Up-Regulation of Keratin 17 Expression in Human HaCaT Keratinocytes by Interferon-y Bernd Bonnekoh,*$ Christina Huerkamp,* Andrea Wevers,* Jiirgen Geisel,t Bela Seb6k,§ Franz-Ch. Bange,H David A. Greenhalgh,^ Erik C. B6ttger,11 Thomas Krieg,* and Gustav Malirie* *Department of Dermatology and tinstitute of Clinical Chemistry, University of Cologne, Koln, Germany; :j,'l)epartments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas, U,S,A,; SDcpartiiicnt of Deniiatology, University Medical School, Pecs, Hungary; and Ulnstitute of Medical Microbiology, Medizinische Hocbschule Hannover, Hannover, Germany
The immortalized human keratinocyte cell line HaCaT was used to assess the effect of interferon-y (IFN-y) on expression of keratin K17. Both IFN-y and K17 have heen implicated in the pathophysiology of psoriasis. "Western and quantitative enzyme-linked immunosorhent assay analyses demonstrated increasing induction of K17 protein hy 48 h exposure to IFN-y at concentrations of 10, 50, and 250 U/ml. At 50 U/ml IFN-y, immunohistochemical analysis revealed numerous K17-positive foci, whereas in situ hyhridization demonstrated K17 message in the majority of cells. In
T
he molecular events that play causal roles in the pathogenesis of psoriasis renmin poorly understood [1]. Clearly, several major mechanisms interact, including the inflammatory response [2] and the reprogramming of keratinocyte differentiation that results in the predominant features of psoriasis, namely epidermal hyperplasia and hyperkeratosis [3]. It has heen suggested that various cytokines may play significant roles in psoriasis pathophysiology, ohviously hy eliciting the important immunologic responses [4|, and also possibly hy eliciting ahnormal keratinocyte differentiation, detectahle by aberrant expression of specific keratinocyte differentiation markers [5]. To address this latter possihility, the effect of interferon-y (IFN-y) on keratin K17 protein expression was assessed //; vitro. IFN-y was chosen for study because it has heen shown to induce certain features of psoriasis /// vitro, such as human leukocyte antigen-DR and IL-10 expression in normal keratinocytes [4,6,7]. Furtherinore, IFN-y activity in vivo has heen demonstrated in psoriatic plaques |8,9], prohably released by infiltrating activated T lymphocytes [2,4], and IFN-y can induce psoriatic-like lesions at skin injection sites [10]. K17 was chosen because this keratin is highly expressed in psoriatic plaques [5], whereas normally K17 is expressed only in internal epithelia, specific skin appendages, or developing interfoUicular epiderinis [11—1.^], Because huinan primary keratinocytes express high levels of K17 ill vitro, which is unaffected by I F N - y treatinent (Bonnekoh et al. Manuscript received June 23, 1994; revised August 12, 1994; accepted for publication September 13, 1994, Reprint requests to: Dr, B, Bonnekoh, Department of Cell Biology, Room 132C, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, Abbreviations: APAAP, alkaline phosphatase—anti-alkaline phosphatase; LDH, lactate deliydrogenase.
