Inhibitory effect of cucurbitacin B on imiquimod-induced skin inflammation.

Psoriasis is a common skin disease, of which pathogenesis involves the increase of inflammatory reaction in epidermal cells. In an attempt to find therapeutics for psoriasis, we found that cucurbitacin B has an inhibitory potential on imiquimod-induced inflammation of keratinocytes. Cucurbitacin B significantly inhibited imiquimod-induced expression of crucial psoriatic cytokines, such as IL-8 and CCL20, via down-regulation of NF-κB and STAT3 signaling pathway in human keratinocytes. In addition, keratinocyte proliferation was markedly inhibited by cucurbitacin B. The potential beneficial effect of cucurbitacin B on psoriasis was further validated in imiquimod-induced psoriasiform dermatitis of experimental animal. Topical application of cucurbitacin B resulted in significant reduction of epidermal hyperplasia and inflammatory cytokines production, and ameliorated the psoriatic symptom. Taken together, these results suggest that cucurbitacin B may be a potential candidate for the treatment of psoriasis.

Inhibitory role of Id1 on TGF-ß-induced collagen expression in human dermal fibroblasts.

Inhibitor of DNA binding 1 (Id1) is a basic helix-loop-helix (bHLH) protein that has a variety of functional roles in cellular events including differentiation, cell cycle and cancer development. In addition, it has been demonstrated that Id1 is related with TGF-ß and Smad signaling in various biological conditions. In this study, we investigated the effect of Id1 on TGF-ß-induced collagen expression in human dermal fibroblasts. When Id1-b isoform was overexpressed, TGF-ß-induced collagen expression was markedly inhibited. Consistent with this result, Id1-b significantly inhibited TGF-ß-induced collagen gel contraction. In addition, Id1-b inhibited TGF-ß-induced phosphorylation of Smad2 and Smad3. Finally, immunohistochemistry showed that Id1 expression was decreased in fibrotic skin diseases while TGF-ß signaling was increased. Together, these results suggest that Id1 is an inhibitory regulator on TGF-ß-induced collagen expression in dermal fibroblasts.

Genkwadaphnin induces reactive oxygen species (ROS)-mediated apoptosis of squamous cell carcinoma (SCC) cells.

Genkwadaphnin is a daphnane diterpene ester molecule isolated from the flower buds of Daphne genkwa. In the present study, we investigated the apoptosis-inducing effect of genkwadaphnin in squamous cell carcinoma (SCC) cells. Apoptosis was triggered in SCC12 cells following genkwadaphnin treatment in a time- and concentration-dependent manner. Genkwadaphnin treatment increased phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Knockdown of JNK and p38 MAPK by recombinant adenovirus expressing microRNA (miR) resulted in significant inhibition of genkwadaphnin-induced apoptosis in SCC12 cells. Finally, pretreatment with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) markedly reduced SCC12 cell apoptosis, concomitant with significant inhibition of MAPK activation. These results indicate that genkwadaphnin has the potential to induce apoptosis in SCC cells, providing information on which to base further research with the aim of developing a cure for SCC.

Regional difference in sebum production by androgen susceptibility in human facial skin.

Androgens are important hormones that influence sebum production from the sebaceous glands. Human facial skin can be categorized as T- and U-zones, which are areas with high and low levels of sebum secretion, respectively. This study was performed to investigate whether there are topographical differences in androgen receptor (AR) expression related to regional variations in facial sebum secretion. The results of in vivo analysis indicated a statistically significant increase in AR expression in the sebaceous gland T-zones compared with the U-zones. In vitro experiments using human primary sebocytes also yielded similar results, with higher levels of AR protein and mRNA expression in T-zones. The results of this study suggested that differences in androgen susceptibility may be an important factor influencing regional differences in sebum production in human facial skin.

Glucocorticoid receptor enhances involucrin expression of keratinocyte in a ligand-independent manner.

In this study, we investigated the role of glucocorticoid receptor (GR) in epidermal keratinocytes. In adult normal human skin, GR was highly expressed in the upper layers of the epidermis. Consistent with normal skin, GR expression was increased after calcium treatment of HaCaT keratinocytes cultured in vitro, suggesting that GR is involved in keratinocyte differentiation process. Overexpression of GR using an adenovirus showed that expression of involucrin, an early differentiation marker of keratinocytes, was markedly increased due to GR overexpression. However, treatment with dexamethasone, a GR agonist, did not increase involucrin expression. Overexpression of GR led to phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK) in the absence of glucocorticoid, suggesting that the GR effect on involucrin expression is related to activation of intracellular signaling cascades. This idea was supported by the fact that GR-mediated involucrin induction was abolished after treatment with JNK and ERK inhibitors. In addition, GR mutants lacking the ligand-binding domain increased involucrin expression concomitantly with increase of ERK phosphorylation. Together, these results suggest that GR modulates involucrin expression of keratinocytes by regulating the intracellular signaling network in a ligand-independent manner.

