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Plakoglobin as a Regulator of Expression Etienne Tokonzaba1, Jiangli Chen1, Xing Cheng2, Zhining Den2, Radhika Ganeshan1, Eliane J. Mu+ller3 and Peter J. Koch1,2,4

Desmosomes are junctions required for the normal development and maintenance of mammalian tissues and organs such as the skin, skin appendages, and the heart. The goal of this study was to investigate how (DSCs), transmembrane components of , are regulated at the transcriptional level. We hypothesized that differential expression of the Dsc2 and Dsc3 is a prerequisite for normal development of skin appendages. We demonstrate that (Pg) in conjunction with lymphoid enhancer-binding factor 1 (Lef-1) differentially regulates the proximal promoters of these two genes. Specifically, we found that Lef-1 acts as a switch activating Dsc2 and repressing Dsc3 in the presence of Pg. Interestingly, we also determined that NF-kB pathway components, the downstream effectors of the ectodysplasin-A (EDA)/ ectodysplasin-A receptor (EDAR)/NF-kB signaling cascade, can activate Dsc2 expression. We hypothesize that Lef- 1 and EDA/EDAR/NF-kB signaling contribute to a shift in Dsc isoform expression from Dsc3 to Dsc2 in placode keratinocytes. It is tempting to speculate that this shift is required for the invasive growth of placode keratinocytes into the dermis, a crucial step in skin appendage formation. Journal of Investigative Dermatology (2013) 133, 2732–2740; doi:10.1038/jid.2013.220; published online 11 July 2013

INTRODUCTION Desmosomes contain transmembrane adhesion molecules Desmosomes are cell adhesion complexes that are assembled (desmosomal , (DSGs) and desmocol- at the plasma membrane where they serve as membrane lins (DSCs)) and associated plaque (reviewed in anchors for intermediate filament proteins (Cheng and Cheng et al., 2005; Dubash and Green, 2011). The main Koch, 2004; Cheng et al., 2005; Dubash and Green, 2011). plaque proteins belong either to the armadillo family of The importance of this cell junction for organ stability and structural and signaling proteins (plakoglobin (Pg) and function is demonstrated by the severe acquired and inherited plakophilins) or the plakin family (desmoplakin). The mouse diseases of the skin, skin appendages (e.g., hair), and the heart genome encodes three Dsc and six Dsg genes. All DSG and that result from impaired function (see, e.g., DSC proteins are synthesized in the epidermis of the skin Amagai and Stanley, 2012; Petrof et al., 2012; Swope et al., andinskinappendages:e.g.,Dsc2 and Dsc3 are present in 2013). These observations in human patients are further the basal and immediate suprabasal layers, whereas Dsc1 is supported by the severe skin and heart phenotypes of mice mainly restricted to the granular layer of the mouse with mutations in genes encoding desmosomal components epidermis (references in Chen et al., 2008). It is thought (see, e.g., Koch et al., 1997, 1998; Chen et al.,2008; that the specific complement of DSG and DSC proteins Ganeshan et al., 2010; Li et al., 2011, 2012). Desmosomes affects the adhesive and potentially the signaling properties also contribute to cell sorting, morphogenetic cell movement, of desmosomes (see, e.g., Schmidt and Koch, 2007; Muller and the formation of the proper histoarchitecture during et al., 2008). However, little is known about the gene- embryonic development (Cheng and Koch, 2004). regulatory mechanisms that control the expression of these desmosomal genes (see, e.g., Oshiro et al., 2003; Smith et al., 1Department of Dermatology, University of Colorado School of Medicine, 2004; Oshiro et al., 2005). In this study, we focused on the Aurora, Colorado, USA; 2Department of Dermatology, Baylor College of regulation of the Dsc2 and Dsc3 genes; their coexpression in 3 Medicine, Houston, Texas, USA; Molecular Dermatology, Vetsuisse Faculty, the deep layers of the mouse epidermis suggests that they may University of Bern, Bern, Switzerland and 4Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, have a role in keratinocyte differentiation and skin appendage Colorado, USA formation (see, e.g., Chidgey et al., 1997; Cheng and Koch, Correspondence: Peter J. Koch, Department of Dermatology, University of 2004). Furthermore, we questioned whether signaling Colorado Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, cascades known to have important roles during skin Colorado 80045, USA. E-mail: [email protected] appendage formation affect the expression of Dsc2 and Abbreviations: ChIP, chromatin immunoprecipitation; DSC, desmocollin; Dsc3, respectively. DSG, ; HF, hair follicle; Lef-1, lymphoid enhancer-binding factor 1; MDCK, Madin Darby canine kidney; Pg, plakoglobin; TCF, T-cell factor Both the WNT and the ectodysplasin-A (EDA)/ ectodyspla- Received 24 January 2013; revised 10 April 2013; accepted 12 April 2013; sin-A receptor (EDAR)/NF-kB pathways have been shown published online 11 July 2013 to have critical roles in hair follicle (HF) formation

