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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Allergy Clin Immunol. Author manuscript; available in PMC 2013 August 6.
Published in final edited form as:
PMCID: PMC3734863
NIHMSID: NIHMS495206

Specificity Protein 1 is Pivotal in the Skin’s Anti-viral Response

Lianghua Bin, MD, PhD,1,# Michael D Howell, PhD,1,2,3,# Byung Eui Kim, MD, PhD,1 Joanne E Streib, BA,1 Clifton F Hall, MS,1 and Donald YM Leung, MD, PhD1,3,5

Abstract

Background

Previous studies have found Sp1 transcription factor in the viral replication machinery and postulated that Sp1 was required for viral replication in host cells.

Objectives

We investigated the role of Sp1 in the skin’s anti-viral responses from the perspective of host defense and its biological relevance in patients with atopic dermatitis and a history of eczema herpeticum (ADEH+).

Methods

Small interfering RNA duplexes were used to knock-down Sp1 in keratinocytes. The expression of vaccinia virus (VV), herpes simplex virus-1 (HSV-1) and other genes were evaluated by real-time PCR, or combined with western blot and immunohistofluorescence staining. A total of 106 human subjects participated in this study.

Results

Both VV and HSV-1 replication were enhanced in Sp1 knocked-down keratinocytes. Sp1 gene expression was significantly decreased in ADEH+ subjects, as compared to ADEH− and non-atopic subjects (P<0.0001); and inversely correlated with VV DNA copy number in human skin explants incubated with VV in vitro (partial correlation r = −0.256, P = 0.009). Gene profiling revealed that the anti-viral genes, PKR and OAS2, were significantly downregulated in Sp1 silenced keratinocytes. Gene expression of PKR and OAS2 were also significantly decreased in skin biopsies from ADEH+, as compared to ADEH- and non-atopic subjects. IFN-γ augmented the anti-viral capacity of Sp1 silenced keratinocytes.

Conclusions

Sp1 knockdown enhances viral replication in keratinocytes by downregulating gene expression of PKR and OAS2. Sp1 deficiency in ADEH+ patients may contribute to their increased propensity to disseminated skin viral infections. IFNγ augmentation may be a potential treatment for ADEH+ patients.

Keywords: specificity protein 1, vaccinia virus, herpes simplex virus 1, double-stranded RNA dependent protein kinase, 2′5′-oligoadenylate synthetase 2, atopic dermatitis, eczema herpeticum, IFNγ

INTRODUCTION

Atopic dermatitis (AD) is the most common chronic skin disease worldwide, affecting up to 30% of children in certain countries.1,2 A small subset of AD patients suffer from disseminated viral skin infections, i.e. eczema herpeticum (EH) after herpes simplex virus infection3 or eczema vaccinatum (EV) after smallpox vaccination with VV vaccine.4 As a result, AD patients are generally excluded from smallpox vaccination. This is a major impediment to mass vaccination with the conventional smallpox vaccine.57 To address this major health care problem, the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) funded the Atopic Dermatitis Vaccinia Network (ADVN) to identify mechanisms which predispose this subset of AD patients to disseminated viral infections.

Specificity protein 1 (Sp1) is a cellular transcription factor involved in diverse cellular functions.810 The recognition elements of Sp1 are frequently found in the promoters of various viral genes, and several lines of evidence have suggested the contribution of Sp1 in transactivation of viral genes.1114 While no report has identified Sp1 recognition elements in the genome of VV, Sp1 has been reported to be co-localized with the DNA replication machinery of VV in host cell cytoplasm.15

To further study the relationship of Sp1 to VV replication, our current experiments examined the effect of Sp1 gene silencing on VV replication in normal human keratinocytes (NHK). To our surprise, we found that Sp1 gene knockdown enhanced VV replication, and Sp1 gene expression was down-regulated in the skin of ADEH+ patients. We therefore explored, in further detail, the role of Sp1 in the skin’s host response against VV. Our findings indicate that enhanced VV replication in Sp1 silenced keratinocytes is associated with attenuation of the PKR/eIF2α signal pathway and decreased gene expression of OAS2. Our current findings suggest that Sp1 deficiency predisposes a subset of AD patients to disseminated viral skin infections.

METHODS

Subjects

Human subjects included 31 non-atopic healthy individuals, 55 ADEH−, 20 ADEH+. The detailed demographics information of human subjects was included in Supplemental Table I. The institutional review board at National Jewish Health approved this study. All subjects provided written informed consent to participate in the study.

Punch biopsies (2 mm) were collected from the uninvolved skin and immediately placed in a 96 well plate with RPMI 1640 (Cellgro, Herndon, VA) supplemented with 10% Fetal Calf Sera (FCS) (Gemini Bio Products, Calabasas, CA) and penicillin (100U/ml)/streptomycin (100ug/ml). Biopsies were cultured in the presence of media alone or 2 × 105 pfu vaccinia virus for 24h. Following the exposure period, media was removed and biopsies were submerged in 10% buffered formalin for immunohistochemical staining or Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) for RNA and DNA isolation.