addition, at low (5 U/ml) concentrations of IFN-y, cell proliferation and protein synthesis decreased, as determined hy ^H-thymidine laheling and *'*C-aniino acid uptake. These data suggest that aherrant K17 expression ohserved in psoriatic lesions may he a consequence of IFN-y overexpression, and that the HaCaT cell line may he a useful in vitro model system to elucidate the underlying mechanisms. Key words: type II interferonfin vitroldifferentiationlpsoriasis. J Invest Dertnatol 104:58-61, 1995
unpuhlished data), we assessed the effect of IFN-y on K17 expression in the HaCaT cell line, HaCaT cells are an immortalized human keratinocyte line [14], well characterized as an in vitro model system for experimental hyperproliferation [15], and have the advantage of low levels of endogenous K17 expression. Previously, by using epithelial cell lines, including HaCaT, to identify IFN-y— inducible genes, investigators identified a 1.6-kb mRNA, designated 39,1 [16], This was later shown to he a typo I keratin with high homology to K17 [12[, Using specific anti-K17 antibodies, we now confirm K17 induction by IFN-y, quantitated at the protein level. Although an in vitro system was used, these data suggest that anomalous IFN-y production in psoriatic lesions can alter the expression of specific keratin differentiation markers. MATERIALS AND METHODS Cell Culture and IFN-y Treatment HaCaT keratinocytes were grown routinely in Dulbecco's modification of Eagle's medium (Flow, Mcckenheini, Germany) supplemented with 10% fetal bovine serum as described |14,1,S|, For IFN-y treatment, cells were exposed at concentrations of 5 to 1250 U/ml IFN-y (Biofcron, Liuipbcim, Germany), For Western analysis, keratins were extracted from confluent cultures after 48 li incubation with IFN-y, In the culture-plate—directed alkaline pliospbatase-aiiti-alkaline pbosphatase enzyme-linked immunosorbent assay (Al'AAP ELISA) tecbnique, 1,5 X 10'' HaCaT cells were seeded per well on a 96-well plate; at confluence 24 h later, the cells were ex|:)osod to IFN-y and ELISA was performed after 48 h. To determine DNA and protein synthesis, 4 X 10'' HaCaT cells were plated per well in i\ 24-wcll multidisli, and 24 b later subconfluent cultures were incubated with IFN-y for 48 li. At this time, cultures were pulse-labeled simultaneously for 2 li with 1,0 ^Ci methyl— 'H-thymidine and 0,,S /iCi '''C-labeled amino acids. The cells were harvested and prepared for lneasurement in a Rackbota scintillation counter (LKB, BetliL-sda, MD) \iS], and total protein was determined by the Bradford assay (liiorad Laboratories, Hercules, CA) [17], In addition, thc effect of higb-dosc IFN-y on HiiCaT cell viability was assessed by trypan blue exclusion and determination of lactate deliydrogenase release (LDH), For the latter, after 48 h HaCaT culture media were processed tor
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photometric determination of LDH activity by a Boelmiiger Mannheim test kit [15]. Western Analysis IFN--)^treated HaCaT cells were trypsinized and pelleted, and keratins were extracted as descrihed [18]. Extracted keratins were soluhilized in 63 niM Tris (pH 7.4)/160 liiM sodium dodecylsulfate/2% /3-mercaptoethanol (v/v), and separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (40 ;xg protein per lane) [19]. After the transfer to nitrocelltilose, the filter was incuhated with mouse monoclonal anti-human K17 antibody (anti-KI 7, clone 17.E3, Progen, Heidelberg, Germany; dilution 1:10) [13], and the bands were visualized hy the standard indirect peroxidase technique [17]. Coomassie-blue-stained gels and K17 inimunohlots were photographed for densitometric analysis and semiquantitated using an LKB 2202 UltraScan laser densitonieter. Quantification of K17 Expression by a Culture-Plate-Directed APAAP ELISA Technique KI 7 expression was analyzed hy a modified ELISA technique, essentially as descrihed previously [20]. Briefly, after lFN-7 treatment the cultures were treated with 0.05'Xi saponin (Serva, Heidelberg, Germany)/50 uiM Tris huffcr, pH 7.4 (w/v), for 10 min at 37°C, and fixed in ice-cold 50% acetone/50% ethanol for 5 min. The cells were then incuhated with anti-K17 antihodies (diluted 1:50) and suhsequently