APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF.

Pancreatic cancer is the worst exocrine gastrointestinal cancer leading to the highest mortality. Recent studies reported that aberrant expression of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is involved in uncontrolled cell growth. However, the molecular mechanism of APE1 biological role remains unrevealed in pancreatic cancer progression. Here, we demonstrate that APE1 accelerates pancreatic cancer cell proliferation through glial cell line-derived neurotrophic factor (GDNF)/glial factor receptor α1 (GFRα1)/Src/ERK axis-cascade signaling. The proliferation of endogenous APE1 expressed-MIA PaCa-2, a human pancreatic carcinoma cell line, was increased by treatment with GDNF, a ligand of GFRα1. Either of downregulated APE1 or GFRα1 expression using small interference RNA (siRNA) inhibited GDNF-induced cancer cell proliferation. The MEK-1 inhibitor PD98059 decreased GDNF-induced MIA PaCa-2 cell proliferation. Src inactivation by either its siRNA or Src inhibitor decreased ERK-phosphorylation in response to GDNF in MIA PaCa-2 cells. Overexpression of GFRα1 in APE1-deficient MIA PaCa-2 cells activated the phosphorylation of Src and ERK. The expression of both APE1 and GFRα1 was gradually increased as progressing pancreatic cancer grades. Our results highlight a critical role for APE1 in GDNF-induced pancreatic cancer cell proliferation through APE1/GFRα1/Src/ERK axis-cascade signaling and provide evidence for future potential therapeutic drug targets for the treatment of pancreatic cancer.

Scavenger Receptor Class A to E Involved in Various Cancers.

Scavenger receptors typically bind to multiple ligands on a cell surface, including endogenous and modified host-derived molecules and microbial pathogens. They promote the elimination of degraded or harmful substances such as non-self or altered-self targets through endocytosis, phagocytosis, and adhesion. Currently, scavenger receptors are subdivided into eight classes based on several variations in their sequences due to alternative splicing. Since recent studies indicate targeting scavenger receptors has been involved in cancer prognosis and carcinogenesis, we will focus on the current knowledge about the emerging role of scavenger receptor classes A to E in cancer progression.

Therapeutic Effects of Synthetic Antimicrobial Peptides, TRAIL and NRP1 Blocking Peptides in Psoriatic Keratinocytes.

Psoriasis is a chronic, recurrent, heterogeneous, cutaneous inflammatory skin disease for which there is no cure. It affects approximately 7.5 million people in the United States. Currently, several biologic agents that target different molecules implicated in the pathogenic processes of psoriasis are being assessed in diverse clinical studies. However, relapse usually occurs within weeks or months, meaning there is currently no cure for psoriasis. Therefore, recent studies have discovered diverse new potential treatments for psoriasis: inhibitors of bacteria such as Staphylococcus aureus, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and neuropilin 1 (NRP1). A promising approach that has recently been described involves modifying antimicrobial peptides to develop new cutaneous anti-bacterial agents that target inflammatory skin disease induced by Staphylococcus. Increased expression of TRAIL and its death receptors DR4 and DR5 has been implicated in the pathogenesis of plaque psoriasis. In addition, TRAIL has the ability to inhibit angiogenesis by inducing endothelial cell death and by negative regulation of VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions. Since NRP1 regulates angiogenesis induced by multiple signals, including VEGF, ECM and semaphorins, and also initiates proliferation of keratinocytes through NF-κB signaling pathway in involved psoriatic skin, targeting NRP1 pathways may offer numerous windows for intervention in psoriasis. In this review, we will focus on the current knowledge about the emerging role of synthetic antimicrobial peptides, TRAIL and NRP1 blocking peptides in the pathogenesis and treatment of psoriasis.

Melanoma Cell Death Mechanisms.

Over recent years, several new molecular and immunogenic therapeutic approaches to melanoma treatment have been approved and implemented in clinical practice. Mechanisms of resistance to these new therapies have become a major problem. Mutation-specific pharmacotherapy can result in simultaneous emergence of resistant clones at many separate body sites despite an initially positive therapeutic response. Additionally, treatments aimed at inducing apoptosis are subject to resistance due to escape through other known mechanisms of regulated cell death (RCD). In this review, we discuss the complexity in pharmacological manipulation of melanoma with c-Kit, BRAF, MEK, and/or mTOR mutant cell lines. This study also addresses melanoma evasion of cell death through modalities of RCD such as apoptosis, autophagy, and necroptosis. This study also examines new combination therapies which have been approved to target both cell cycle dysregulation and cell death pathways. Lastly, we recognize the importance of immunomodulation though manipulation of the body's natural killing mechanisms with CTLA4, PD1, and CSF1 inhibition. As we begin to recognize tumor cell activation of alternate pathways, evasion of programmed cell death, and manipulation of the tumor microenvironment, it is increasingly important to grasp the complexity of personalized therapy in melanoma treatment.