Cells and Virus source

Normal human keratinocytes (NHK) were purchased from Cascade Biologics (Portland, OR) and maintained in serum free EpiLife Medium containing 0.06 mM CaCl2. The Wyeth/ACAM2000 strain of VV was obtained from the Centers for Disease Control and Prevention. HSV-1(VR-733) was purchased from American Type Culture Collection (Manassas, VA).

Small interfering RNAs (siRNAs) silencing experiment and virus infection

Sp1 and Silencer Negative control 1 siRNA duplexes were purchased from Ambion (Austin, TX). Sequences for targeting Sp1 were as follows: Sp1 #1 siRNA: 5′-GCAACAUGGGAAUUAU GAA-3′; Sp1 #2 siRNA: 5′-GGCAGACCUUUACAACUCA-3′; Sp1 #3 siRNA: 5′-CCACAAGCCCAAACAAUCA-3′. PKR and OAS2 siRNA duplexes were purchased from Dharmacon (Lafayette, CO). Sequences for targeting OAS2 were: 5′-AGAGGCAACUCCGAUGGUA-3′; 5′-AAGAGAAGCCAACGUGACA-3′; 5′-GGGAUAA GCUGAAGUUCUG-3′; 5′-GUUGGUUUAUCCAGGAAUA-3′. Sequences for targeting PKR were: 5′-GUAAGGGAACUUUGCGAUA-3′; 5′-GCGAGAAACUAGACAAA GU-3′; 5′-CGACCUAACAAUCUGAAA-3′; 5′-CCACAUGAUAGGAGGUUUA-3′.

NHK cells were plated in 24 well plates at 1×105 per well the day before transfection. Cells were transfected with 5 pmol of siRNA duplexes per well using lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). To knock-down Sp1 at different levels, 0.05 pmol, 0.5 pmol and 5 pmol of Sp1 #1 siRNA duplexes per well were transfected into NHK cells. Forty-eight hours after transfection, cells were infected with VV at various MOIs from 0.01 to 0.1 and HSV 1 at MOI 0.05. We also used different concentrations of PKR and OAS2 siRNA duplexes to knock-down PKR and OAS2 at different levels.

Plaque assays

To assess the production of infectious viral particles, cells and culture supernatants were harvested together after 24h of VV incubation. Cells were disrupted by three freeze-thaw cycles. Viral yields were determined by titration plaque assay as previously described.16

RNA isolation and PCR analysis

Total RNA was isolated from skin biopsies and cells using Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) and RNeasy Mini Kits (Qiagen, Valencia, CA) according to the manufacturer’s guidelines. DNA was extracted from skin biopsies after RNA extraction according to the manufacturer’s instruction. RNA was reverse transcribed into cDNA and analyzed by real time RT-PCR using an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA) as previously described.17 Primers and probes for human Sp1, OAS1, OAS2, OAS3, PKR and 18S were purchased from Applied Biosystems (Foster City, CA). The primer sequences for VV transcripts were prepared as previously described.18 The primer sequences for HSV-1 transcripts were prepared as previously described.19 Quantities of all target genes in test samples were normalized to the corresponding 18S levels.

Western blot analysis

Whole-cell extracts were prepared in the presence of 1% (vol/vol) of protease inhibitor cocktail and 1% (vol/vol) of phosphatase inhibitor cocktail (Sigma-Aldrich). The cell nuclear compartment was extracted from cells using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL) according to manufacturer’s instructions. Protein was then separated using SDS-PAGE and then transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The blots were then blocked and incubated with primary and secondary antibodies. Rabbit anti-human Sp1, and rabbit anti-human eIF2α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-β-actin (Sigma Chemical Co., St. Louis, MO) and monoclonal anti-human TATA box binding protein (TBP) (Abcam Inc., Cambridge, MA) were used as loading controls for whole cell lysates and nuclear protein, respectively. Rabbit anti-phospho-eIF2α monoclonal antibody was purchased from Epitomics Inc. (Burlingame, CA). Blots were developed with ECL Detection Reagents (GE Healthcare Bio-Sciences, Piscataway, NJ). We analyzed the density of protein bands using software Alpha Imager 2200 (Cell Biosciences, Inc., Santa Clara, CA).

Immunofluorescence staining for human Sp1

Immunofluorescence staining for human Sp1 with paraffin-embedded tissues followed the conventional staining protocol as previously described.20 Paraffin-embedded tissues were cut at 5 micrometer thickness on frosted microscope slides. Using toluene and a series of ethanol washes, the slides were deparaffinized and then rehydrated. Immunofluorescence staining was visualized with confocal microscopy (Leica, Wetzlar, Germany). Images were collected at x 630, and levels of MFI were measured with Slidebook 4.1 (Intelligent Imaging Innovations). The MFI was determined for each exposure group and was reported as mean MFI ± s.e.m.