with rabhit anti-mouse immuiioglohulins (Dakopatts, Copenhagen, Denmark; 1:50), followed by addition of the APAAP complex (Dakopatts; 1:100). The plates were developed by incubating with 200 /xl/well of a solution containing 79 mg p-iiitroplienyl phosphate (Signia Chemical Co., St. Lonis, Mo.) and 33 mg levamisole (Signia) dissolved in 58 ml of Tris-NaCl (0.87% NaCl, 0.15% Tri.s-HCl, 0.49% Tri.s-hase pH 8.8 [w/v]) and 21 ml of 0.2 M 2-amino-2-methyl-l,3-propa-diol (Serva). The reaction was stopped after 30 min hy addition of 50 yxl 3 M NaOH per well. The concentration of the p-uitroplienol product was measured by ahsorhance at 405 nm nsing an inimunoreader, and results were related to the cell numher per well (relative units/10'' cells). Immunohistochemical Analysis and Jii Situ Hybridization HaCaT keratinocytes grown on Lah-Tek chamher slides (Nunc, Naperville, IL) were incubated for 48 h with 10, 50, 250, or 12.S0 U/ml IFN-y, and then fixed with ice-cold 50"/a ethanol and 50%i acetone (v/v). Immunohistoehemistry was performed using anti-K17 antihodies (dilution 1:40), and the reaction was visualized hy the APAAP technique descrihed above (Dakopatts). At 48 h, parallel HaCaT cultures were fixed with 4°i parafoniialdehyde, and in sitit hyhridization was performed as described [21]. Briefly, a K17-specific, 240-hp anti-sense oligonucleotide was used (K17 sequence position 184-424 |12]), and the corresponding sense sequence served as a negative control probe. In t'iiro transcription was performed with 40 fiCi "'^S-UTP (Amersham, Braunschweig, Germany). Slides were incuhated for 3 h at 50°C using 10'' cpm laheled anti-sense or sense RNA prohe per section, in a mixture of 55% (v/v) formamide, 2 X sodium citrate/sodium chloride huffer (SSC) (1 X SSC = 0.15 M NaCl, 15 mM Na-citiate, pH 7.0), salmon sperm DNA (1 /J.g/fil), yeast tRNA (1 /xg//j,l), and hoviue serum alhuniin (2 /xg//Ml). To remove unliyhridized prohe, slides were washed with 2 X SSC/50% formamide at 52°C and digested with RNase A (1 /xg/ml). Autoradiography was performed at 4°C using NTli2-lilm emulsion (Kodak, Stuttgart, Germany) for 7 or 14 d, respectively. Statistical Analysis Statistical probabilities were obtained hy the U test with a level of significance of 0.05. Multiple comparisons required Boiiferroni's adjustment [15]. This statistical analysis was applied to the data from seven independent Western hlots, six separate ELISA experiments, and three independent proliferation assays, representing 36 cultures for each IFN-y concentration assessed. RESULTS AND DISCUSSION To investigate whether specific cytokines could influence the expression of keratinocyte differentiation markers implicated in psoria.sis, we incuhated the immortalized keratinocyte line HaCaT with IFN-7. As shown previously, lFN-y induced a 1.6-kh mRNA with high homology to K17 [16], and given that K17 expression is a marker for the psoriatic state |.S|, IFN-y-tieated HaCaT cells were assessed for K17 protein induction hy .specific antibodies. Western analysis (Fig lA) demonstrated that the low hasal level of K17 expression ohserved in confluent cultures increased on treatment of HaCaT cells with low (10 U/inl) doses of IFN-7 for 48 h. Repeated Western hlots demonstrated induction of K17 in a dose-dependent manner until doses exceeded 250 U/ml IFN-7 (Fig lA). Semiquantitative densitometric analysis of Western hlots
INDUCTION OF KERATIN 17 BY INTERFERON-y 59
A 46kD50
10
250
1250
IFN-7 (U/ml)
100
1000
IFN-y (U/ml) Figure 1. Western and quantitative analyses of K17 induction by IFN-y. A) Western blot of keratins extracted from confluent HaCaT cells exposed for 48 h to the concentrations of IFN-y indicated, probed with an anti-K17 antihody. Note induction of K17 protein over 10, 50, and 250 U/ml of IFN-y hut little obvious increase at higher concentrations. B) Culture-plate APAAP ELISA analysis to quantitate K17 expression per 10'' cells, measured hy relative ahsorhance at 405 nm compared to untreated controls. Data arc pooled from six separate experiments at each IFN-7 concentration. Note a two- to threefold induction of K17 up to 250 U/ml and a continued increase at 1250 U/ml, hut with a significant increase in the standard deviation.