2732 Journal of Investigative Dermatology (2013), Volume 133 & 2013 The Society for Investigative Dermatology E Tokonzaba et al. Transcriptional Regulators of DSC

(Schmidt-Ullrich et al., 2001; Zhang et al., 2009). The canonical –1,113 –888 Wnt cascade ultimately affects target gene expression via TCF/Lef Dsc2 TCF/Lef Dsc3 b-catenin/T-cell factor (TCF)/lymphoid enhancer-binding factor (Lef) complexes. Pg, which is sequence related to Wt CTTTGAA Wt CATTGAA b-catenin, has also been shown to act as a transcription Mut CTTTGGC Mut CATTGGC factor, both in conjunction and independent of TCF/Lef factors Q5 (see e.g., Simcha et al., 1998; Zhurinsky et al., 2000; Shtutman TCF-4 IgG Input Lef-1 IgG Input TCF-4 IgG Input Lef-1 IgG Input et al., 2002; Maeda et al., 2004; Teuliere et al., 2004; Dsc2 Dsc3 Williamson et al., 2006; Galichet et al., 2012). Similar to Dsc2 Mut Dsc3 Mut b-catenin, Pg does not possess a DNA-binding domain, i.e., it requires cofactors (such as TCF/Lef factors) to bind to promoter 5 5 sequences. 4.5 4.5 * 4 4 It is well established that Pg, independent of b-catenin 3.5 3.5 3 3 signaling, is a key regulator of various cellular pro- 2.5 2.5 cesses such as migration (Rieger-Christ et al., 2005; Yin 2 2 1.5 1.5 et al., 2005; Franzen et al., 2012), proliferation (Li et al., 1 1 0.5 0.5

2012), apoptosis (Dusek et al., 2007), and gene expression activity luciferase Relative 0 activity luciferase Relative 0 (see above). Dsc2 Dsc2 Mut Dsc3 Dsc3 Mut Here we demonstrate that Pg, in conjunction with Lef-1, Figure 1. Identification and functional characterization of T-cell factor/ can control Dsc2 and Dsc3 gene expression: in the presence lymphoid enhancer-binding factor (TCF/Lef)–binding sites in the desmocollin of Lef-1, Pg activates Dsc2 and represses Dsc3 expression. 2 (Dsc2)anddesmocollin 3 (Dsc3) promoters. (a, b) Schematic representation Furthermore, we show that NF-kB proteins, the downstream of putative transcription factor–binding sites (TCF/Lef) in the proximal Dsc promoters. The arrows indicate translation start sites (ATG; A is defined as effectors of EDA/EDAR signaling (Schmidt-Ullrich et al., position þ 1). The DNA sequences of wild-type (Wt) and mutant (Mut) 2001), activate Dsc2 as well. Both the EDA/EDAR/NF-kB transcription factor–binding sites are shown. (c, d) Chromatin and the TCF/Lef signaling have crucial roles in the early steps immunoprecipitation (ChIP) assays demonstrating TCF/Lef binding to the of HF formation. Our results predict that these two pathways predicted target sites in the Dsc promoters. Note that the point mutations shift expression from Dsc3 to Dsc2 in placode keratinocytes introduced into the TCF/Lef target sequences (Dsc2 Mut, Dsc3 Mut) abrogate invading the dermis, a process that might be required binding of the transcription factors. Input, chromatin used for for effective keratinocyte migration and thus appendage immunoprecipitation; IgG, immunoprecipitation (IP) with unspecific IgG. (e, f) Reporter assays in mouse keratinocytes. Note that inactivation of the formation. To the best of our knowledge, these results TCF/Lef-binding sites in the Dsc3 construct increases reporter activity provide previously unreported evidence that the catenin/ significantly. Error bars, SD. Star indicates a statistically significant result TCF/Lef and NF-kB signaling cascades control Dsc gene (Po0.05). expression in keratinocytes. TCF/Lef regulation of the Dsc2 and Dsc3 promoters RESULTS We cloned 2 kb of the genomic DNA sequences immediately Dsc gene regulation in skin appendage formation upstream of the Dsc2 and Dsc3 translation start codons It is thought that changing the molecular composition of (Figure 1a and b). These DNA fragments were cloned into a desmosomes is one mechanism used by keratinocytes to promoter-less Luciferase reporter vector designed to assess adapt their cell adhesion system to their specific environment. transcriptional activity of promoter fragments (reporter assays). For example, in the early stages of skin appendage formation, Both the Dsc2 and Dsc3 constructs showed reporter activity in desmocollins are downregulated in placode keratinocytes primary mouse keratinocytes (data not shown). Next, we invading the dermis (Nanba et al., 2000, 2001). However, introduced point mutations abrogating DNA binding of little is known regarding gene-regulatory mechanisms TCF/Lef factors to their target sites into both promoters that control Dsc gene expression in skin. To begin to (Figure 1a and b). The wild-type, but not the mutant TCF/Lef identify gene-regulatory pathways controlling Dsc gene sites, showed binding of Lef-1 and TCF-4 as demonstrated by expression, we cloned and sequenced the proximal chromatin immunoprecipitation (ChIP) assays (Figure 1c and promoters of mouse, Dsc2 and Dsc3. These two Dsc genes d). TCF3 behaved identical to TCF-4 in all assays performed in are expressed in the basal layers of the interfollicular this study. Consequently, only the Lef-1 and TCF-4 data are epidermis, the cellular compartment that maintains the shown. All ChIP assays and biochemical experiments were epidermis and that forms HFs. conducted in canine MDCK (Madin Darby canine kidney) DNA sequence analysis revealed the presence of putative cells, whereas reporter assays and gene expression studies TCF/Lef (Figure 1a and b) and NF-kB (see below) target sites in were conducted in primary mouse keratinocytes. MDCK cells the Dsc2 and Dsc3 promoters. Considering that the Wnt express Dsc2 and Dsc3 (data not shown). Owing to pathway, which affects gene expression via catenin/TCF/Lef differences in the promoter DNA sequences of dog and transcription factors, and the NF-kB pathways have been mouse and the use of species-specific antibodies, we were shown to have major roles in HF formation, we decided to able to investigate –DNA and protein–protein interac- focus on the role of these two signaling cascades in Dsc gene tions in MDCK cells without the interference of endogenous regulation. canine genes and proteins.