Statistical analysis

All statistical analysis was conducted using Graph Pad prism, version 4.03 (San Diego, CA) or SAS version 9.13 or greater. Comparisons of expression levels were performed using analysis of variance (ANOVA) techniques and independent sample t tests as appropriate. Log10 transformations were applied when necessary to satisfy statistical assumptions. Pearson correlations and partial correlations were computed using log10 transformed values for Sp1 mRNA expression and VV viral copies. Partial correlations were computed to describe the association between VV viral copies and Sp1 mRNA expression while adjusting for the influence of r diagnostic group. Partial correlations were computed with multiple linear regression models using the SAS REG procedure. Dose-response inferences were made using linear regression models with log10 of dose (either Sp1 siRNA or IFNγ doses) as the independent variable. Differences were considered significant at P<0.05.

RESULTS

Gene Silencing of Sp1 enhances VV replication in keratinocytes

To investigate the importance of Sp1 in skin keratinocyte responses against VV, we inhibited Sp1 expression in cultured NHK cells using a small interfering RNA (siRNA) technique. Sp1 protein expression was significantly inhibited following transfection with three different specific siRNA duplexes targeting three independent mRNA sequences of Sp1 (Fig 1A). Specificity protein 3 (Sp3), a protein from the same family of transcription factors as Sp1,9 expression didn’t change under the same gene silencing conditions, suggesting the knock-down of Sp1 was specific. We didn’t find any morphological changes of NHK cells following Sp1 silencing. By using a MTS assay to determine cell proliferation, we found the cell growth of Sp1 silenced NHK cells didn’t change significantly as compared to scrambled siRNA silenced NHK cells(data not shown). After gene silencing, we inoculated the cells with VV at a multiplicity of infection (MOI) of 0.1 and incubated for 24 hours (h). The infected cells were then harvested for RNA isolation and reverse transcribed into complementary DNA (cDNA). VV replication was evaluated by real-time quantitative PCR. We found that VV replication was significantly enhanced in NHK cells silenced by three independent Sp1 siRNA duplexes as compared to cells transfected with scrambled siRNA control duplexes (Fig. 1B; P<0.01), indicating a specific effect of Sp1 silencing and not an “off-target” effect. We used a functional approach to further evaluate whether Sp1 silenced NHK cells support increased production of infectious VV particles using a viral plaque assay. As shown in Fig. 1C, after 24h, the yield of infectious virus was significantly increased in Sp1 silenced NHK cells compared to cells transfected with scrambled siRNA duplexes. We also inoculated Sp1 and scrambled siRNA silenced NHK cells with various MOIs, and we found that the enhancement of VV replication in Sp1 silenced NHK cells was consistent with different MOIs (data not shown). Extending the observation to another virus, we found that Sp1 silencing also led to significantly increased herpes simplex virus (HSV)-1 replication in keratinocytes compared to transfection with scrambled siRNA (Fig. 1D). These data demonstrate that Sp1 plays a critical role in anti-viral cellular responses.

Figure 1
Inhibition of Sp1 enhances viral replication in NHK cells

Sp1 gene expression is significantly decreased in ADEH+ patients

Next, we determined the potential clinical relevance of our in vitro observation that inhibiting Sp1 gene expression enhances VV replication in keratinocytes. Eczema herpeticum has been reported to clinically resemble EV,21 suggesting that ADEH+ patients would be good clinical models for mechanistic investigation of both EV and EH. We therefore examined gene expression of Sp1 in skin biopsies from ADEH+ subjects comparing them to ADEH− and non-atopic healthy subjects. As shown in Fig. 2A, Sp1 gene expression was decreased in skin biopsies from ADEH− subjects (mean: 13.58±2.50; n=55) compared to non-atopic subjects (mean: 27.48 ± 5.26; n=31) (P<0.01). However, Sp1 expression was significantly more decreased in ADEH+ (mean: 3.39±1.23; n=20) (P<0.0001) as compared to non-atopic subjects. Most importantly, Sp1 gene expression was significantly lower in ADEH+ skin as compared to ADEH− skin (P<0.05).

Figure 2
Sp1 gene expression is decreased in skin biopsies from ADEH+ patients

In order to determine which cell types in the skin express reduced Sp1 protein levels, we performed immunofluorescence staining of skin explants from ten subjects in each group. Sp1 staining intensity was significantly decreased in the keratinocytes of the epidermis from ADEH+ subjects compared to keratinocytes of ADEH− (Fig. 2B; Fig. 2C; P<0.05) and non-atopic subjects (Fig. 2C; P<0.001).

