Introduction

Darier disease (DD) is an autosomal-dominant skin disorder characterized by warty papules and plaques in the seborrheic area, palmo-plantar pits, and distinctive nail abnormalities. The typical histological features are loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization (dyskeratosis), occasionally accompanied by a wide range of neuropsychiatric problems, including epilepsy and depression. In 1999, the human ATP2A2 gene encoding the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) was identified as the defective gene in DD, because several mutations were found in this gene in DD patients (Sakuntabhai et al., 1999b). According to several studies regarding SERCA2 proteins carrying the mutations found in the ATP2A2 gene in DD patients, it is believed that DD is caused in most cases by SERCA2 haploinsufficiency (Ahn et al., 2003; Dhitavat et al., 2003; Dode et al., 2003; Sato et al., 2004; Foggia et al., 2006). These observations indicate that elucidating the mechanism of the ATP2A2 gene expression would provide insight into restoring the decreased function of SERCA2 in DD keratinocytes.

To date, there have been no reports on the mechanisms for transcriptional regulation of the human ATP2A2 gene in keratinocytes, although the gene product, SERCA2, is important for normal Ca²⁺ homeostasis of keratinocytes, and decreased expression and/or activity results in occurrence of DD. In this study, we analyzed the regulatory mechanisms of human ATP2A2 gene transcription in primary normal human keratinocytes (NHK) using luciferase reporter assay and electrophoretic mobility shift assay (EMSA), and identified a transcription factor Sp1, as a nuclear protein binding to critical cis-elements of the ATP2A2 promoter in vitro. Furthermore, we confirmed that Sp1 actually binds to the promoter region on chromatin in living keratinocyte cells by chromatin immunoprecipitation (ChIP) assay and that downregulation of Sp1 expression by small interfering RNA (siRNA) resulted in suppression of ATP2A2 promoter activity and transcription levels.

Results

Determination of transcriptional regulatory region of the human ATP2A2 promoter

In order to determine the regulatory region required for expression of the human ATP2A2 gene, a series of reporter plasmids carrying various length of the 5'-flanking region of the human ATP2A2 gene were constructed. Each of these reporter plasmids was introduced into NHK and luciferase activity derived from NHK cells transfected with each plasmid was measured. The -679/+61 region showed significant promoter activity (approximately 95-fold) when compared with the basic control (Table 1). Deletions of -622/-529, -528/-419, and -418/-307 caused significant decreases in promoter activity. Furthermore, the shortest region, -90/+61, still possessed apparent promoter activity. These results suggest that the -622/-529, -528/-419, -418/-307, and -90/+61 regions contain cis-enhancing elements.

Table 1 - Determination of the transcriptional regulatory region of the human ATP2A2 promoter by luciferase assay.

Full table

We generated additional reporter plasmids in an attempt to further identify the cis-enhancing elements located in above-mentioned regions. The deletions between -679 and -528 is shown in Table 2a. A deletion of 22 bp from -550 to -528 resulted in a marked reduction of promoter activity, thus, suggesting that one of cis-enhancing elements is present in -550/-529. Similarly, deletions from -488 to -472 (Table 2b), from -390 to -362 (Table 2c), and from -42 to -21 (Table 2d) caused significant decreases in promoter activity. These results indicate that cis-enhancing elements of the human ATP2A2 promoter are present in four regions; -550/-529, -488/-472, -390/-362, and -42/-21.

Table 2 - Mapping of cis-enhancing elements in 5'-flanking region of human ATP2A2 gene.

Full table

Identification of transcription factor binding to cis-enhancing elements of human ATP2A2 gene

