Activating transcription factor 3 (ATF3) expression is increased in erythema multiforme and is regulated by IFN-c in human keratinocytes

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1 DOI:1.1111/j x Activating transcription factor 3 () expression is increased in erythema multiforme and is regulated by IFN-c in human keratinocytes Brian P. Pollack 1,2,3, Bishu Sapkota 1 and Paul L. Haun 4 1 Department of Dermatology, Emory University School of Medicine, Atlanta, GA, USA; 2 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA; 3 Veteran s Administration Medical Center, Decatur, GA 333, USA; 4 Medical College of Georgia, Augusta, GA 3912, USA Correspondence: Brian P. Pollack, Emory University School of Medicine, Department of Dermatology, 11 Woodruff Circle Suite 51, Atlanta, GA 3322, USA. Tel.: , Fax: ; bppolla@emory.edu Abstract: Activating transcription factor 3 () is a member of the ATF cyclic AMP responsive element-binding protein (CREB) family of transcription factors and is involved in the regulation of immune responses, apoptosis, DNA repair and oncogenesis. The epidermal expression of in the setting of cutaneous inflammation has not been well characterized. To examine the expression of in the setting of inflammatory skin disease, protein expression was analysed by immunohistochemistry (IHC). We found diffuse epidermal protein expression in skin biopsies of erythema multiforme (EM). Given the role of interferon (IFN)-c in erythema multiforme, we sought to examine the impact of IFN-c on expression in human keratinocytes. IFN-c induced mrna and protein in primary human keratinocytes and HaCaT cells. Thus, epidermal expression can be increased in the setting of inflammatory skin diseases and is regulated by IFN-c in human keratinocytes. Key words: activating transcription factor 3 epidermis erythema multiforme gene expression interferon-gamma keratinocyte Accepted for publication 19 October 29. Please cite this paper as: Activating transcription factor 3 () expression is increased in erythema multiforme and is regulated by IFN-c in human keratinocytes. Experimental Dermatology 21; 19: e31 e313. Background Activating transcription factor 3 () is a member of the ATF cyclic AMP response element-binding protein (CREB) family of transcription factors that has been implicated in several processes relevant to skin disease and epidermal function such as: apoptosis (1), DNA repair (2), immune response regulation (3,4) and oncogenesis (5,6). In most tissues examined, basal levels are low but can be induced in response to cytokines, DNA damaging agents and other inducers of cellular stress responses (7). The impact of expression depends on the cellular context and both pro-apoptotic (1,8) and pro-survival (9,1) functions have been reported. The epidermal expression and function of in human skin has not been well characterized. Erythema multiforme (EM) is an acute (and possibly recurrent) mucocutaneous inflammatory skin disease that is thought to represent an immunologic reaction to either an infection or medication (11). However, a specific cause is not always identified (12). The generation of auto-reactive T cells that target the epidermis plays an important role in the pathogenesis of EM (13). These auto-reactive T cells release inflammatory cytokines such as interferon (IFN)-c and tumor necrosis factor (TNF)-a (14) that can induce apoptosis and other changes in epidermal keratinocytes. Understanding the factors that regulate the response of epidermal keratinocytes to these cytokines will provide important insight into disease pathogenesis and may help identify additional therapeutic approaches. Questions addressed What is the epidermal expression pattern of in inflamed and non-inflamed human skin? Does IFN-c increase mrna and protein in human epidermal keratinocytes? Experimental design Immunohistochemistry Cases of EM and other skin biopsies were stained for expression in the Winship Cancer Institute Pathology Core laboratory using standard techniques and commercially available reagents as described in Supporting information. 31 ª 29 John Wiley & Sons A/S, Experimental Dermatology, 19, e31 e313