Absence of a human DnaJ protein hTid-1S correlates with aberrant actin cytoskeleton organization in lesional psoriatic skin.

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.

Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1.

The scaffolding adaptor protein p62/SQSTM1 (p62) has been shown to be an autophagy receptor that acts as a link between the ubiquitination and autophagy machineries. However, the roles of autophagy and p62 in human keratinocytes are not well understood. In this study, we show that keratinocyte autophagy negatively regulates p62 expression, which is essential for the prevention of excessive inflammation and the induction of cathelicidin in human keratinocytes. Stimulation of TLR2/6 or TLR4 in primary human keratinocytes robustly activated autophagy pathways and up-regulated p62 expression through induction of NADPH oxidases 2 and 4 and the generation of reactive oxygen species. MyD88 and TNFR-associated factor 6, key signaling molecules that mediate TLR activation, played an essential role in the induction of autophagy and p62 expression. Additionally, blockade of autophagy significantly increased the generation of inflammatory cytokines and expression of p62 in primary human keratinocytes. Notably, silencing hp62 through RNA interference resulted in a significant decrease in NF-κB activation, inflammatory cytokine production, cathelicidin expression, and cell proliferation (as well as cyclin D1 expression) in keratinocytes. Epidermal expression of p62 was further found to be significantly higher in psoriatic skin than in skin affected by atopic dermatitis or from healthy controls. Collectively, our data provide new insights into the roles of autophagy and p62 in controlling cutaneous inflammation.

Autologous platelet-rich plasma: a potential therapeutic tool for promoting hair growth.

BACKGROUND: Recently, autologous platelet-rich plasma (PRP) has attracted attention in various medical fields, including plastic and orthopedic surgery and dermatology, for its ability to promote wound healing. PRP has been tested during facelift and hair transplantation to reduce swelling and pain and to increase hair density. OBJECTIVE: To investigate the effects of PRP on hair growth using in vivo and in vitro models. METHODS: PRP was prepared using the double-spin method and applied to dermal papilla (DP) cells. The proliferative effect of activated PRP on DP cells was measured. To understand the mechanisms of activated PRP on hair growth, we evaluated signaling pathways. In an in vivo study, mice received subcutaneous injections of activated PRP, and their results were compared with control mice. RESULTS: Activated PRP increased the proliferation of DP cells and stimulated extracellular signal-regulated kinase (ERK) and Akt signaling. Fibroblast growth factor 7 (FGF-7) and beta-catenin, which are potent stimuli for hair growth, were upregulated in DP cells. The injection of mice with activated PRP induced faster telogen-to-anagen transition than was seen on control mice. CONCLUSIONS: Although few studies tested the effects of activated PRP on hair growth, this research provides support for possible clinical application of autologous PRP and its secretory factors for promotion of hair growth.

Apurinic/apyrimidinic endonuclease 1 is a key modulator of keratinocyte inflammatory responses.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes.

Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish.

Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.

Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes.

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

Promoter methylation changes in ALOX12 and AIRE1: novel epigenetic markers for atherosclerosis.

BACKGROUND: Atherosclerosis is the main cause of cardiovascular diseases such as ischemic stroke and coronary heart disease. Gene-specific promoter methylation changes have been suggested as one of the causes underlying the development of atherosclerosis. We aimed to identify and validate specific genes that are differentially expressed through promoter methylation in atherosclerotic plaques. We performed the present study in four steps: (1) profiling and identification of gene-specific promoter methylation changes in atherosclerotic tissues; (2) validation of the promoter methylation changes of genes in plaques by comparison with non-plaque intima; (3) evaluation of promoter methylation status of the genes in vascular cellular components composing atherosclerotic plaques; and (4) evaluation of promoter methylation differences in genes among monocytes, T cells, and B cells isolated from the blood of ischemic stroke patients. RESULTS: Upon profiling, AIRE1, ALOX12, FANK1, NETO1, and SERHL2 were found to have displayed changes in promoter methylation. Of these, AIRE1 and ALOX12 displayed higher methylation levels in plaques than in non-plaque intima, but lower than those in the buffy coat of blood. Between inflammatory cells, the three genes were significantly less methylated in monocytes than in T and B cells. In the vascular cells, AIRE1 methylation was lower in endothelial and smooth muscle cells. ALOX12 methylation was higher in endothelial, but lower in smooth muscle cells. Immunofluorescence staining showed that co-localization of ALOX12 and AIRE1 was more frequent in CD14(+)-monocytes than in CD4(+)-T cell in plaque than in non-plaque intima. CONCLUSIONS: Promoter methylation changes in AIRE1 and ALOX12 occur in atherosclerosis and can be considered as novel epigenetic markers.