In order to identify the transcription factor(s) binding to cis-enhancing elements in the human ATP2A2 promoter, we performed EMSA using nuclear extracts prepared from keratinocyte cells. When EMSA was performed with probe oligonucleotides for -391/-359 or -41/-19, no specific bands, which disappeared in the presence of wild-type competitors and did not disappear in the presence of mutant competitors, were observed (data not shown). In contrast, we identified a specific band in each EMSA using probe oligonucleotides for -550/-524 or -488/-465 (arrows in Figure 1a and 1b, respectively). We identified several possible recognition motifs for transcription factors, including Sp1 and AP-2, in these two regions using the motif analysis program TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) (Figure 4). This band completely disappeared by adding anti-Sp1 antibody (Ab) to the EMSA reaction mixture (lane 2 in Figure 1a), but remained even in the presence of anti-Sp3 and anti-AP-2 Abs (lanes 3 and 4 in Figure 1a). Similarly, anti-AP-2 and anti-AP-2 Abs did not affect this band shift (data not shown). A specific band with the -488/-465 probe also disappeared by addition of anti-Sp1 Ab but was still observed in the presence of anti-Sp3, -AP-2, -AP-2, and -AP-2 Abs (lanes 2–6 in Figure 1b). These results demonstrate that Sp1 binds to the human ATP2A2 promoter via Sp1-binding motifs in -550/-524 and -488/-465 in vitro.

Figure 1.

Identification of transcription factor binding to cis-enhancing elements in human ATP2A2 promoter by EMSA. EMSA was performed with FITC-labeled probe corresponding to (a) -550/-528 or (b) –488/-465 with nuclear extract from NHK.

Full figure and legend (94K)

Figure 4.

Nucleotide sequence of the 5'-flanking region of the human ATP2A2 gene. The transcription initiation site is indicated as +1, based on the 5'-end of the human ATP2A2 mRNA sequence (accession number NM_170665). Possible transcription factor binding sites found in cis-enhancing elements identified by the publicly available MOTIF search services are underlined, and Sp1-binding sites confirmed by EMSA are boxed. This genomic region corresponds to 109203135/109203873 of chromosome 12 published in NCBI human genomic DNA sequence site. Three regions underlined with dotted black line are identified to be highly conserved sequences by UCSC (chromosome 12: 109203326–109203493, lod=178; chromosome 12: 109203225–109203285, lod=41; chromosome 12: 109203813–109203827, lod=28).

Full figure and legend (216K)

In vivo binding of Sp1 to promoter region of human ATP2A2 gene

ChIP assay was performed in an effort to examine whether Sp1 protein binds to human ATP2A2 promoter region in keratinocytes in vivo. A markedly higher amount of chromatin containing the ATP2A2 promoter region was immunoprecipitated by anti-Sp1 Ab when compared with control IgG (Figure 2a). In contrast, the amount of chromatin immunoprecipitated by anti-Sp3 Ab was comparable with that by control IgG (Figure 2a). Both anti-Sp1 and anti-Sp3 Abs did not immunoprecipitate cis control region of ATP2A2 gene specifically (Figure 2b). These results suggest that Sp1 but not Sp3, a similar transcription factor belonging to same family, binds to the ATP2A2 promoter region of chromatin in human keratinocytes.

Figure 2.

Sp1 binds to human ATP2A2 promoter region in vivo. Binding between the human ATP2A2 promoter (a) or cis control region of ATP2A2 gene (b) and Sp1 or Sp3 was analyzed by ChIP assay using anti-Sp1, anti-Sp3, or isotype control Ab (mouse IgG1 and mouse IgG2a as controls for anti-Sp1 and anti-Sp3, respectively). Binding was quantitatively analyzed by real-time PCR. Relative input units are calculated from cycle threshold values as described in "Materials and Methods". Results are expressed as meansSD of triplicate samples. Representative results of three independent experiments are shown.

Full figure and legend (21K)

Knockdown of Sp1 expression by siRNA downregulates promoter activity and transcription of ATP2A2