The regulation of combined treatment-induced cell death with recombinant TRAIL and bortezomib through TRAIL signaling in TRAIL-resistant cells.

BACKGROUND: Multiple trials have attempted to demonstrate the effective induction of cell death in TRAIL-resistant cancer cells, including using a combined treatment of recombinant TRAIL and various proteasome inhibitors. These studies have yielded limited success, as the mechanism of cell death is currently unidentified. Understanding this mechanism's driving forces may facilitate the induction of cell death in TRAIL-resistant cancer cells. METHODS: Three kinds of recombinant soluble TRAIL proteins were treated into TRAIL-resistant cells and TRAIL-susceptible cells, with or without bortezomib, to compare their respective abilities to induce cell death. Recombinant TRAIL was treated with bortezomib to investigate whether this combination treatment could induce tumor regression in a mouse syngeneic tumor model. To understand the mechanism of combined treatment-induced cell death, cells were analyzed by flow cytometry and the effects of various cell death inhibitors on cell death rates were examined. RESULTS: ILz:rhTRAIL, a recombinant human TRAIL containing isoleucine zipper hexamerization domain, showed the highest cell death inducing ability both in single treatment and in combination treatment with bortezomib. In both TRAIL-resistant and TRAIL-susceptible cells treated with the combination treatment, an increase in cell death rates was dependent upon both the dose of TRAIL and its intrinsic properties. When a syngeneic mouse tumor model was treated with the combination of ILz:rhTRAIL and bortezomib, significant tumor regression was seen as a result of the effective induction of cancer cell death. The combination treatment-induced cell death was both inhibited by TRAIL blocking antibody and caspase-dependent. However, it was not inhibited by various ER stress inhibitors and autophagy inhibitors. CONCLUSIONS: The combination treatment with ILz:rhTRAIL and bortezomib was able to induce cell death in both TRAIL-susceptible and TRAIL-resistant cancer cells through the intracellular TRAIL signaling pathway. The efficiency of cell death was dependent on the properties of TRAIL under the environment provided by bortezomib. The combination treatment-induced cell death was not regulated by bortezomib-induced ER stress response or by autophagy.

Therapeutic Inhibitors against Mutated BRAF and MEK for the Treatment of Metastatic Melanoma.

Melanoma is one of the most aggressive cancers in the world and is responsible for the majority of skin cancer deaths. Recent advances in the field of immunotherapy using active, adoptive, and antigen-specific therapeutic approaches, have generated the expectation that these technologies have the potential to improve the treatment of advanced malignancies, including melanoma. Treatment options for metastatic melanoma patients have been dramatically improved by the FDA approval of new therapeutic agents including vemurafenib, dabrafenib, and sorafenib. These kinase inhibitors have the potential to work in tandem with MEK, PI3K/AKT, and mTOR to inhibit the activity of melanoma inducing BRAF mutations. This review summarizes the effects of the new therapeutic agents against melanoma and the underlying biology of these BRAF inhibitors.

The Alpha-Melanocyte-Stimulating Hormone Suppresses TLR2-Mediated Functional Responses through IRAK-M in Normal Human Keratinocytes.

Alpha-melanocyte stimulating hormone (α-MSH) is a highly conserved 13-aa neuropeptide derived from pro-opiomelanocortin by post-translational processing, which has been reported to exhibit potent anti-inflammatory activity and a wide range of immunosuppressive activities in the skin. However, the regulatory effect of α-MSH is not completely clear in cutaneous innate immunity. In this study, we investigate the functional regulation of α-MSH in TLR2-mediated inflammatory responses in normal human keratinocytes (HKs). α-MSH pretreatment down-regulated the Staphylococcus aureus LTA-induced expression of both TLR2 and IL-8 as well as NF-κB nuclear translocation in HK cells. The inhibitory effect of α-MSH was blocked by agouti signaling protein (ASP), an α-MSH receptor-1 antagonist. To investigate the mechanism of this response in more detail, siRNA of IRAK-M, a negative regulator of TLR signaling, was utilized in these studies. The α-MSH suppressive effect on IL-8 production and NF-κB transactivation was inhibited by IRAK-M siRNA transfection in HK cells. These results indicate that α-MSH is capable of suppressing keratinocyte TLR2-mediated inflammatory responses induced by S. aureus-LTA, thus demonstrating another novel immunomodulatory activity of α-MSH in normal human keratinocytes.