VV replication is inversely correlated with Sp1 expression level

Although Sp1 gene expression was reduced in both ADEH− and ADEH+ patients as compared to non-atopic subjects, the reduction of Sp1 gene expression was significantly greater in ADEH+ patients as compared to ADEH− patients. This suggested that the level of Sp1 expression might determine the ability of keratinocytes to control VV replication. We therefore inhibited Sp1 gene expression to various levels in NHK cells by transfection with various concentrations of siRNA duplexes. Cells were then inoculated with VV at a MOI of 0.01 for 24h. We found that Sp1 protein levels kept reducing in NHK cells following transfection with increasing concentration of Sp1 siRNA duplexes (Fig. 3A upper panel), and VV replication in NHK cells increased in a dose dependent manner with increasing concentrations of transfected Sp1 siRNA duplexes (Fig. 3A lower panel; P<0.01 from linear model relating log10 VV expression to log10 Sp1 siRNA dose).

Figure 3
Sp1 expression level is inversely correlated with VV replication

We then investigated whether Sp1 gene expression level correlated with viral replication in skin explants from human subjects. Skin explants from ADEH+, ADEH−, and non-atopic healthy subjects were incubated with VV for 24h; RNA and DNA were then isolated from the skin explants for evaluation of Sp1 gene expression and VV viral copies, respectively. As shown in Figure 3B, the number of VV viral copies in skin explants was inversely correlated with Sp1 gene expression levels (subject group adjusted, partial correlation r = −0.256, P = 0.009).

The PKR/eIF2α anti-viral pathway is impaired in Sp1 silenced NHK and ADEH skin

We searched for mechanism(s) that could lead to enhanced viral replication in Sp1 silenced NHK. The mechanism of the PKR/eIF2α pathway in host anti-viral responses is well known to act by inhibition of viral translation.2224 A previous study has shown that Sp1 binds to the gene promoter of PKR, and over-expression of Sp1 in mammalian cells induces activation of the PKR promoter.25 Thus, we further investigated gene expression and protein phosphorylation of gene components in this signaling pathway. We found that PKR was significantly down-regulated in Sp1 silenced NHK cells compared to cells transfected with scrambled siRNA duplexes (Fig 4A, P<0.01).

Figure 4
PKR/eIF2α pathway is impaired in Sp1 deficient NHK cells and skin of ADEH+ patients

After viral infection, PKR was activated by a viral replication intermediate of double-stranded RNA. Activated PKR then catalyzes the phosphorylation of eIF-2α. Phosphorylation of eIF2α increases its affinity for guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF-2B), blocking the ability of eIF-2B to catalyze nucleotide exchange, and subsequently inhibiting protein synthesis.26 We therefore determined the protein expression and phosphorylation of eIF2α in Sp1 silenced keratinocytes. We found that eIF2α was constitutively phosphorylated in NHK cells (see Fig. 4B). Protein phosphorylation of eIF2α was slightly enhanced after VV inoculation of NHK cells transfected with scrambled siRNA duplexes 2h post infection and persisted to 8h post infection. However, eIF2α phosphorylation was decreased in Sp1 silenced NHK cells during this same time course of VV infection as compare to scrambled siRNA silenced control cells. These data demonstrate that the PKR/eIF2α signaling pathway is impaired in Sp1 silenced NHK cells.

We knocked-down PKR in NHK cells to a similar expression level as in Sp1 silenced NHK cells. We then inoculated VV (MOI of 0.01) to those cells and VV replication was evaluated. As shown in Figure 4C, VV replication is significantly enhanced in PKR silenced NHK cells but the enhancement is not as great as in Sp1 silenced NHK cells, suggesting the decrease in PKR expression is only a part of the mechanism resulting in enhanced VV replication in Sp1 silenced keratinocytes and other mechanisms may contribute to the viral replication enhancement as well.

Because Sp1 is significantly lower in skin biopsies from ADEH+ patients, we searched for evidence of attenuation of the PKR/eIF2α signal pathway in human subjects. Indeed, we found that PKR gene expression was also significantly decreased in the skin biopsies from ADEH+ patients as compared to ADEH− patients (Fig. 4D; P<0.001) and non-atopic subjects (Fig 4D; P<0.0001). These data support the concept that the PKR/eIF2α signal pathway is functionally decreased in the skin of ADEH+ patients with Sp1 deficiency.

OAS2 is significantly decreased in Sp1 silenced keratinocyte and ADEH+ skin

To search for additional mechanism(s) independent of PKR, which might account for increased propensity to disseminated viral skin infections, we used a gene microarray technique to identify candidate gene(s) associated with impaired anti-viral responses in Sp1 silenced NHK. We searched for genes that had greater than a 1.5 fold change in expression and also belonged to the gene ontology (GO) group designated “immune system process” (GO accession number: 0002376). Out of approximately 972 genes, a total of 143 probes representing 67 genes were identified to have a greater than 1.5 fold change of gene expression in Sp1 silenced keratinocytes compared to cells transfected with scrambled siRNA (Supplemental Fig 1). Among these genes, we found that 2′5′-oligoadenylate synthetase 2 (OAS2), a member of the OAS family involved in host innate immune responses, had a two fold reduction in gene expression.2730 The effect of OAS2 on VV replication has not been previously reported. We therefore further explored the role of OAS2 in controlling VV replication.