ChIP assay revealed that a significant amount of Sp1 occupied human ATP2A2 promoter in keratinocytes. To evaluate the effects of Sp1 on ATP2A2 expression, Sp1 siRNA or control was introduced into keratinocytes, and the Sp1 mRNA in each transfectant was determined using quantitative real-time PCR. Sp1 siRNA reduced Sp1 mRNA level by 85% under the condition of approximately 60–90% of transfection efficiency (Figure 3a), whereas control siRNA had no effect on Sp1 mRNA levels (Figure 3b). Reduced Sp1 protein level was also observed by Western blot analysis (Figure 3c). Co-transfection of keratinocytes with Sp1 siRNA and reporter plasmid confirmed that the luciferase activity driven by the ATP2A2 promoter was significantly lower in Sp1-siRNA transfectant than that in control-siRNA transfectants (Figure 3d). In contrast, the presence of Sp1 siRNA did not affect the luciferase activity derived from control transfectant carrying pGL3-Basic (Figure 3d). When ATP2A2 mRNA levels were measured by real-time PCR, significant suppression of ATP2A2 transcription (approximately 60%) was observed in Sp1-siRNA transfectants (Figure 3e). These results indicate that Sp1 positively regulates the expression of human ATP2A2 in keratinocytes by transactivating the promoter.

Figure 3.

Knockdown of Sp1 expression by siRNA results in downregulation of ATP2A2 promoter activity. (a) Fluorescence microscopy of FITC-labeled control oligonucleotide-introduced keratinocytes. After 24 hours culture from transfection of oligonucleotides, cells were washed and visualized by phase microscopy to visualize cells (left) and by fluorescence microscopy using a green fluorescent protein filter (right). Bar=100 m. (b) Sp1 mRNA level in Sp1 siRNA-treated keratinocytes analyzed by real-time PCR. (c) Sp1 protein level in Sp1 siRNA-treated keratinocytes analyzed by Western blotting. (d) Transcription activity of human. ATP2A2 promoter in Sp1 siRNA-introduced keratinocytes. The ratio of luciferase activity in each transfectant to that in cells transfected with pGL3-Basic and control siRNA is given as relative activity. (e) ATP2A2 mRNA level in Sp1 siRNA-introduced keratinocytes analyzed by real-time PCR.

Full figure and legend (93K)

Discussion

ATP2A2 encodes the SERCA2, which belongs to a family of P-type membrane-bound ATPases and transports Ca²⁺ from the cytoplasm into reservoirs in the endoplasmic reticulum. As the early evidence showing mutations in human ATP2A2 gene to be the cause of DD (Sakuntabhai et al., 1999b), various mutations, including missense mutations, nonsense mutations, and mutations causing frame-shifts and abnormal splicing, have been identified in the ATP2A2 gene in DD patients (Jacobsen et al., 1999; Ruiz-Perez et al., 1999; Sakuntabhai et al., 1999a; Ringpfeil et al., 2001; Takahashi et al., 2001; Yang et al., 2001). Several studies on SERCA2 mutations found in DD patients suggest that reduced expression of SERCA2 owing to haploinsufficiency is the main cause of DD (Ahn et al., 2003; Dhitavat et al., 2003; Dode et al., 2003; Sato et al., 2004; Foggia et al., 2006). These observations prompted us to analyze the expression mechanism of the human ATP2A2 gene in keratinocytes, as we believe that elucidation of this mechanism will be useful for further understanding the cause of DD and for developing new therapeutic methods for DD.

In this study, we used reporter assay and EMSA to identify a transcription factor Sp1, which binds to critical cis-enhancing elements on the human ATP2A2 promoter. Furthermore, we confirmed the in vivo binding of Sp1 to the ATP2A2 promoter region on chromatin in keratinocytes by ChIP assay and the involvement of Sp1 in ATP2A2 transcription in keratinocytes using siRNA. Although Sp1 is a widely expressed transcription factor, Sp1 has been confirmed to be involved in the transcriptional regulation of keratinocyte-specific genes, including transglutaminase 3 (Lee et al., 1996), loricrin (Jang and Steinert, 2002), involucrin (Eckert et al., 2004; Crish et al., 2006), keratin 5 (Kaufman et al., 2002), peptidylarginine deiminase type III (Dong et al., 2006), Np63 (Romano et al., 2006), and profilaggrin (Markova et al., 2006). We have also found that Sp1 is involved in the promoter activity of the Hailey–Hailey disease (HHD)-related gene human ATP2C1 in keratinocytes (Kawada et al., 2005). In that study, the amount of nuclear Sp1 was elevated by stimulation of normal keratinocytes with increased concentrations of Ca²⁺ in culture medium (Kawada et al., 2005). On the other hand, it is reported that extracellular Ca²⁺ induces growth suppression and differentiation of NHK (Hennings et al., 1980). Furthermore, upregulation of Sp1 activity induces the expression of the differentiation-specific molecule transglutaminase type 1 in keratinocytes (Wong et al., 2005). These observations suggest that Sp1 plays an important role in keratinocyte-specific gene expression induced or enforced by differentiation of keratinocytes.