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Next, we assessed whether loss of functional TCF/Lef sites

affected reporter activity in primary keratinocytes. As shown in Lef-1 IgG Input Pg IgG Input Lef-1 IgG Input Pg IgG Input Figure 1, Dsc2 promoter activity was unchanged when Dsc2 Dsc3 wild-type and mutant reporter constructs lacking a functional TCF/Lef site were compared (Figure 1e). Dsc3 promoter activity was significantly increased in the mutant construct, Pg+Lef-1 indicating that this site is repressive (Figure 1f). 10 * 10 9 9 8 8 Pg * b-Catenin expression has no effect on the proximal Dsc2 and 7 7 6 6 Dsc3 promoters 5 5 TCF/Lef sites are the primary target sites of b-catenin/TCF/Lef 4 4 3 3 Pg Pg+Lef-1

transcription factor complexes, the principal effectors of Pg+TCF-4 Baseline 2 2 Pg+TCF-4 the Wnt signaling pathway. ChIP assays demonstrated that 1 Baseline 1 Relative luciferase activity luciferase Relative 0 activity luciferase Relative 0 b-catenin can bind to the Dsc3 but not the Dsc2 promoter Dsc2 Dsc3 (Supplementary Figure S1 online; these experiments were conducted in MDCK cells expressing TCF-4 as a cofactor, data not shown). To determine whether b-catenin can influ- Pg+Lef-1 ence Dsc promoter activity, we transfected the Dsc reporter 8 * 8 constructs with different combinations of TCF/Lef factors into 7 7 Pg

6 Pg keratinocytes. We did not observe a statistically significant 6 Baseline 5 5 * * * effect of these b-catenin/TCF/Lef factors on Dsc2 or Dsc3 4 4 reporter activity (Supplementary Figure S1 online). A slight 3 3 Pg+Lef-1 2 Baseline Baseline repression of Dsc3 reporter activity by b-catenin and Lef-1 was 2 Baseline 1 1 often observed but did not reach statistical significance. 0 Relative luciferase activity luciferase Relative activity luciferase Relative 0

Pg is a key regulator of Dsc2 and Dsc3 promoter activity Dsc2 Dsc2 Mut Dsc3 Dsc3 Mut Pg has been shown to regulate gene expression in keratino- cytes and other epithelial cell types (see references in the Introduction section). To determine whether Pg can affect Dsc Pg+Lef-1 promoter activity, we first assessed the ability of this protein to 4 4 Pg * * bind the Dsc2 and Dsc3 promoter fragments. As shown in 3 3 2 2 Lef-1 Baseline Pg+Lef-1 Baseline Lef-1 Figure 2a and b, Pg binds to the Dsc2 promoter but not the Pg Dsc3 promoter in the presence of Lef-1. Reporter assays 1 1