Induction of alopecia areata in C3H/HeJ mice using polyinosinic-polycytidylic acid (poly[I:C]) and interferon-gamma.

Alopecia areata (AA) is a chronic, relapsing hair-loss disorder that is considered to be a T-cell-mediated autoimmune disease. Several animal models for AA have been created to investigate the pathophysiology and screen for effective therapeutic targets. As C3H/HeJ mice develop AA spontaneously in a low frequency, a novel animal model is needed to establish an AA-like condition faster and more conveniently. In this study, we present a novel non-invasive AA rodent model that avoids skin or lymph-node cell transfer. We simply injected C3H/HeJ mice subcutaneously with interferon-gamma (IFNγ) along with polyinosinic:polycytidylic acid (poly[I:C]), a synthetic dsRNA, to initiate innate immunity via inflammasome activation. Approximately 80% of the IFNγ and poly(I:C) co-injected mice showed patchy AA lesions after 8 weeks. None of the mice displayed hair loss in the IFNγ or poly(I:C) solely injection group. Immunohistochemical staining of the AA lesions revealed increased infiltration of CD4+ and CD8+ cells infiltration around the hair follicles. IFNγ and poly(I:C) increased the expression of NLRP3, IL-1ß, CXCL9, CXCL10, and CXCL11 in mouse skin. Taken together, these findings indicate a shorter and more convenient means of AA animal model induction and demonstrate that inflammasome-activated innate immunity is important in AA pathogenesis.

Targeted deletion of Crif1 in mouse epidermis impairs skin homeostasis and hair morphogenesis.

The epidermis, which consists mainly of keratinocytes, acts as a physical barrier to infections by regulating keratinocyte proliferation and differentiation. Hair follicles undergo continuous cycling to produce new one. Therefore, optimum supply of energy from the mitochondria is essential for maintaining skin homeostasis and hair growth. CRIF1 is a mitochondrial protein that regulates mitoribosome-mediated synthesis and insertion of mitochondrial oxidative phosphorylation polypeptides into the mitochondrial membrane in mammals. Recent studies reveal that conditional knockout (cKO) of Crif1 in specific tissues of mice induced mitochondrial dysfunction. To determine whether the mitochondrial function of keratinocytes affects skin homeostasis and hair morphogenesis, we generated epidermis-specific Crif1 cKO mice. Deletion of Crif1 in epidermis resulted in impaired mitochondrial function and Crif1 cKO mice died within a week. Keratinocyte proliferation and differentiation were markedly inhibited in Crif1 cKO mice. Furthermore, hair follicle morphogenesis of Crif1 cKO mice was disrupted by down-regulation of Wnt/ß-catenin signaling. These results demonstrate that mitochondrial function in keratinocytes is essential for maintaining epidermal homeostasis and hair follicle morphogenesis.

Double-stranded RNA induces inflammation via the NF-κB pathway and inflammasome activation in the outer root sheath cells of hair follicles.

Alopecia areata (AA), a chronic, relapsing, hair-loss disorder, is considered to be a T cell-mediated autoimmune disease. It affects approximately 1.7% of the population, but its precise pathogenesis remains to be elucidated. Despite the recent attention focused on the roles of inflammasomes in the pathogenesis of autoinflammatory diseases, little is known about inflammasome activation in AA. Thus, in this study, we investigated the pattern of NLRP3 inflammasome activation in the outer root sheath (ORS) cells of hair follicles. We found that interleukin (IL)-1ß and caspase-1 expression was increased in hair follicle remnants and inflammatory cells of AA tissue specimens. After stimulation of ORS cells with the double-stranded (ds)RNA mimic polyinosinic:polycytidylic acid (poly[I:C]), the activation of caspase-1 and secretion of IL-1ß were enhanced. Moreover, NLRP3 knockdown decreased this poly(I:C)-induced IL-1ß production. Finally, we found that high-mobility group box 1 (HMGB1) translocated from the nucleus to the cytosol and was secreted into the extracellular space by inflammasome activation. Taken together, these findings suggest that ORS cells are important immunocompetent cells that induce NLRP3 inflammasomes. In addition, dsRNA-induced IL-1ß and HMGB1 secretion from ORS cells may contribute to clarifying the pathogenesis and therapeutic targets of AA.