In three independent experiments, we examined OAS2 gene expression levels in Sp1 silenced keratinocytes using NHK cells from three different donors. We confirmed that OAS2 gene expression was significantly down-regulated in Sp1 silenced keratinocytes, but not OAS1 or OAS3 (Fig. 5A–C). These results suggested that Sp1 silencing regulated the gene expression of OAS2, but didn’t affect the other two OAS family members although they are all closely located as a gene cluster.

Figure 5
OAS2 is decreased in Sp1 silenced keratinocytes and ADEH+ patients

To confirm the biological relevance of OAS2 deficiency, we investigated OAS2 gene expression in skin biopsies from ADEH−, ADEH+ and non-atopic subjects by real-time PCR. We found that OAS2 gene expression is indeed significantly down-regulated in the skin of patients with ADEH+ as compared to ADEH− and non-atopic subjects (Fig. 5D).

OAS2 is a part of mechanism resulting in enhanced VV replication in Sp1 silenced keratinocytes

To demonstrate the functional significance of OAS2 deficiency, we also selectively silenced OAS2 gene expression in NHK cells then inoculated them with VV at an MOI of 0.01 for 24h. We found that VV replication was indeed enhanced in OAS2 silenced NHK cells as compared to cells transfected with scrambled siRNA. However, enhancement of VV replication in OAS2 silenced NHK cells was not as great as in Sp1 silenced cells although the OAS2 expression level was lower in OAS2 silence cells than Sp1 silenced cells (P<0.01) (Fig. 6A–B). These results suggest that down-regulation of OAS2 is a part of the mechanism involved in the enhancement of VV replication in Sp1 deficient cells other than down-regulation of PKR.

Figure 6
OAS2 reduction is part of the mechanism resulting in enhanced VV replication in Sp1 silenced keratinocytes

Interferon (IFN)γ treatment significantly augments the anti-viral capacity of Sp1 silenced keratinocytes

Since IFNγ treatment can induce OAS2 gene expression and enhance phosphorylation of eIF2α protein, we tested whether IFNγ treatment could by-pass the effect of Sp1 deficiency in keratinocytes. We treated Sp1 silenced NHK and scrambled siRNA transfected cells with different concentrations of IFNγ for 8h, and then inoculated the cells with VV at MOI of 0.01 for 24h. We found that OAS2 was significantly induced by IFNγ in both Sp1 silenced NHK cells and control cells transfected with scrambled siRNA, in a dose-dependent manner (from linear models relating log10 of relative OAS2 expression to log10 dose of IFNγ, for both cell types P<0.01). The induction of OAS2, however, in Sp1 deficient NHK was not as great as in cells transfected with scrambled siRNA (Fig. 7A). This result suggests that Sp1 only regulates the constitutive gene expression of OAS2 but does not affect its induction by IFNγ. We also evaluated protein phosphorylation of eIF2α in Sp1 silenced NHK cells and control cells transfected with scrambled siRNA after IFNγ treatment. We found that IFNγ treatment enhanced the phosphorylation level of eIF2α protein in both Sp1 silenced NHK and scrambled siRNA transfected control cells, however, eIF2α protein phosphorylation level was lower in Sp1 silenced keratinocytes than cells transfected with scrambled siRNA under both stimulated and unstimulated conditions (Fig. 7B). Accordingly, VV replication evaluated by real-time PCR was significantly inhibited in IFNγ treated Sp1 deficient NHK and control cells transfected with scrambled siRNA in a dose dependent manner (Fig. 7C) (P< 0.01 from linear models relating log10 relative VV expression to log10 IFNγ dose). However, the inhibition of VV replication in Sp1 deficient cells was not as great as in cells transfected with scrambled siRNA (Fig. 7C). For example, when treated with 1 ng/ml of IFNγ, VV replication decreased almost 90 fold in scrambled siRNA silenced NHK cells, while there was only about 8.5 fold reduction of VV replication in Sp1 silenced NHK cells (Fig. 7C). Nevertheless, IFNγ treatment did significantly augment the anti-viral response of Sp1 deficient cells.

Figure 7
IFNγ treatment significantly augments anti-viral responses in Sp1 deficient keratinocytes

DISCUSSION

Since Sp1 binding sites are frequently found in various viral gene promoters, the potential role of Sp1 in promoting virulence has been suggested by numerous studies using modified virus with deletion of Sp1 binding sites of certain viral genes. To the best of our knowledge, the current study is the first report that demonstrates, from the perspective of immune host defense, that Sp1 is critical to anti-viral responses in skin keratinocytes. Furthermore, this report is the first to demonstrate that OAS2 is a target gene of the Sp1 transcription factor and down-regulation of OAS2 is part of the mechanism that results in enhanced viral replication in Sp1 deficient keratinocytes. Sp1 target genes encompass many key players involved in cell growth, angiogenesis, and genomic stability. Indeed, over-expression of Sp1 has been found in several cancers, and its expression level correlates with tumor grade/stage and poor prognosis.3135 Aside from over-expression in patients with various types of cancer, promoter mutations in Sp1 target genes have been implicated in various human diseases.36, 37 The current study, however, is the first to identify a human disease, ADEH, which is associated with decreased gene expression of Sp1. The in vivo relevance of Sp1 deficiency is supported by our observation that downstream effects of Sp1 (i.e. activation of OAS2 and PKR) are both affected in the skin of patients with ADEH+.