Gene expression 0 Gene expression 0 demonstrated that Pg can affect both promoters, although its Dsc2 Dsc3 effects are dependent on Lef-1 (Figure 2c and d); in the presence of Lef-1, Pg activates the Dsc2 promoter, whereas Figure 2. Effects of plakoglobin (Pg) on desmocollin (Dsc) reporter activities. (a, b) Chromatin immunoprecipitation (ChIP) assays demonstrating direct Dsc3 promoter activation occurs in the absence of ectopic binding of Pg to the Dsc2 promoter in the presence of lymphoid enhancer- TCF/Lef expression. These results suggest that Lef-1 can act as binding factor 1 (Lef-1). Note that Pg does not bind the Dsc3 promoter in the a switch that shifts activity from the Dsc3 to the Dsc2 presence of Lef-1 (b) or T-cell factor 4 (TCF-4; data not shown). Input, promoter in the presence of Pg. chromatin used for immunoprecipitation (IP); IgG, IP with unspecific IgG. Neither Lef-1, TCF-3, nor TCF-4 had any effect on the Dsc2 (c–f) Reporter assays in keratinocytes. (c) Coexpression of Pg and Lef-1 or Dsc3 promoter in single transfection experiments (data not markedly increases Dsc2 reporter activity, whereas all other combinations of Pg and TCF/Lef factors have no effect. (d)TheDsc3 promoter is activated in the shown). These results are expected given that keratinocytes presence of Pg. This activation is reversed in cells coexpressing Pg and express endogenous TCF/Lef factors, but show very low TCF-4 or Lef-1. (e) Dsc2 promoter activation by Pg and Lef-1 is dependent cytoplasmic and nuclear Pg levels (Williamson et al., 2006), on the presence of a functional TCF/Lef-binding site. Mut, mutant. (f)Pg conditions that favor low Dsc2 and Dsc3 reporter activity. overexpression and loss of a functional TCF/Lef–binding site increase Dsc3 As shown in Figure 2e, Dsc2 promoter activation by Pg and reporter activity to a similar extent. Note that expression is normalized to the Lef-1 was dependent on the presence of a functional TCF/Lef- baseline expression of the WT promoter (set to 1). (g, h) Endogenous Dsc gene binding site. In the case of the Dsc3 promoter, we found no expression in keratinocytes transfected with different combinations of Pg and TCF/Lef factors. Error bars, SD. Stars indicate statistically significant results additive or synergistic effects of Pg and the TCF/Lef mutation (Po0.05). (Figure 2f). These findings are consistent with the hypothesis that Pg asserts its activating effect on Dsc2 by binding to the TCF/Lef site, whereas this was not the case for the Dsc3 promoter. confirming the validity of our conclusions for the regulation We next assessed the effects of Pg and Lef-1 coexpression of these genes in keratinocytes. on the endogenous Dsc2 and Dsc3 gene activity in primary To gain further insights into the mechanisms by which Pg keratinocytes by quantitative reverse transcriptase–PCR. As and Lef-1 control Dsc gene expression, we assessed whether shown in Figure 2g and h, both genes responded to ectopic Pg can bind to Lef-1. As shown in Figure 3a, Lef-1 and Pg Pg/Lef expression as predicted by our reporter assays, can form a complex as shown by coimmunoprecipitation

2734 Journal of Investigative Dermatology (2013), Volume 133 E Tokonzaba et al. Transcriptional Regulators of DSC Gene Expression