Hyaluronic Acid Decreases Lipid Synthesis in Sebaceous Glands.

Hyaluronic acid (HA) is the major glycosaminoglycan in the extracellular matrix and has been implicated in several functions in skin cells. However, evidence is lacking regarding the HA signaling in sebaceous glands, and its potential role needs to be clarified. We investigated the role of HA in lipid production in sebaceous glands in an experimental study of human sebocytes followed by a clinical study. We first examined the effects of HA on sebaceous glands in hamsters and intradermal injection of HA into hamster auricles decreased both the size of sebaceous glands and the level of lipid production. We demonstrated that human skin sebaceous glands in vivo and sebocytes in vitro express CD44 (HA binding receptor) and that HA downregulates lipid synthesis in a dose-dependent manner. To evaluate the clinical relevance of HA in human skin, 20 oily participants were included in a double-blind, placebo-controlled, split-face study, and the HA-treated side showed a significant decrease in sebum production. The results of this study indicate that HA plays a functional role in human sebaceous gland biology and HA signaling is an effective candidate in the management of disorders in which sebum production is increased.

Altered immune cell proportions in the radiodermatitis induced hairless mice-1 (HR-1).

Accidental radiation exposures or radiation therapy can cause internal and external damage including radiodermatitis. Even though radiodermatitis is one of the dose limiting factors in radiotherapy, the immunological nature of it is not yet been clearly understood. In this study, we have examined the alteration in immune cell population during the radiodermatitis process. A radiodermatitis model was established in HR-1 mice by locally exposing a posterior dorsal region to 10 Gy X-ray/day for 4 consecutive days. Collagen accumulation, redness, erythema, and dry desquamation of the skin were detected after X-irradiation. The size and total cell number of the spleen decreased immediately after X-irradiation, compared to those of the sham-irradiated mice, and recovered to the normal levels two weeks later. Reduction and recovery of the bone marrow cell population preceded a similar change of the spleen cell population. The proportion of CD4+ T cell increased, while the proportion of CD8+ T cell decreased ahead of the obvious skin damage, in both lymph node and spleen of the irradiated mice. Interestingly, the proportion of splenic monocytes/macrophages was expanded gradually at a similar kinetics with the aggravation of the radiodermatitis. The infiltration of the CD11b+ monocyte/macrophage to the X-irradiated skin also coincided with the development of radiodermatitis. These altered proportions of immune cells may play important roles in radiodermatitis.

Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues.

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

The expression pattern and functional role of REIC/Dkk-3 in the development of cutaneous squamous cell carcinoma.

BACKGROUND: The exact physiological function of REIC/Dkk-3 in the development of squamous cell carcinoma(SCC) remains unclear. OBJECTIVE: We aimed to investigate the expression pattern and functional role of REIC/Dkk-3 in the development of SCC. METHODS: We stained normal skin, actinic keratosis (AK) and SCC tissue with REIC/Dkk-3. The proliferation and migration of SCC 12 over-expressed with REIC/Dkk-3 were observed. For in vivo study, SCC12 cells in PBS, SCC12 cells containing LacZ, and REIC/Dkk-3-transduced SCC 12 cells were injected intra-dermally into the left and right backside flanks of SCID mice respectively, and tumor growth was evaluated. RESULTS: REIC/Dkk-3 staining was detected throughout the full epidermis in normal skin, focally positive in AK. Negative or very low stain of REIC/Dkk-3 was observed in SCC in situ, keratoacanthoma, and SCC. REIC/Dkk-3 mRNA level in SCC was very low compared with that in normal skin tissue. REIC/Dkk-3 significantly decreased the proliferation and migration of SCC12 cells comparing with control (p<0.05). Cyclin D1 and CDK4/6 expression was slightly lower and p21 was very higher in REIC/Dkk-3-overexpressed group than in the LacZ group. Fewer ITGA6 cells were found in the REIC/Dkk-3 overexpressed group than in the LacZ control (p<0.01). Mean tumor volume was smallest in the REIC/Dkk-3 overexpressed group (p<0.01) 21days after the intradermal injection of SCC12 cells. CONCLUSION: REIC/Dkk-3 could be involved early in SCC development and have inhibitory effect on the development of SCC.