Suppression of Propionibacterium acnes Infection and the Associated Inflammatory Response by the Antimicrobial Peptide P5 in Mice.

The cutaneous inflammation associated with acne vulgaris is caused by the anaerobic bacterium Propionibacterium acnes through activation of the innate immune system in the skin. Current standard treatments for acne have limitations that include adverse effects and poor efficacy in many patients, making development of a more effective therapy highly desirable. In the present study, we demonstrate the protective effects of a novel customized α-helical cationic peptide, P5, against P. acnes-induced inflammatory responses in vitro and in vivo. Application of P5 significantly reduced expression of two inflammatory cytokines IL-8 and TNF-α in P. acnes-treated primary human keratinocytes, where P5 appeared to act in part by binding to bacterial lipoteichoic acid, thereby suppressing TLR2-to-NF-κB signaling. In addition, in a mouse model of acne vulgaris, P5 exerted both anti-inflammatory and antimicrobial effects against P. acnes, but exerted no cytotoxic effects against skin cells. These results demonstrate that P5, and perhaps other cationic antimicrobial peptides, offer the unique ability to reduce numbers P. acnes cells in the skin and to inhibit the inflammation they trigger. This suggests these peptides could potentially be used to effectively treat acne without adversely affecting the skin.

The expression of p21 is upregulated by forkhead box A1/2 in p53-null H1299 cells.

The expression of the cell cycle inhibitor p21 is increased in response to various stimuli and stress signals through p53-dependent and independent pathways. We demonstrate in this study that forkhead box A1/2 (FOXA1/2) is a crucial transcription factor in the activation of p21 transcription via direct binding to the p21 promoter in p53-null H1299 lung carcinoma cells. In addition, histone deacetylase inhibitor trichostatin A (TSA)-mediated upregulation of p21 expression was repressed by knockdown of FOXA1/2 in H1299 cells. Consequently, these results suggest that FOXA1/2 is required for p53-independent p21 expression.

Mycophenolate antagonizes IFN-γ-induced catagen-like changes via ß-catenin activation in human dermal papilla cells and hair follicles.

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce ß-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3ß, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-ß2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased ß-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3ß and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-ß2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/ß-catenin signaling, and that MPA stabilizes ß-catenin by inhibiting GSK3ß leading to increased ß-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.

Colonization and infection of the skin by S. aureus: immune system evasion and the response to cationic antimicrobial peptides.

Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus virulence factors. As part of the innate immune system in the skin, cationic antimicrobial peptides (CAMPs) such as the ß-defensins and cathelicidin contribute to host cutaneous defense, which prevents harmful microorganisms, like S. aureus, from crossing epithelial barriers. Conversely, S. aureus utilizes evasive mechanisms against host defenses to promote its colonization and infection of the skin. In this review, we focus on host-pathogen interactions during colonization and infection of the skin by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). We will discuss the peptides (defensins, cathelicidins, RNase7, dermcidin) and other mediators (toll-like receptor, IL-1 and IL-17) that comprise the host defense against S. aureus skin infection, as well as the various mechanisms by which S. aureus evades host defenses. It is anticipated that greater understanding of these mechanisms will enable development of more sustainable antimicrobial compounds and new therapeutic approaches to the treatment of S. aureus skin infection and colonization.

Effect of repetitive lysine-tryptophan motifs on the bactericidal activity of antimicrobial peptides.

Previous studies identified lysine- and tryptophan-rich sequences within various cationic antimicrobial peptides. In the present study, we synthesized a series of peptides composed of lysine (K)-tryptophan (W) repeats (KW)( n ) (where n equals 2, 3, 4 or 5) with amidation of the C-terminal to increase cationicity. We found that increases in chain length up to (KW)(4) enhanced the peptides' antibacterial activity; (KW)(5) exhibited somewhat less bactericidal activity than (KW)(4). Cytotoxicity, measured as lysis of human red blood cells, also increased with increasing chain length. With (KW)(5), reduced antibacterial activity and increased cytotoxicity correlated with greater hydrophobicity and self-aggregation in the aqueous environment. The peptides acted by inducing rapid collapse of the bacterial transmembrane potential and induction of membrane permeability. The mode of interaction of the peptides and the phosphate groups of lipopolysaccharide was dependent upon the peptides' ability to permeate the membrane. Longer peptides [(KW)(4) and (KW)(5)] but not shorter peptides [(KW)(2) and (KW)(3)] strongly bound and partially inserted into negatively charged, anionic lipid bilayers. These longer peptides also induced membrane permeabilization and aggregation of lipid vesicles. The peptides had a disordered structure in aqueous solution, and only (KW)(4) and (KW)(5) displayed a folded conformation on lipid membranes. Moreover, (KW)(4) destroyed and agglutinated bacterial cells, demonstrating its potential as an antimicrobial agent. Collectively, the results show (KW)(4) to be the most efficacious peptide in the (KW)( n ) series, exhibiting strong antibacterial activity with little cytotoxicity.