PgKT3+Lef-1 + + – Lef-1 ––+ IP:IgG – + – Pg

IP: anti-Lef-1 + – + Lef-1 IgG Input 0.0 μg 1.5 μg Pg 2.5 μg Dsc3

Lef-1 Pg+Lef-1 NT Pg+Lef-1 Pg NT CNN CNC CNCNCN

Lef-1 KT3

Lamin B1 Lamin B1

α-Tubulin α-Tubulin

Pg Lef-1 Pg Lef-1

Figure 3. Plakoglobin (Pg) colocalizes in the nucleus with lymphoid enhancer-binding factor 1 (Lef-1) and disrupts T-cell factor (TCF)/Lef transcription factor binding to the desmocollin 3 (Dsc3) promoter. (a) Co-immunoprecipitation (Co-IP) assays demonstrating an interaction between Pg and Lef-1. (b)Chromatin immunoprecipitation (ChIP) assays demonstrating that increasing amounts of Pg (measured in mg plasmid transfected) interfere with the binding of Lef-1 to the Dsc3 promoter. Input, chromatin used for immunoprecipitation; IgG, IP with unspecific IgG. (c, d) Western blot analysis of Madin Darby canine kidney (MDCK) cells transfected with Pg and Lef-1 (transfection constructs shown on top; NT, not transfected). The nuclear (N) and cytoplasmic (C) distribution of the proteins is shown. Antibodies used to detect Lef-1 and the KT3-tagged plakoglobin construct are shown on the left sides of the blots. Our Lef-1 antibody does not detect endogenous Lef-1 expression in MDCK cells. Lamin B1 (nuclear fraction) and a-tubulin (cytoplasmic fraction) antibodies were used as controls. (e) Immunofluorescence microscopy of MDCK cells transfected with Pg and Lef-1. The antibodies used for staining are indicated. Note the nuclear colocalization of Pg and Lef-1 in several cells (arrows). Bar ¼ 50 mm. experiments using Lef-1 antibodies. Most interestingly, ChIP Lef-1 in keratinocytes can suppress the Dsc3 gene. We competition experiments suggested that the interaction of Pg therefore compared the distribution of DSC3 and Lef-1 in and Lef-1 interferes with the ability of Lef-1 to bind to the transgenic mice expressing a nuclear LacZ reporter under Dsc3 promoter (Figure 3b). Considering that TCF/Lef-1 com- the control of the Dsc3 promoter (Dsc3-LacZ mice). These plexes can act as transcriptional repressors (see, e.g., Hoverter animals were designed to identify cells and tissues that express and Waterman, 2008), these results raise the possibility that Pg low levels of Dsc3, or cells in which antigen masking could activate Dsc3 expression by interfering with the binding prevented protein detection via antibodies. In all develop- of a TCF/Lef repressor complex to the Dsc3 promoter. Further mental stages and tissues examined so far, DSC3 antibody support for this hypothesis is provided by western blot staining and LacZ transgene activity perfectly overlapped experiments (Figure 3c and d), demonstrating that ectopically (data not shown). expressed Pg can interfere with the nuclear accumulation of We conducted whole-mount b-galactosidase staining Lef-1 and thus potentially suppress the formation of a repressor experiments using Dsc3-LacZ embryos (Figure 4a). At embryo- complex at the Dsc3 promoter. On the other hand, Lef-1 is nic day 15.5 (E15.5), b-galactosidase activity was prominent required to shuttle Pg into the cell nucleus, where it activates in whisker pads (vibrissae follicles), in mammary gland buds, the Dsc2 promoter as shown in Figure 3d and e. The data and in developing HFs over the entire body surface (Figure 4a, summarized above demonstrate that Lef-1 and Pg localize and data not shown). Strikingly, the overall DSC3-LacZ to the nucleus, which is predicted to lead to an activation of expression pattern appeared similar to the staining patterns the Dsc2 gene and a suppression of the Dsc3 gene. observed in transgenic embryos of the same age that expressed a TCF/Lef-regulated promoter driving a LacZ reporter Topology of Lef-1 and Dsc3 expression in developing hair (Figure 4b; Bat-Gal; Maretto et al., 2003). However, a follicles is mutually exclusive histochemical analysis revealed that the expression patterns Our results so far suggest that Lef-1 can act as a switch of the Wnt pathway components (Lef-1; TCF-3/-4; data not between Dsc2 and Dsc3 expression and that the presence of shown) and the DSC3-LacZ transgene did not overlap at the

www.jidonline.org 2735 E Tokonzaba et al. Transcriptional Regulators of DSC Gene Expression

Dsc3-LacZ BAC β-Gal BAT-gal β-Gal

Dsc3-LacZ BAC β-Gal Ab Lef-1 Ab

Dsc3Lef-1 Lef-1 Dsc3

Dsc3Lef-1

Figure 4. Wnt activity and desmocollin 3 (Dsc3) expression during mouse appendage development. (a) Whole-mount in situ staining for b-galactosidase (b-gal) activity of a transgenic mouse (Dsc3-LacZ BAC, embryonic day 15.5 (E15.5)) expressing b-gal under the control of the Dsc3 promoter. Note that whisker pads, hair follicles, and mammary glands (not shown) are strongly stained. (b) Whole-mount b-gal staining of a BAT-gal transgenic mouse (Maretto et al., 2003) at E15.5. Note the similar staining patterns of the transgenic mice shown in (a)and(b). (c) Immunohistochemical staining of a skin section from a Dsc3-LacZ BAC mouse at E15.5 with (c) b-gal antibodies (Abs) and (d) Lef-1 Abs. The LacZ transgene contains a nuclear localization signal. Note that b-gal and Lef-1 expression appears to be mutually exclusive. Arrows point to Lef-1-expressing cells in a placode and a dermal papilla of a forming hair follicle. (e, g) Immunofluorescence staining of a developing hair follicle in wild-type mouse skin at E16.5 with antibodies against Lef-1 and DSC3. The area demarcated by the dotted box is shown at higher magnification in (h). (h) The large arrow points to DSC3-positive keratinocytes in the basal cell layer. The short arrow points to DSC3-negative and Lef-1-positive cells in a forming hair placode. Dotted lines in e and h indicate basement membrane area. Bar ¼ 50 mm.