2 Induction of by IFN-c The impact of IFN-c on mrna and protein levels as well as mrna stability were analysed in HaCaT cells and primary human epidermal keratinocytes using standard molecular approaches as described in Supporting information. Results protein expression within human epidermis We found conspicuous nuclear epidermal expression of (score of 2 or 3) in 6 8 (75%) skin biopsies from patients diagnosed with EM (Fig. 1a, First row and Figure S1, first and third row). Addition of an -blocking peptide completely eliminated the staining (Fig. 1a, first versus second row and Fig. S1, first versus second row and third versus fourth row). Thus, can be expressed (a) + Blocking peptide (b) EM 1 E M2 EM 3 EM 1 E M2 EM 3 BCC SCC SCC Figure 1. protein expression in human skin. (a) Top panels: Formalin-fixed paraffin-embedded (FFPE) human skin biopsies of erythema multiforme (EM) were analysed by immunohistochemistry (IHC) using antibodies against. Bottom panels: The same tissue as those in the panels directly above were analysed using the same IHC staining protocol and conditions with the addition of an blocking peptide. (b) FFPE skin biopsies from normal (excisional) skin (), basal cell carcinoma (BCC), and two different squamous cell carcinomas (SCC) were analysed by IHC for protein expression. The scale bar represents 15 lm. in vivo within human epidermal keratinocytes in the setting of inflammatory skin disease. In contrast to EM, other skin biopsies had less intense or only focal epidermal expression of. was expressed at the periphery of sebaceous lobules in skin biopsies that had little detectable expression elsewhere (Fig. 1b, top row second and third panel from left). Within a basal cell carcinoma (BCC), expression of was seen in cells at the periphery of a tumor nodule as well as focally within the overlying epidermis (Fig. 1b, bottom row, first panel from left). In contrast, we found little or only focal staining in two squamous cell carcinomas analysed (Fig. 1b, bottom row, second and third panels from left). We also analysed two biopsies of malignant melanoma (MM) and found only focal epidermal staining (Fig. S2). Preliminary analysis of other inflammatory skin diseases suggests that increased epidermal expression is not unique to EM as elevated levels were found in skin biopsies from patients with cutaneous lupus erythematosus (LE), cutaneous T cell lymphoma (CTCL) and psoriasis (Supporting information, Fig. S3). IFN-c induces mrna and protein in human keratinocytes We examined whether IFN-c alone is sufficient to induce the expression of in human keratinocytes as has been demonstrated in other cell types, specifically, murine macrophages (15) and a human monocyte cell line (16). Primary human epidermal keratinocytes (PHKs), and the immortalized keratinocyte cell line HaCaT were used for these experiments. HaCaT cells were chosen because they respond to IFN-c (17) and maintain the ability to differentiate (18). While a modest induction of mrna and protein was seen 4 and 8 h following IFN-c treatment, by 24 and 48 h the induction was more pronounced (Fig. 2a,b). We confirmed the induction of in PHKs. mrna was induced following IFN-c treatment in a dosedependent manner in PHKs (Fig. 2c) and this increased mrna was associated with an increase in protein (Fig. 2d). The magnitude of the induction was much smaller in PHKs. The difference in the induction of mrna observed between PHKs and HaCaT cells was at least partially due to increased basal mrna levels in PHKs prior to IFN-c treatment (Supporting information, Fig. S4a). IFN-c increases mrna stability IFN-c increased the stability of mrna (Fig. 2e). In contrast, no effect on mrna stability was seen 24 h after 4 mj cm 2 of ultraviolet radiation B (UVB) which can induce mrna and protein and stabilize the ª 29 John Wiley & Sons A/S, Experimental Dermatology, 19, e31 e

3 (a) 3 HaCaT mrna 25 2 Histone H Time (h) (c) Time (h) mrna induction in PHKs (d) 7 UnTx IFN- γ GAPDH 1 UnTx Dose of IFN-γ (ng/ml) (e) 1 mrna stability.8 *.6 * UnTx IFN-γ.4 * UVB.2 Fold induction Fold induction Relative mrna Time (h) Figure 2. Induction of by IFN-c in human keratinocytes. (a) mrna induction in HaCaT cells. Cells were treated with IFN-c (25 ng ml) and total RNA isolated just prior to treatment (T =)or following IFN-c treatment at the times indicated along the x axis. RNA was analysed by RT-PCR and DC T calculated relative to GAPDH and DDC T calculated relative to time T =. Error bars represent SEM of two or three experiments. (b) protein expression was analysed in HaCaT cells following IFN-c treatment (25 ng ml). HaCaT whole cell lysates were prepared just prior to IFN-c (T =)orat4,8,24and48h