demonstrated an approximately twofold increase in KI 7 expression levels over basal levels at 10 U/ml, increasing to approximately threefold at 250 U/ml. These significant results are in agreement with the two- to fivefold induction of the 1.6-kb K17 mRNA ohserved previously by treatment of HaCaT cells with IFN-7 [16]. To quantitate K17 indtiction accurately, a culture-plate APAAP ELISA was designed. Using this sensitive technique, an approximately threefold induction of K17 was observed over a dose range of 10 to 250 U/ml (Fig IB), and statistical analysis of 36 dishes per concentration in six separate experiments gave a significant p value of 0.05. Unlike Western analysis, this ELISA-based technique demonstrated an increase in K17 expression above 250 U/ml, albeit with a large variation in standard deviation at high IFN-7 doses (Fig IB). This variation may be due, in part, to the effects of IFN-7 cytotoxicity, which was observed morphologically and by tiypan blue exclusion analysis at concentrations greater than 400 U/ml (not shown). W e attempted to quantify the effects of cytotoxicity by assessing LDH release. Although this assay detected a wide variation of LDH release in repeated control dishes and IFN-7 treatments up to 250 U/ml, the increase in LDH release above 400 U/ml was found to be statistically significant (p = 0.05, not shown). The continued increase in K17 protein levels expressed as a function of cell nuniber observed in the ELISA (Fig IB) is intriguing and raises the possibility that, as keratin transcripts are relatively stable [22] and IFN-7 exerts an antipioliferative effect (below), this result derives from translation of accumulated K17
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THE JOURNAL OF rNVESTIGATIVE DERMATOLOGY
Figure 2. Imtnunohistochemical analysis of K17 expression. A) Kl 7 expression in confluent HaCaT cells treated with 50 U/ml IFN-y for 48 h. Note tbe focal nature of tbe intense K17 expression compared to the low levels in surrounding cells. Numerous foci were also obtained at doses of 10 and 250 U/ml. B) Low-level K17 expression in control, untreated continent HaCaT cells. Bar, 3.5 ixtn.
. That a post-transcriptional mechanism may exist in tbe regnlation of K17 protein expression is strengtbened by a comparison of K17 ininiunohistochcmical analysis (Fig 2) and in sittt hybridization (Fig 3). An anti-K17 antibody analysis of HaCaT cells treated witb 50 U/ml IFN-7 revealed numerous K17-positive foci throughout the confluent monolayer (Fig 2A). Tbese foci expressed bigb levels of K17 protein compared to surrounding cells (Fig 2A) or untreated controls (Fig 2B). This result was also obtained at 10 and 250 U/ml IFN-y (not sbown). Conversely, an itt situ analysis (Fig 3) of parallel cultures revealed relatively uniform induction of K17 transcripts (Fig iA, darkficld; Fig 3B, lightficld). Tbis discrepancy may involve cross-bybridization of tbe K17 in .citti probe witb otber keratins associated witb byperproliferation and psoriasis, sncb as K16 or K6. To rule tbis out, we tested the specificity of tbe K17 probe by in sitti analysis of frozen sections
Figure 3. In situ hybridization analysis of IFN-y-induced Kl7 mRNA. Darkfield (A) and ligbtfield (B) in situ analyses sbow higb levels of uniform K17-.specific message throughout the confluent monolayer after treatment with 50 U/ml IFN-y. Darkfield ( Q and ligbtfield (D) analyses demonstrate low levels of K17 transcripts in untreated HaCaT cells.