As described above, several genes are under the control of Sp1 in keratinocytes. Therefore, in the knockdown experiment in this study, we cannot completely exclude the possibility that reduction of ATP2A2 expression may be due to the indirect effect of reduced expression of Sp1, which suppresses the expression of various target genes. Although further detailed analysis will be required to clarify this issue, we believe that ATP2A2 expression is downstream of Sp1 and that direct regulation by Sp1 indicated by reporter assay, EMSA, and ChIP assay is one of the mechanism of ATP2A2 gene regulation.

When nucleotide sequence of human ATP2A2 promoter is analyzed by UCSC (http://genome.ucsc.edu/cgi-bin/hgTracks), three regions (Figure 4) are identified as highly conserved sequences. Interestingly, two of three regions at 5'-site contain Sp1-binding sites identified in this study, suggesting a possibility that Sp1 regulates ATP2A2 gene expression not only in human but also in other species. The last one at just upstream of transcription start site may be important for basal transcriptional machinery including RNA polymerase.

ATP2A2 was also expressed in other tissues, including the brain, and DD has been reported to be associated with certain neuropsychiatric illness. Considering that Sp1 is a ubiquitous transcription factor and regulates transcription of several genes in brain (Gui et al., 2006; Ramos et al., 2007), Sp1 may control ATP2A2 expression in brain as well as keratinocytes. Alternatively, other transcription factors including other Sp1-factors may regulate ATP2A2 transcription in brain, as the case of NFB element in superoxide dismutase-2 gene, which was dominantly occupied by Sp3 and Sp4 in neurons, whereas Sp1, Sp3, and NFB bound this site in astroglia (Mao et al., 2006). Regardless, further study is required to reveal the involvement of Sp1 in ATP2A2 gene expression in other tissues.

In keratinocytes from HHD patients, Sp1 protein levels in the nucleus are not increased by higher concentrations of extracellular Ca²⁺ because of incomplete Ca²⁺ response due to malfunction of ATP2C1 (Kawada et al., 2005). Abnormalities in Ca²⁺-pumps such as ATP2C1 and ATP2A2 may result in incomplete expression of Sp1-mediated genes (including ATP2C1 and ATP2A2 themselves) in the Ca²⁺-induced differentiation stage of keratinocytes. It is possible that the defects in cell-to-cell adhesion observed in DD and HHD are typically present in the suprabasal layers of the epidermis where keratinocytes are in the final stage of differentiation. Therefore, restoration of Sp1 activity to normal level in a Ca²⁺-signaling independent manner may be a useful approach for treatment of DD and HHD.

To date, factors including transforming growth factor- (Sakaguchi et al., 2005), PGE₂ (Kanda et al., 2005; Matlhagela et al., 2005), 12-O-tetradecanoylphorbol 13-acetate, (Matlhagela et al., 2005), and 17-estradiol (Kanda and Watanabe, 2005) have been reported to induce activation of Sp1, thus suggesting that the mechanism of Sp1 activation via these factors is important for normalization of reduced Sp1-mediated gene expression levels in DD- and HHD-keratinocytes. Previously, S100C/A11-mediated post-translational regulation of Sp1 was reported in the analysis of Ca²⁺-induced growth inhibition of keratinocytes (Sakaguchi et al., 2003), where an increase in intracellular Ca²⁺ activates protein kinase C, which leads to phosphorylation of S100C, resulting in release of free Sp1 from nucleolin, which provides a site for switching transcriptional activation of Sp1 through protein–protein interaction. Therefore, we are further analyzing the effects of other factors, such as transforming growth factor- on activation of Sp1 in order to rescue the reduced expression of ATP2A2 and ATP2C1, as well as other genes observed in differentiated keratinocytes, as a possible therapeutic approach for DD and HHD.