Suppression of IRF4 by IRF1, 3, and 7 in Noxa expression is a necessary event for IFN-γ-mediated tumor elimination.

IFN-γ plays a critical role in tumor immunosurveillance by affecting either immune cells or tumor cells; however, IFN-mediated effects on tumor elimination are largely unknown. In this study, we showed that IFN regulatory factors (IRF) modulated by IFNs up- and downregulated Noxa expression, a prodeath BH3 protein, in various cancer cells. Inhibition of Noxa expression using short hairpin RNA in tumor cells leads to resistance against lipopolysaccharide (LPS)-induced tumor elimination, in which IFN-γ is known as a critical effecter in mice. Chromatin immunoprecipitation analysis in both CT26 cells and SP2/0 cells, sensitive and resistant to LPS-induced tumor elimination, respectively, revealed that the responsiveness of IRF1, 3, 4, and 7 in the Noxa promoter region in response to IFN-γ might be crucial in LPS-induced tumor elimination. IRF1, 3, and 7 were upregulated by IFN-γ and activated Noxa expression, leading to the death of Noxa wild-type baby mouse kidney (BMK) cells but not of Noxa-deficient BMK cells. In contrast, IRF4 acts as a repressor for Noxa expression and inhibits cell death induced by IRF1, 3, or 7. Therefore, although IFN-γ alone are not able to induce cell death in tumor cells in vitro, Noxa induction by IFN-γ, which is regulated by the balance between its activators (IRF1, 3, and 7) and its repressor (IRF4), is crucial to increasing the susceptibility of tumor cells to immune cell-mediated cytotoxicity.

Antimicrobial and anti-inflammatory effects of Cecropin A(1-8)-Magainin2(1-12) hybrid peptide analog p5 against Malassezia furfur infection in human keratinocytes.

The lipophilic fungus Malassezia furfur (M. furfur) is a commensal microbe associated with several chronic diseases such as pityriasis versicolor, folliculitis, and seborrheic dermatitis. Because M. furfur-related diseases are difficult to treat and require prolonged use of medications, the treatment for M. furfur-related skin diseases is supposed to gain control over M. furfur growth and the inflammation associated with it, as well as to prevent secondary infections. In this study, we investigated the antifungal and anti-inflammatory effects of cecropin A(1-8)-magainin 2(1-12) hybrid peptide analog P5 on M. furfur. The minimal inhibitory concentration of P5 against M. furfur was 0.39 µM, making it 3-4 times more potent than commonly used antifungal agents such as ketoconazole (1.5 µM) or itraconazole (1.14 µM). P5 efficiently inhibited the expression of IL-8 and Toll-like receptor 2 in M. furfur-infected human keratinocytes without eukaryotic cytotoxicity at its fungicidal concentration. Moreover, P5 significantly downregulated NF-κB activation and intracellular calcium fluctuation, which are closely related with enhanced responses of keratinocyte inflammation induced by M. furfur infection. Taken together, these observations suggest P5 may be a potential therapeutic agent for M. furfur-associated human skin diseases because of its distinct antifungal and anti-inflammatory action.

Protein phosphatase 5 is necessary for ATR-mediated DNA repair.

Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

The cell death-inducing activity of the peptide containing Noxa mitochondrial-targeting domain is associated with calcium release.

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide-induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs.

Effect of Leucine and Lysine substitution on the antimicrobial activity and evaluation of the mechanism of the HPA3NT3 analog peptide.