from psoriatic plaques. This produced a suprabasal pattern of K17 expression consistent witb tbat observed previously [5] and different from tbe basal expression pattern produced by an in sittt probe specific for K6 transcripts. Furtbermore, K17 probe specificity was confirmed by detection of bigb K17 niRNA expression in basal cell carcinoma and absent expression in nonnal interfoUicular epidermis (Wevers A, Bonnekoh B, manuscript in preparation). Tberefore, tbis comparison of tbe in situ and immnnobistocbeinistry data supports tbe existence of post-transcriptional regulation of Kl 7 protein expression. The data also suggest that K17 protein induction by IFN-'y requires otber factors, a conclusion suggested previously by investigation of K6, K16, and K17 expression in short-term, split-thickness skin organ culture [23], In tbis study, we did not assess whetber the effects of IFN-y are specific to K17 expression or other differentiation markers of disease state, or affect byperproliferation in general. Recently, expression of K6 and K16 has been observed after injection of IFN-y in huinan skin [24], and altbougb tbis expression followed a significant lag time, its presence suggests tbat IFN-y can exert a regulatory iiiRnence on a variety of keratin markers. However, it is unlikely tbat tbe iit vitro HaCaT .system wonld be useful to assay K6 or K16 induction because one of tbe major and early effects of low-dose IFN-y was inbibition of HaCaT proliferation and protein syntbesis (Fig 4), and K6 and K16 bave been well cbaracterized as markers of proliferation [25]. Down-regulation of HaCaT growth and protein synthesis was detected at IFN-y doses as low as 5 U/ml (Fig 4) and coincident with tbe induction of K17. Tbis decrease in proliferation bas also been observed in primary keratinocytes [26]. Again, wbetber IFN-y togetlier witb otber cytokines plays a role in tbe decision-making process of proliferation to differentiation will require further study, such as assessment of the expression of differentiation markers, e.g., Kl, KIO, filaggrin, and loricrin. In addition, in future studies this system may be useful to identify which of the signaling pathways are involved, and may identify a role for the Jak-STAT pathway, a direct nuclear signaling pathway associated witb IFN-y [27]. Finally, as witb any in vitro system, one sbould use caution wben extrapolating data to an itt vivo situation, especially one as complicated as psoriasis. However, tbis study bas sbown tbat HaCaT cells can be a relevant test system to evaluate keratin gene expression in
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INDUCTION OF KERATIN 17 BY INTERFERON-y
5. protein content 'H-thymidine incorporation I
6.
I "C-amino acid incorporation
7. 8.
9. 10.
11.
12.
Con.
15
44
133
400
1200
IFN-y (U/ml)
13.
14.
content determined by the Bradford assay. Note tliat even at doses as low as 5 U/ml, IFN-7 had a significant effect, wliich leveled off at approximately 60% to 70% control values at 133 U/ml.
15.
16. 17.
response to cytokine treatment, and, at least iti this instance, demonstrated a direct link between IFN-y and K17 expression. Nate Added in Proof: Following submission of our manuscript, Jiang ct al {Mol Cell Biol) 14:4759-4769, 1994) described a molecular mechanism of Iyniphocyte-keratinocyte signaling in the epidermis, and identified a site in the Kt7 promoter that conferred responsiveness to IFN-y via binding of the STAT-91 transcription factor.
18.
19. 2t).
21.
22. B.B. iims supported by the "Deutsche Foisclumgsgemeinschafi" (Bo 1194/2-1), and B.S. mas a fellow of the Alexniiiler-voii-Hiimlnihlt foundation. HaCaT keiatiuocytes were a kiud xift from Dr. N.E. Fusenig of the German Cancer Research Center, Heidelberg. The technical assistance of Mrs. V. Schhssberg and Mrs. G. Franck is gratefully appreciated.
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