Materials and Methods

Cell culture

NHK derived from normal newborn foreskin were purchased from KURABO (Osaka, Japan) and cultured in a serum-free standard medium Epilife-KGM (KURABO) containing 0.06 mM Ca²⁺, 10 g/ml insulin, 0.1 ng/ml hEGF, 0.5 g/ml hydrocortisone, 50 g/ml gentamicin, 50 ng/ml amphotericin-B, and 0.4% bovine pituitary extract. Second-to-fourth-passage keratinocytes in monolayer at 60–90% confluence were used. All experiments were performed according to the approved manual of the institutional review board of Juntendo University School of Medicine, Japan, adhered to the Declaration of Helsinki Principles.

Plasmid construction

The 5'-flanking region of approximately 740 bp including a 5'-untranslated (UT) region of the human ATP2A2 gene was amplified by PCR from human genomic DNA, which was purified from peripheral blood using a DNA quick kit (Dainippon Pharmaceutical, Osaka, Japan). The following synthesized oligonucleotides were used as primers: 5'-agatctCCCACCCTGCGTCTGCAGGTGGGTGGGTCAGAG-3', and 5'-aagcttACGGGCTCTCCCCTCCTCCTCTTGCGGCCGCTT-3' (replaced nucleotides for introduction of BglII and HindIII sites are shown in lowercase letters). This genomic region corresponds to 109203135/109203873 of chromosome 12 published in (NCBI) National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The amplified DNA fragment was inserted into the BglII/HindIII site of pGL3-Basic (Promega, Madison, WI). Other reporter plasmids carrying various length of the ATP2A2 promoter were constructed by PCR in a similar manner or via a newly introduced BglII site at the appropriate positions in the promoter using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The nucleotide sequences of the human ATP2A2 promoter in all reporter plasmids were confirmed by sequencing analysis using an ABI PRISM377 DNA sequencer (Applied Biosystems, Foster City, CA).

Luciferase assay

NHK cells (1 10⁵) cultured in six-well plates were transfected with 2 g reporter plasmid, and 250 ng pRL-null (Promega), which was an internal control for transfection efficiency, by Fugene6 (Roche Diagnostics, Indianapolis, IN). After 20–24 hours of culture, cells were harvested and the luciferase activity of each cell lysate was measured using a Dual-luciferase assay kit (Promega) and a luminescence detector, Micro Lumat Plus (Berthold, Postfach, Germany) as described previously (Akizawa et al., 2003).

EMSA

Probes were prepared by annealing two synthesized 5'-FITC-labeled oligonucleotides (Invitrogen, Carlsbad, CA) having complementary sequences. The sequences of the sense strand of probes were as follows: 5'-GGGAGGGGGCGGGGCCTGCGCGGCAGC-3' for –550/-524, 5'-GGAGGGGGCGGGGCCGCGCCGCCC-3' for –488/-465, 5'-CCGATAAATGCTATTAGAGCAGCCGCCGCGGA-3' for –391/-359, and 5'-TCGGGGCCGCGCGAGGGGCGGTT-3' for –41/-19. Nuclear proteins were extracted from NHK as described previously (Nishiyama et al., 1999; Maeda et al., 2003). Each probe (5 pmol) was mixed with nuclear extract containing 5 g of protein in the buffer (Nishiyama et al., 1999) and was applied to a 4% polyacrylamide gel. To identify the transcription factors binding to the probe, 1 g of Ab against Sp1 (clone no. 1C6, cat no. sc-420X, mouse IgG1), Sp3 (clone no. F-7, cat no. sc-28305X, mouse IgG2a), AP-2 (clone no. 3B5, cat no. sc-12726X, mouse IgG2b), AP-2 (clone no. H-87, cat no. sc-8976X, rabbit IgG) or AP-2 (clone no. 6E4/4, cat no. sc-12762X, mouse IgG1) was added to the mixture. After electrophoresis for 2 hours at 250 V, gels were placed in a fluorescence detector, FluoroImager 595 (Molecular Dynamics, Sunnyvale, CA).