In this study, a HPA3NT3-analog (FKKLKKLFKKILKLK-NH2) peptide was designed. In this analog, two Trp residues (positions 12, 14) were replaced with Leu, and Arg and Asn (positions 3, 13) were replaced with Lys to investigate the role of amino acid substitution and increased cationicity on antimicrobial and hemolytic activities. In fungal and Gram-negative bacterial cells, HPA3NT3-analog activity was unchanged or slightly enhanced when compared to the HPA3NT3 peptide. In addition, a twofold decrease in activity against Gram-positive bacteria was observed. The HPA3NT3-analog also induced less hemolysis (4.2%) than the HPA3NT3 peptide (71%) at 200 microM. Circular dichroism (CD) spectra revealed that the HPA3NT3-analog peptide had an unordered structure in buffer and egg yolk L-2-phosphatidyl choline (EYPC), but adapted an alpha-helical conformation in 50% 2,2,2-trifluoroethanol (TFE) and negatively charged egg yolk L-2-phosphatidyl glycerol (EYPG), while the parent peptide showed an ordered structure in the EYPC. Additionally, the HPA3NT3-analog peptide induced the leakage of calcein from egg yolk L-2-phosphatidyl ethanolamine (EYPE)/EYPG (7:3 w/w) large unilamellar vesicles (LUVs); however, the activity was slightly weaker than that of the HPA3NT3 peptide. The molecular dynamics (MD) structures revealed that the amino acid substitutions induced a significant variation in peptide structure. These results suggest that the substitutions of Arg and Asn with Lys and two Trp with Leu resulted in small changes in HPA3NT3-analog activity and significant decreases in hemolytic activity.

p19(ras) amplifies p73beta-induced apoptosis through mitochondrial pathway.

p73 and p53 have been known to play an important role in cellular damage responses such as apoptosis. Although p73 is a structural and functional homolog of p53 tumor suppressor gene, much less is known about the mechanism of p73-induced apoptotic cell death. In this study, we demonstrate that p19(ras) interaction with p73beta amplifies p73beta-induced apoptotic signaling responses including Bax mitochondrial translocation, cytochrome c release, increased production of reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential (DeltaPsi(m)). Furthermore, endogenous expression of p19(ras) and p73beta is significantly increased by Taxol treatment, and Taxol-enhanced endogenous p73beta transcriptional activities are further amplified by p19(ras), which markedly increased cellular apoptosis in p53-null SAOS2 cancer cell line. These results have important implications for understanding the molecular events of p19(ras) to p73 functions in cancer cells.

TIP60 represses transcriptional activity of p73beta via an MDM2-bridged ternary complex.

TIP60, a histone acetyl transferase, acts as a p53 coactivator by interfering with MDM2-mediated degradation of p53. However, little is known about its functional regulation of p73, which has structural features similar to p53. In this study we found that TIP60 represses apoptosis, which is induced by exogenous and endogenous p73beta. TIP60 also negatively regulated the expression of p73beta downstream target genes such as p21 and Bax. Moreover, the specific repression of p73beta-mediated transactivation by TIP60 was independent of p53 expression and not due to histone deacetylase recruiting transcriptional machinery. Transcriptional activities of both p73 splicing variants, p73alpha and p73beta, were also repressed by TIP60. Furthermore, TIP60 markedly enhanced p73beta binding affinity to MDM2 and physically associated with MDM2 through its zinc finger domain, which is specifically localized in the nucleus. Therefore, we demonstrate that TIP60 forms a ternary complex with p73beta, which is directly bridged by MDM2. It is important to note that our findings contribute to a functional linkage between TIP60 and p73beta through MDM2 in the transcriptional regulation of cellular apoptosis.

Expression of Toll-like receptors in verruca and molluscum contagiosum.

Recent studies indicate that several Toll-like receptors (TLRs) are implicated in recognizing viral structures and instigating immune responses against viral infections. The aim of this study is to examine the expression of TLRs and proinflammatory cytokines in viral skin diseases such as verruca vulgaris (VV) and molluscum contagiosum (MC). Reverse transcription-polymerase chain reaction and immunostaining of skin samples were performed to determine the expression of specific antiviral and proinflammatory cytokines as well as 5 TLRs (TLR2, 3, 4, 7, and 9). In normal human skin, TLR2, 4, and 7 mRNA was constitutively expressed, whereas little TLR3 and 9 mRNA was detected. Compared to normal skin (NS), TLR3 and 9 mRNA was clearly expressed in VV and MC specimens. Likewise, immunohistochemistry indicated that keratinocytes in NS constitutively expressed TLR2, 4, and 7; however, TLR3 was rarely detected and TLR9 was only weakly expressed, whereas 5 TLRs were all strongly expressed on the epidermal keratinocytes of VV and MC lesions. In addition, the mRNA expression of IFN-beta and TNF-alpha was upregulated in the VV and MC samples. Immunohistochemistry indicated that IFN-beta and TNF-alpha were predominantly localized in the granular layer in the VV lesions and adjacent to the MC bodies. Our results indicated that VV and MC skin lesions expressed TLR3 and 9 in addition to IFN-beta and TNF-alpha. These viral-induced proinflammatory cytokines may play a pivotal role in cutaneous innate immune responses.