cellular level. In fact, Wnt activity (Lef-1 expression) and NF-jB regulation of the Dsc2 gene Dsc3 expression were mutually exclusive (Figure 4c and d). Our initial analysis of the Dsc promoters indicated the We observed that LacZ-positive (Dsc3-expressing) cells were presence of putative NF-kB (Rel)-binding sites in both Dsc found in the suprabasal layer on top of the newly forming HFs. promoters. We thus performed ChIP assays and reporter Lef-1, on the other hand, was observed in the leading edge of assays to determine the effect of Rel factors and a dominant- keratinocytes growing down into the dermis and throughout negative (DN) construct blocking NF-kB signaling (IkB-DN) the basal cell layer. These findings were confirmed by staining on Dsc promoter activity. The results shown in Figure 5a–c E16.5 wild-type mouse epidermis with Lef-1 and DSC3 demonstrate that c-Rel specifically binds to and activates the antibodies (Figure 4e–h). Strong Lef-1 antibody staining proximal Dsc2 promoter, whereas we did not observe any correlated with reduced or absent staining for DSC3. Unfortu- effect of the NF-kB factors on the proximal Dsc3 promoter nately, we were not able to assess the distributions of DSC2, as (Figure 5d). We then transfected primary keratinocytes antibodies that recognize mouse DSC2 are not available. Pg with the NF-kB factors and assessed endogenous Dsc gene antibodies stained cell–cell borders in placode keratinocytes expression (Figure 5e and f). We confirmed the specific (data not shown). Nuclear Pg staining was not observed, activation of the endogenous Dsc2 gene by ectopic expression possibly because of epitope masking or low nuclear Pg levels. of c-Rel.

2736 Journal of Investigative Dermatology (2013), Volume 133 E Tokonzaba et al. Transcriptional Regulators of DSC Gene Expression

–217 –108 Rel Rel Dsc2 c-Rel RelA IgG Input c-Rel+RelA Dsc2 GGAGTTCCC GGAAAAGCCC

10 c-Rel 10 9 * 9 8 8 7 7 6 6 5 5 4 4 c-Rel+RelA 3 3 RelA Baseline c-Rel c-Rel+RelA I κβ –DN RelA 2 Baseline

2 I κβ –DN 1 1 Relative luciferase activity luciferase Relative Relative luciferase activity luciferase Relative 0 0 Dsc2 Dsc3 c-Rel 4 * 4 3.5 3.5 3 3 2.5 2.5

2 c-Rel+RelA 2 RelA Baseline I κβ –DN RelA Baseline 1.5 1.5 c-Rel c-Rel+RelA I κβ –DN 1 1

Gene expression 0.5 Gene expression 0.5 0 0 Dsc2 Dsc3

Eda Tcf/Lef Tcf/Lef

c-Rel Pg/Lef-1 Tcf-4/Tcf-3/Lef-1

Up

Dsc2 Dsc3 Down

Figure 5. Effects of NF-jB transcription factors on the proximal desmocollin 2 (Dsc2)anddesmocollin 3 (Dsc3) promoters. (a) Chromatin immunoprecipitation (ChIP) assays demonstrating that both c-Rel and Rel A bind to a Dsc2 promoter fragment that contains two predicted Rel-binding sites. Input, chromatin used for immunoprecipitation (IP); IgG, IP with unspecific IgG. (b) Schematic representation of putative NF-kB transcription factor–binding sites in the proximal Dsc2 promoter. The arrows indicate translation start sites (ATG; A is defined as position þ 1). (c) c-Rel markedly increases the reporter activity of the Dsc2 construct, whereas Rel A does not. The IkB-DN construct encodes a dominant-negative (DN) inhibitor of the NF-kB pathway, i.e., this construct can block endogenous NF-kB activity. The promoter activities in the absence of ectopically expressed transcription factors are defined as relative expression level 1. (d)TheDsc3 reporter construct does not show any significant changes in activity in response to NF-kB transcription factors. (e, f) Endogenous Dsc gene expression in mouse keratinocytes. (e) c-Rel ectopic expression significantly increases endogenous Dsc2 expression. (f) None of the NF-kB transcription factors affects endogenous Dsc3 expression. (g) Simplified model of signaling pathways active in hair follicle placodes and their proposed effects on Dsc2 and Dsc3 gene expression. Note that open arrows symbolize the upregulation and downregulation of gene expression, respectively. Error bars, SD. Stars indicate statistically significant results (Po0.05). Lef-1, lymphoid enhancer-binding factor 1; Tcf, T-cell factor.

