Methods.

H7 (8 mM), a serine/threonine kinase inhibitor, less effectively counteracted the upregulation ofR-PTP-k mRNA expression, indicating that the serine/threonine phosphorylation of the TGF-breceptors may be inhibited. In addition, the TGF-b1-induced levels of R-PTP-k mRNA expressionwere not altered by treatment of genistein (20 mg/ml), a tyrosine kinase inhibitor. These datastrongly suggest a critical relationship between a TGF-b1-induced upregulation of R-PTP-kmRNAexpression and an inhibition of keratinocyte growth by TGF-b1.

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FIG. 3. Effect of kinase and phosphatase inhibitors on R-PTP-k mRNA induction. Cells were incubated for 24 hwith 2 ng/ml TGF-b1 or with genistein (20 mg/ml), sodium orthovanadate (100 mM), or H7 (8 mM) alone or incombination with TGF-b1. Total RNA was assayed by Northern blot analysis for hybridization to R-PTP-k andGAPDH cDNA probes.

DISCUSSION

TGF-b1 is a well-known growth inhibitory modulator for a variety of cell types includingepithelial, endothelial, lymphoid, and many myeloid cells (15-18). In the human keratinocytes,TGF-b1 acts as a potent growth inhibitor (9). The mechanism underlying the growth arrest inkeratinocytes by TGF-b is poorly understood. Recent studies indicated that TGF-b1 rapidlysuppressed c-myc expression, which plays an important role in cell proliferation, in mousekeratinocytes. Antisense c-myc oligonucleotides were also effective in the inhibition of theirproliferation, suggesting that TGF-b1 inhibition of c-myc expression may be critical to thegrowth arrest by TGF-b1 (10). In addition, previous studies proposed that the protein productof the retinoblastoma gene (pRB) is a likely candidate for mediating the TGF-b1-inducedgrowth arrest (19). The involvement of protein serine/threonine and tyrosine phosphataseactivations was recently suggested in the inhibition of keratinocyte growth by TGF-b1 (14).We have previously observed that human R-PTP-k, a receptor-type protein tyrosine phospha-tase, was restrictedly expressed in normal human keratinocytes and epidermal cell lines amonga series of cell lines tested (manuscript in preparation). These findings, together with our dataobserved, led us to hypothesize that TGF-b1 upregulation of R-PTP-k expression may relatein a direct or indirect manner to growth inhibition in epidermal cells.

In the present study, therefore, we have investigated the role of R-PTP-k in the mechanismof TGF-b1 growth inhibition in keratinocytes using a human keratinocyte cell line HaCaT.Our studies clearly demonstrated that treatment of the HaCaT cells with the TGF-b1 resultedin the significant increase of R-PTP-k mRNA expression in vitro, providing an evidence insupport of a causative role for TGF-b1 induction of R-PTP-k expression in the growth inhibi-tory response. Although there are not yet data demonstrating a specific tyrosine phosphatasein the growth arrest induced by TGF-b1, an association of the TGF-b1 effect of growth arrestwith the regulation of protein phosphatase activation was recently demonstrated in the humankeratinocytes (14). Consistent with the recent data, the expression of R-PTP-k, was upregulatedfollowing the addition of TGF-b1 to human keratinocyte HaCaT cells. Compared with thetreatment of HaCaT cells with TGF-b1 alone, the significant induction of R-PTP-k mRNAexpression in the presence of cycloheximide and TGF-b1 was observed in our studies, sug-gesting that labile factors either enhance a degradation of its message or repress its transcription(Fig. 2). Moreover, the tyrosine phosphatase inhibitor sodium orthovanadate nearly completely

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abolished the TGF-b1-induced expression of R-PTP-k. Thus, our results provide an evidencethat activation of the tyrosine phosphatase pathway in keratinocytes can lead to the generationand maintenance of signals for the inhibition of growth by TGF-b1. In addition, H7, a serine/threonine kinase inhibitor, effectively decreased the TGF-b1-induced expression of R-PTP-k,probably by inhibiting serine/threonine phosphorylation involved in the initial signal transduc-tion pathway of TGF-b1 via TGF-b1 receptors. We have previously demonstrated that gen-istein, a tyrosine kinase inhibitor, can inhibit the IL-4-induced proliferation in normal humankeratinocytes through the reduction of c-myc gene expression, implicating that tyrosine kinasesmay be involved in the IL-4-induced proliferation (13). As demonstrated in Fig. 2, however,TGF-b1-induced keratinocyte growth arrest was associated with the tyrosine dephosphorylationrather than tyrosine phosphorylation. It seems that differential signal transduction pathway mayoccur in the general mechanisms of growth stimulation and inhibition in human keratinocytes.

In conclusion, together with the activation of protein phosphatases in TGF-b1 inhibition ofkeratinocyte growth, our studies present a strong evidence that expression of R-PTP-k isnecessary for inhibition of keratinocyte growth by TGF-b1, and that the direct inducing effectof TGF-b1 is not dependent on de novo protein synthesis of an intermediate based on theenhancement of R-PTP-k mRNA expression by cycloheximide.

ACKNOWLEDGMENTThis work was supported by a grant (NB061S) from the Ministry of Science and Technology of Korea.

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transforming growth factor-β1 inhibits human keratinocyte proliferation by upregulation of a...

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