To better understand the impact of Sp1 expression deficiency in keratinocytes, we did a gene array assay to study the gene expression profiling of Sp1 silenced NHK cells as compared to scrambled siRNA silenced NHK cells. Aside from looking for dysregulated genes associated with the immune process, we also searched for genes that were dysregulated following Sp1 silencing and involved in skin barrier integrity as ADEH patients have skin barrier defects. We found that multiple genes from kallikrein (KLK) family, including KLK6, KLK12, KLK10, KLK8, KLK5 and KLK7, were up-regulated following Sp1 silencing in NHK cells. Kallikrein genes encode proteins with serine protease activities which are important for the desquamation process in epidermis.38 Elevated Kallikrein 5 activity was found to induce AD in a mouse model of Netherton syndrome.39 Three keratins, KRT 13, KRT 19 and KRT15, are significantly down-regulated following Sp1 silencing by 8.2, 7.5, and 3.8 fold, respectively. We believe the altered expression of these genes could profoundly change skin barrier integrity. The functional study of Sp1 in skin barrier integrity is currently underway in our lab.

In the presence of IFN or viral infection, OAS1, OAS2, and OAS3, three functional genes from the human OAS gene family, can be activated to catalyze the oligomerization of ATP into 2′,5′-linked oligoadenylate (2–5A). 2–5A binds to and activates the latent RNase L, which in turn degrades viral RNA to inhibit viral replication in host cells.40 Previous studies demonstrated that various human OAS proteins can be differentially induced in different types of cells and appear in different subcellular locations and enzymatic parameters, suggesting that these proteins might have distinct biological functions.27, 28 Searching up-stream of 2kb DNA sequences from the starting code for Sp1 binding site, we found there are three putative Sp1 binding sites in OAS2 gene promoter within 300bp up-stream of its starting code, suggesting that Sp1 may directly bind to OAS2 promoter to maintain its constitutive expression. In contrast, we didn’t find Sp1 binding sites in the promoter of OAS1 and OAS3, suggesting that Sp1 may only be involved in regulation of OAS2. This notion is supported by our data that the constitutive gene expression of OAS2, but not OAS1 and OAS3, is significantly decreased in Sp1 deficient NHK cells.

Our lab previously demonstrated that production of the anti-microbial peptide, cathelicidin, was deficient in skin biopsies from patients with AD and demonstrated that this deficiency contributed to the susceptibility of AD patients to viral infection.20 Although cathelicidin deficient mice had increased VV replication at the site of viral inoculation, they didn’t develop disseminated skin VV infection.18 This suggested that mechanisms other than cathelicidin deficiency were involved in the etiology of disseminated viral infections in ADEH+ patients. In the current study, we didn’t find down-regulation of cathelicidin in Sp1 silenced keratinocytes (data not shown). This suggests that cathelicidin is not directly regulated by Sp1 in keratinocytes. Cathelicidin appeared to act extracellularly by damaging the viral envelope.18 This was a different but complementary mechanism to IFN-inducible genes which acted intracellularly to inhibit viral transcription and translation. Evidence collected by our lab and others strongly suggest that both intracellular and extracellular defects in anti-viral host response are involved in mechanisms leading to the increased propensity of viral infections in patients with ADEH+.

Our study demonstrated treatment with exogenous IFNγ can significantly enhance both OAS2 gene expression and protein phosphorylation of eIF2α in Sp1 silenced keratinocytes. Although the enhancement is less than in scrambled siRNA transfected cells, this result suggests that the effect of deficiency in Sp1 can still be substantially corrected by IFNγ treatment. Of interest, a previous study has demonstrated that IFNγ production is deficient in ADEH+ patients as compared to ADEH− patients.41 Our study therefore suggests that the subset of patients with ADEH+ may benefit from IFNγ treatment or an immune adjuvant which augments IFNγ expression.

Taken together, we have demonstrated in this study that Sp1 silencing leads to enhanced VV and HSV-1 replication. The mechanisms resulting in this enhancement are associated with reduced gene expression of OAS2 and an attenuated PKR/eIF2α pathway in human keratinocytes. Furthermore, we also report the novel finding that the Sp1, OAS2 and PKR antiviral pathways are decreased in the skin of ADEH+ patients, highlighting the clinical significance of the current study. Molecules identified by this study may be potential biomarkers for identification of the subpopulation of AD at risk for EV and EH. More importantly, the current report also identifies new molecular targets that may make smallpox vaccination safer for patients with AD at risk for life threatening viral infections.