DISCUSSION the regulation of Dsc2 and Dsc3, the two DSCs expressed in It has been shown that deregulated expression or loss of the deep epidermis, the compartment that maintains the skin desmosomal cadherins, including Dsc, canleadtoabnormal and which has a crucial role in skin appendage development. differentiation of the epidermis and defects in HFs (see, Little is known regarding gene-regulatory pathways that e.g., Chidgey et al., 2001; Elias et al., 2001; Merritt et al., control the expression of Dsc genes in processes such as 2002; Hardman et al., 2005), suggesting that tight control of epidermal differentiation and HF formation. Epidermal appen- Dsc gene expression is required for normal epidermal dage formation requires extensive remodeling of cell adhesion development and homeostasis. In this study, we focused on systems, including desmosomes (Kurzen et al., 1998). The first

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step in the development of these appendages is placode NF-kB pathway, which interact with the Wnt pathway, a formation. Keratinocytes in these structures segregate from process crucial for HF formation during embryogenesis (Zhang the surrounding epidermis and then begin to invade the et al., 2009). The observation that both catenin/TCF/Lef and dermis. Nanba et al. (2000, 2001) have demonstrated that NF-kB signaling are required for pelage hair development, and DSCs are downregulated both in the hair and mammary our in vitro data demonstrating differential regulation of Dsc gland placodes. However, the antigen specificity of the genes by these two pathways, suggest the possibility that both antibody used by these authors was not determined, i.e., it signaling cascades partially function through modulating was not known which of the DSC proteins was actually desmosomal cell adhesion. downregulated. Given the results presented in this study, it is On the basis of the observation summarized above, we likely that the main desmosomal isoform suggest the following mechanisms for the regulation of Dsc2 downregulated in this process is Dsc3. and Dsc3 gene expression during the early stages of skin The expression of classical cadherins is also switched during appendage formation: Wnt activation in the early placode hair follicle formation. E-cadherin is downregulated in HF leads to an increase accumulation of Lef-1 in placodes. Lef-1, placodes, whereas P-cadherin is upregulated. Furthermore, it potentially in conjunction with other corepressors (Hoverter was shown that forced expression of E-cadherin inhibits HF and Waterman, 2008; Arce et al., 2009), binds to the Dsc3 formation (Jamora et al., 2003), demonstrating that control of promoter and inhibits expression, thus leading to a reduction cadherin isoform expression is crucial for HF formation. in the desmosomal adhesion receptors present at the plasma Given our results, it is tempting to speculate that an membrane. In turn, this could lead to a transient increase analogous switch occurs from Dsc3 (TCF/Lef factor-mediated of cytoplasmic Pg, which is usually bound to the carboxy- repression) to Dsc2 (Pg/Lef-1-mediated induction) in placode terminal domain of DSC3. Given the abundance of Lef-1 in keratinocytes (Figure 5g). Unfortunately, owing to a lack of placode keratinocytes, Pg would then form a transcription appropriate antibodies, we currently do not have the tools complex with Lef-1 and activate the Dsc2 promoter. This required to assess DSC2 expression in mouse epidermis. chain of events would lead to a shift from DSC3 to DSC2 as Nevertheless, expression studies in human embryonic epider- the main DSC synthesized in placode keratinocytes, which mis indicated increased DSC2 and reduced DSC3 expression would be consistent with the proposed distribution of these in bulbous hair pegs, suggesting that the above postulated two proteins in skin placodes (our data and Kurzen et al., switch from Dsc3 to Dsc2 expression is likely to occur in 1998). Activation of the EDA/EDAR pathway, via its NF-kB mammalian skin (Kurzen et al., 1998). effectors, would then further facilitate the shift from DSC3 to Pg appears to activate both the Dsc2 and the Dsc3 DSC2.ItispossiblethatthischangefromDSC3toDSC2is promoter. Previous studies have suggested that Pg can signal better suited to support invasive keratinocytes growth. In this by changing the levels of signaling active b-catenin in cells. context, it is noteworthy that we have previously shown in Our experiments failed to demonstrate a role of b-catenin in a mouse model that invasive growth of skin squamous regulating the two Dsc genes, suggesting that the signaling we cell carcinoma is associated with a specific loss of Dsc3 observed is a specific function of Pg. expression in tumor cells (Chen et al., 2011). Similar results The Dsc2 promoter requires Lef-1 for activation and this have also been reported in other types of cancer, such as regulation is dependent on the TCF/Lef-binding site in the breast cancer (Klus et al., 2001). Dsc2 promoter. Interestingly, this effect is specific for Lef-1 as Further experiments will be required to unequivocally prove TCF3 and TCF4 do not appear to be able to substitute for this model. It is tempting to speculate that changes in Lef-1. It is noteworthy that Lef-1 expression facilitates nuclear desmosomal cell adhesion might affect the signaling pool accumulation of Pg, a prerequisite for activation of the of plakoglobin and thus, in a feedback loop, control the Dsc2 gene. expression of genes that encode central desmosomal compo- Pg activated the Dsc3 promoter without direct binding. nents. This represents a previously unreported and exciting Interestingly, ectopic expression of TCF/Lef factors completely conceptthatcannowbetestedin vivo. blocked Pg-mediated activation of the Dsc3 promoter. This suggests that Pg interferes with the activity of a repressor MATERIALS AND METHODS complex containing TCF/Lef factors that inhibits the Dsc3 Animal protocols promoter. A likely mechanism that could explain this observa- Animal experiments were approved by the institutional animal tion is that binding of Pg to TCF/Lef proteins causes a care and use committee of the University of Colorado Denver depletion of the TCF/Lef pool, thus preventing these factors (UC Denver; Denver, CO). from forming an inhibitory complex on the Dsc3 promoter. Previous experiments in mouse models suggested that Pg is Generation of Dsc promoter constructs and luciferase reporter not required for the formation of HFs (Teuliere et al., 2004; Li assays et al., 2012). These results suggest the existence of alternative A total of 2 kb promoter sequences immediately upstream of the Dsc2 mechanisms that can regulate a switch of Dsc isoforms, and Dsc3 translation start codons were cloned into pGL3-basic vector specifically the activation of Dsc2.NF-kB signaling might be (Promega, Madison, WI), which contains a promoter-less luciferase such an alternative mechanism. Our experiments revealed a reporter cassette. TCF/Lef-1 mutations were generated by PCR using role of c-Rel, a NF-kB protein, in activating the Dsc2 gene. primers DSC2-5F/5R and DSC3-2F/2R (Supplementary Table S1 NF-kB proteins are downstream effectors of the EDA/EDAR/ online). The following expression vectors were used: CMV-bgal