Quantitative ChIP assay

ChIP assay was performed as described previously using a ChIP Assay Kit (Upstate, Lake Placid, NY) with a modification for use of protein G (Upstate) instead of protein A (Maeda et al., 2006). Anti-Sp1 Ab, anti-Sp3 Ab, and mouse IgG1 (no. 553485) and IgG2a (no. 553454) (BD Pharmingen, San Diego, CA) were used as controls in ChIP assay. The amounts of Sp1 and Sp3 bound to the promoter region of the human ATP2A2 gene were quantified using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Briefly, fluorescence was evaluated in each well of 96-well plates (Applied Biosystems) containing TaqMan Universal PCR Master Mix (Applied Biosystems), and immunoprecipitate was monitored and cycle threshold values in amplification plots were calculated using 7500 SDS software (Applied Biosystems). The ratio of specific DNA fragments in each immunoprecipitate against input DNA was calculated from cycle threshold values. Nucleotide sequences of primers and a TaqMan probe are as follows: to detect the –580/-364 region of the human ATP2A2 gene, forward primer (5'-CCTCGATCCGGGTTCCTAG-3' for -580/-562), reverse primer (5'-GGCGGCTGCTCTAATAGCAT-3' for -364/-384), and TaqMan probe (5'-FAM-CTGGCGCCCACGC-MGB-3' for -513/-525). For the cis control region (+54456/+54536), forward primer (5'-TGGAGCTTGGATTCCTATGGA-3' for +54456/+54476), reverse primer (5'-CATGGTGATGACAGACAGGTAGGT-3' for +54536/+54513), and TaqMan probe (5'-FAM-TCTGCTGAACATACCTC-MGB-3' for +54484/+54500).

Knockdown of Sp1 expression by siRNA

In order to suppress the expression of Sp1 in keratinocytes, siRNA was used. Ten microliters of 20 M Sp1 siRNA, non-silencing control, or FITC-labeled control oligonucleotide (Validated Stealth RNAi DuoPak no. 12936-62; Invitrogen) was introduced into 1 10⁵ of keratinocytes with lipofectamine (Invitrogen) according to the manufacturer's instructions. Transfection efficiency of keratinocytes treated with FITC-labeled control oligonucleotides was analyzed by using a fluorescence microscopy BZ-8000 (Keyence Co., Woodcliff Lake, NJ).

Quantification of Sp1 and ATP2A2 mRNA by real-time PCR

The amounts of Sp1 and ATP2A2 mRNA were quantified using a 7500 Real-Time PCR System with Assays-on-Demand gene expression products (Applied Biosystems) of human Sp1 (Hs00412720), human ATP2A2 (Hs00155939), and endogenous controls (Human glyceraldehyde-3-phosphate dehydrogenase; no. 4326317E). Expression levels of Sp1 and ATP2A2 are given relative to those of glyceraldehyde-3-phosphate dehydrogenase based on cycle threshold values as described previously (Nishiyama et al., 2004).

Western blot analysis

Cell lysates (1 10⁶ cells) from each transfectant were subjected to electrophoresis on a 7.5%. SDS polyacrylamide gel. The anti-Sp1 Ab (clone no. 1C6) or anti-actin Ab (clone no. C-2, cat no. sc-8432, mouse IgG1; Santa Cruz Biotechnology, Santa Cruz, CA) was used as the primary Ab. Alexa Fluor 680 goat anti-mouse IgG (no. A-21058, Molecular Probes Inc., Eugene, OR) was used as the secondary Ab. Infrared fluorescence on membranes was detected by Odyssey infrared imaging system (Model ODY-9201-SC, LI-COR Inc., Lincoln, NE).

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

We thank the members of Atopy Research Center, Department of Dermatology, and Department of Immunology for their helpful discussions regarding this paper. We are grateful to Drs Miyuki Takagi, Tomonobu Ito, Naomi Shimokawa, William Ng, Tatsuo Fukai, Ms Mutsuko Hara, Ms Kanako Fukuayama, and Mr Hokuto Yokoyama for technical assistance, and Ms Michiyo Matsumoto for secretarial assistance. This work was supported by a Grant-in-Aid for Scientific Research (c) (to CN and SI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.