Key Messages

  1. Sp1 promotes host anti-viral response by regulating gene expression of PKR and OAS2.
  2. Sp1, PKR and OAS2 are significantly decreased in the skin of ADEH patients.
  3. IFNγ treatment augments anti-viral response in Sp1 deficient cells.

Supplementary Material

01

Supplemental Figure 1:

Heatmap presentation of genes in GO group of 0002376 that have greater than 1.5 fold change between Sp1 siRNA and scrambled siRNA.

Acknowledgments

This work was supported by NIH/NIAID contracts N01 AI 40029 and N01 AI40033, and NIAMS grant AR41256, and also in part by Colorado CTSA grant 1 UL1 RR025780 from NCRR/NIH. Dr. Bin’s salary was supported in part by the Eugene F. and Easton M. Crawford Pediatric Research Fellowship Fund at National Jewish Health. We also are grateful to Maureen Sandoval for her help in preparation of this manuscript.

ABBREVIATIONS

18S
18s ribosomal RNA
AD
Atopic dermatitis
ADEH−
AD patients without a history of eczema herpeticum
ADEH+
Atopic dermatitis and a history of eczema herpeticum
ADVN
Atopic Dermatitis Vaccinia Network
cDNA
Complementary DNA
EH
Eczema herpeticum
EV
Eczema vaccinatum
h
hours
eIF2α
Eukaryotic initiation factor 2α
FCS
Fetal calf sera
GO
Gene ontogeny
HSV-1
Herpes simplex virus 1
IFN
Interferon
MFI
Mean fluorescence intensity
MOI
Multiplicity of infection
NHK
Normal human keratinocytes
NIH
National Institutes of Health
NIAID
National Institute of Allergy and Infectious Diseases
OAS2
2′5′-oligoadenylate synthetase 2
PKR
Double-stranded RNA dependent protein kinase
siRNA
Small interfering RNA
Sp1
Specificity protein 1
TBP
TATA box binding protein
VV
Vaccinia virus

Footnotes

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no financial conflict of interests.