after IFN-c treatment. Proteins were separated by SDS PAGE and transferred to PVDF membranes which were then probed with either or histone H4 (loading control) antibodies. (c) Primary human epidermal keratinocytes (PHKs) were treated with IFN-c at the concentrations indicated along the x axis and total RNA isolated 24 h later. DC T was calculated relative to GAPDH mrna and DDC T calculated relative to untreated cells. Error bars represent SEM for three experiments. (d) protein induction in PHKs was analysed by Western blot. PHKs were treated with IFN-c (25 ng ml) or left untreated (UnTx) and whole cell lysates isolated 24 h after IFN-c treatment. Proteins were separated by SDS PAGE and transferred to PVDF membranes which were then probed with antibodies against or GAPDH (loading control). (e) HaCaT cells were exposed to IFN-c (25 ng ml, black boxes), UVB irradiation (4 mj cm 2, grey triangles) or were left untreated (UnTx, white diamonds). Twenty-four hours later, transcription was inhibited with actinomycin-d (Act-D; 1 lg ml) and total RNA isolated just prior to Act-D (time point T =) or 2, 4 or 6 h after the addition of Act-D as indicated along the x axis. RNA was analysed using real-time RT-PCR and DC T calculated relative to GAPDH and DDC T calculated relative to time point T = (just prior to Act-D) for the untreated, IFN-c or UVB treated cells. Error bars represent ± SEM for three experiments. Asterisks represent P- values <.5 as determined using Student s t-test comparing UnTx and IFN-c treated samples at each time point. message of other UVB-induced genes (19,2). Thus, IFN-c can increase mrna levels by increasing new transcription (Supporting information, Figure S4b) and increasing the stability of mrna. (b) Conclusions is increasingly being recognized as an important transcription factor because of its ability to regulate immune responses, apoptosis and oncogenesis [recently reviewed in reference (21)]. Our study revealed that epidermal levels are increased in EM and that IFN-c can increase via transcriptional and non-transcriptional mechanisms in human keratinocytes. The differences in basal mrna levels between PHKs and HaCaT cells may be due to mutation in p53 that exists in HaCaT cells (22) as is a known transcriptional target of p53 (23). However, despite p53 mutation, is still induced by IFN-c in HaCaT cells. IFN-c and TNF-a have been shown to contribute to the pathogenesis of EM (24). IFN-c (or TNF-a) produced by auto-reactive T cells recruited to EM skin lesions, impacts keratinocyte gene expression and survival (24). may serve as an important effector of these cytokines since regulates genes that influence apoptosis (1,6) and immune responses (4). While our studies suggest that IFN-c may contribute to the increased epidermal expression of in EM, it is important to note that TNF-a, an important cytokine in EM (14) and also an inducer of (25), may also contribute to the increased expression observed in these studies. Thus, increased expression may be due to either IFN-c or TNF-a in herpesassociated EM and drug-induced EM, respectively (14). At this point, it is unclear whether epidermal is pro- or anti-apoptotic and both roles have been ascribed to in keratinocytes (1,2,26). In conclusion, our findings demonstrate that epidermal levels can be increased in human inflammatory skin diseases and suggest that may play a role in the response of epidermal keratinocytes to inflammatory cytokines. Acknowledgements We appreciate the critical comments from Drs. Robert A. Swerlick, Jack L. Arbiser, Kellie J. White, Daniel J. Brat, Jeremy M. Boss, Carlos S. Moreno, Craig A. Elmets and William Lewis. This work was supported by a research grant from the American Skin Association (B.P.P) and a Physician Scientist Career Development grant from the Dermatology Foundation (B.P.P). Supported by a Physician-scientist Career Development Award from the Dermatology Foundation. Conflicts of interest The authors declare no financial conflicts of interest. References 1 Turchi L, Aberdam E, Mazure N et al. Cell Death Differ 28: 15: Turchi L, Fareh M, Aberdam E et al. Cell Death Differ 29: 16: Gilchrist M, Thorsson V, Li B et al. Nature 26: 441: Gilchrist M, Henderson W R Jr, Clark A E et al. J Exp Med 28: 25: Wang A, Arantes S, Yan L et al. BMC Cancer 28: 8: ª 29 John Wiley & Sons A/S, Experimental Dermatology, 19, e31 e313

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