2738 Journal of Investigative Dermatology (2013), Volume 133 E Tokonzaba et al. Transcriptional Regulators of DSC Gene Expression

(Dennis Roop, UC Denver); pcDNA1.1p65 and pcDNA-Ikb-DN Quantitative real-time quantitative reverse transcriptase–PCR (Rune Toftga˚rd, Karolinska Institute, Sweden); pcmv4c-Rel (Warner Real-time reverse transcriptase–PCR was performed using a Greene, University of California, San Francisco, San Francisco, CA) LightCycler 480 from Roche (Indianapolis, IN), following the manu- (Doerre et al., 1993); pCS2 DNPb-catenin (Pamela Cowin, New York facturer’s recommendations. Dsc2 and Dsc3 complementary University, New York, NY) (Imbert et al., 2001); pRcCMV were amplified with ‘‘assay-on-demand probes’’ Mm00516355_m1 plakoglobin expression vector tagged with a KT3 epitope (Ansgar and Mm00492270_m1, respectively, from Applied Biosystems. A Smith, University of Marburg, Marburg, Germany); pCS2-hLef-1 (Rolf glyceraldehyde-3-phosphate dehydrogenase probe set from Applied Kemler, Max-Planck Institute, Freiburg, Germany) (Huber et al., Biosystems was used as an internal control. The data shown are based 1996); and pGLOWMYC-hTcf-4 (Hans Clevers, University Hospital, on three independent experiments. Utrecht, The Netherlands) (Korinek et al., 1997). Plasmids were transiently transfected into the canine kidney epithelial cell line CONFLICT OF INTEREST MDCK (ATCC CCL-34) and mouse keratinocytes (MPEK-BL6; The authors state no conflict of interest. Cellntec, Bern, Switzerland), using the Nucleofector Technology ACKNOWLEDGMENTS (Lonza, Walkersville, MD). Reporter activities were measured with We thank Charlene O’Shea for expert technical assistance, Maranke Koster and the ‘‘Chemiluminescent Reporter Gene Assay System’’ (Applied Jason Dinella for critical reading of the manuscript, and Sarah Millar Biosystems, Bedford, MA) using a Glomax Multi Detection System (University of Pennsylvania) for providing Bat-Gal mice with permission from (Promega, Madison, WI). The results shown represent average Stefano Piccolo (University of Padova, Padova, Italy). Research reported in this publication was supported by the National Institute of Arthritis and Muscu- expression levels from three independent experiments, each loskeletal and Skin Diseases of the National Institutes of Health under award conducted in triplicate (error bars in all figures: SD, P-values of number 5RO1AR053892. The content is solely the responsibility of the authors o0.05 were considered statistically significant). and does not necessarily represent the official views of the National Institutes of Health. ChIP experiments ChIP assays were performed essentially as described previously SUPPLEMENTARY MATERIAL (Koster et al., 2007). The following antibodies were used: Pg Supplementary Material is linked to the online version of the paper at http:// (Cell Signaling, Boston, MA), b-catenin (Santa Cruz Biotechnology, www.nature.com/jid Santa Cruz, CA), Lef-1 (Millipore, Billerica, MA), and TCF-4 (Millipore). 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