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References

1. Bieber T. Atopic dermatitis. N Engl J Med. 2008;358:1483–94. [PubMed]
2. Boguniewicz M, Leung DY. Recent insights into atopic dermatitis and implications for management of infectious complications. J Allergy Clin Immunol. 2010;125:4–13. [PMC free article] [PubMed]
3. Beck LA, Boguniewicz M, Hata T, Schneider LC, Hanifin J, Gallo R, et al. Phenotype of atopic dermatitis subjects with a history of eczema herpeticum. J Allergy Clin Immunol. 2009;124:260–9. [PMC free article] [PubMed]
4. Vora S, Damon I, Fulginiti V, Weber SG, Kahana M, Stein SL, et al. Severe eczema vaccinatum in a household contact of a smallpox vaccinee. Clin Infect Dis. 2008;46:1555–61. [PubMed]
5. Bicknell WJ. The case for voluntary smallpox vaccination. N Engl J Med. 2002;346:1323–5. [PubMed]
6. Drazen JM. Smallpox and bioterrorism. N Engl J Med. 2002;346:1262–3. [PubMed]
7. Fauci AS. Smallpox vaccination policy--the need for dialogue. N Engl J Med. 2002;346:1319–20. [PubMed]
8. Kadonaga JT, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987;51:1079–90. [PubMed]
9. Li L, He S, Sun JM, Davie JR. Gene regulation by Sp1 and Sp3. Biochem Cell Biol. 2004;82:460–71. [PubMed]
10. Wierstra I. Sp1: emerging roles--beyond constitutive activation of TATA-less housekeeping genes. Biochem Biophys Res Commun. 2008;372:1–13. [PubMed]
11. Dynan WS, Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983;35:79–87. [PubMed]
12. Rajcani J, Andrea V, Ingeborg R. Peculiarities of herpes simplex virus (HSV) transcription: an overview. Virus Genes. 2004;28:293–310. [PubMed]
13. Sune C, Garcia-Blanco MA. Sp1 transcription factor is required for in vitro basal and Tat-activated transcription from the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1995;69:6572–6. [PMC free article] [PubMed]
14. Wagner EK, Guzowski JF, Singh J. Transcription of the herpes simplex virus genome during productive and latent infection. Prog Nucleic Acid Res Mol Biol. 1995;51:123–65. [PubMed]
15. Oh J, Broyles SS. Host cell nuclear proteins are recruited to cytoplasmic vaccinia virus replication complexes. J Virol. 2005;79:12852–60. [PMC free article] [PubMed]
16. Howell MD, Wollenberg A, Gallo RL, Flaig M, Streib JE, Wong C, et al. Cathelicidin deficiency predisposes to eczema herpeticum. J Allergy Clin Immunol. 2006;117:836–41. [PMC free article] [PubMed]
17. Nomura I, Goleva E, Howell MD, Hamid QA, Ong PY, Hall CF, et al. Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes. J Immunol. 2003;171:3262–9. [PubMed]
18. Howell MD, Jones JF, Kisich KO, Streib JE, Gallo RL, Leung DY. Selective killing of vaccinia virus by LL-37: implications for eczema vaccinatum. J Immunol. 2004;172:1763–7. [PubMed]
19. Weidmann M, Meyer-Konig U, Hufert FT. Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR. J Clin Microbiol. 2003;41:1565–8. [PMC free article] [PubMed]
20. Howell MD, Gallo RL, Boguniewicz M, Jones JF, Wong C, Streib JE, et al. Cytokine milieu of atopic dermatitis skin subverts the innate immune response to vaccinia virus. Immunity. 2006;24:341–8. [PubMed]
21. Boyd DA, Sperling LC, Norton SA. Eczema herpeticum and clinical criteria for investigating smallpox. Emerg Infect Dis. 2009;15:1102–4. [PMC free article] [PubMed]
22. Garcia MA, Meurs EF, Esteban M. The dsRNA protein kinase PKR: virus and cell control. Biochimie. 2007;89:799–811. [PubMed]
23. Balachandran S, Roberts PC, Brown LE, Truong H, Pattnaik AK, Archer DR, et al. Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection. Immunity. 2000;13:129–41. [PubMed]
24. Dever TE. Gene-specific regulation by general translation factors. Cell. 2002;108:545–56. [PubMed]
25. Das S, Ward SV, Tacke RS, Suske G, Samuel CE. Activation of the RNA-dependent protein kinase PKR promoter in the absence of interferon is dependent upon Sp proteins. J Biol Chem. 2006;281:3244–53. [PubMed]
26. Proud CG. Protein phosphorylation in translational control. Curr Top Cell Regul. 1992;32:243–369. [PubMed]
27. Rebouillat D, Hovanessian AG. The human 2′,5′-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties. J Interferon Cytokine Res. 1999;19:295–308. [PubMed]
28. Hovanessian AG, Justesen J. The human 2′-5′ oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2′-5′ instead of 3′-5′ phosphodiester bond formation. Biochimie. 2007;89:779–88. [PubMed]
29. Silverman RH. A scientific journey through the 2–5A/RNase L system. Cytokine Growth Factor Rev. 2007;18:381–8. [PMC free article] [PubMed]
30. Lin RJ, Yu HP, Chang BL, Tang WC, Liao CL, Lin YL. Distinct antiviral roles for human 2′, 5′-oligoadenylate synthetase family members against dengue virus infection. J Immunol. 2009;183:8035–43. [PubMed]
31. Chiefari E, Brunetti A, Arturi F, Bidart JM, Russo D, Schlumberger M, et al. Increased expression of AP2 and Sp1 transcription factors in human thyroid tumors: a role in NIS expression regulation? BMC Cancer. 2002;2:35. [PMC free article] [PubMed]
32. Jiang Y, Wang L, Gong W, Wei D, Le X, Yao J, et al. A high expression level of insulin-like growth factor I receptor is associated with increased expression of transcription factor Sp1 and regional lymph node metastasis of human gastric cancer. Clin Exp Metastasis. 2004;21:755–64. [PubMed]
33. Kitadai Y, Yasui W, Yokozaki H, Kuniyasu H, Haruma K, Kajiyama G, et al. The level of a transcription factor Sp1 is correlated with the expression of EGF receptor in human gastric carcinomas. Biochem Biophys Res Commun. 1992;189:1342–8. [PubMed]
34. Safe S, Abdelrahim M. Sp transcription factor family and its role in cancer. Eur J Cancern. 2005;41:2438–48. [PubMed]
35. Wang L, Wei D, Huang S, Peng Z, Le X, Wu TT, et al. Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clin Cancer Res. 2003;9:6371–80. [PubMed]
36. Bond GL, Hu W, Bond EE, Robins H, Lutzker SG, Arva NC, et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell. 2004;119:591–602. [PubMed]
37. Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, et al. Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease. Science. 2002;296:2238–43. [PubMed]
38. Brattsand M, Stefansson K, Lundh C, Haasum Y, Egelrud T. A proteolytic cascade of kallikreins in the stratum corneum. J Invest Dermatol. 2005;124:198–203. [PubMed]
39. Briot A, Deraison C, Lacroix M, Bonnart C, Robin A, Besson C, et al. Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome. J Exp Med. 2009;205:1135–47. [PMC free article] [PubMed]
40. Hovanessian AG. On the discovery of interferon-inducible, double-stranded RNA activated enzymes: the 2′-5′ oligoadenylate synthetases and the protein kinase PKR. Cytokine Growth Factor Rev. 2007;18:351–361. [PubMed]
41. Peng WM, Jenneck C, Bussmann C, Bogdanow M, Hart J, Leung DY, et al. Risk factors of atopic dermatitis patients for eczema herpeticum. J Invest Dermatol. 2007;127:1261–3. [PubMed]