Bcl-3 induced by IL-22 via STAT3 activation acts as a potentiator of psoriasis-related gene expression in epidermal keratinocytes.

IL-22 induces STAT3 phosphorylation and mediates psoriasis-related gene expression. However, the signaling mechanism leading from pSTAT3 to the expression of these genes remains unclear. We focused on Bcl-3, which is induced by STAT3 activation and mediates gene expression. In cultured human epidermal keratinocytes, IL-22 increased Bcl-3, which was translocated to the nucleus with p50 via STAT3 activation. The increases in CXCL8, S100As and human ß-defensin 2 mRNA expression caused by IL-22 were abolished by siRNA against Bcl-3. Although CCL20 expression was also augmented by IL-22, the knockdown of Bcl-3 increased its level. Moreover, the combination of IL-22 and IL-17A enhanced Bcl-3 production, IL-22-induced gene expression, and the expression of other psoriasis-related genes, including those encoding IL-17C, IL-19, and IL-36γ. The expression of these genes (except for CCL20) was also suppressed by the knockdown of Bcl-3. Bcl-3 overexpression induced CXCL8 and HBD2 expression but not S100As expression. We also compared Bcl-3 expression between psoriatic skin lesions and normal skin. Immunostaining revealed strong signals for Bcl-3 and p50 in the nucleus of epidermal keratinocytes from psoriatic skin. The IL-22-STAT3-Bcl-3 pathway may be important in the pathogenesis of psoriasis.

Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A).

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-ß1, TGF-ß2, and TGF-ß3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-ß family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell interactions, and the results suggest that stromal cells and epithelial cells may act in concert in the cornea.

Human antigen R-mediated mRNA stabilization is required for ultraviolet B-induced autoinduction of amphiregulin in keratinocytes.

All members of the EGF family are produced as transmembrane precursors that are proteolytically processed into soluble forms by disintegrin and metalloproteinases (ADAMs) for autocrine/paracrine pathways. In turn, the ligand-activated EGF receptor (EGFR) induces the expression of EGF family members, so-called "autoinduction." However, it is not well understood how this autoinduction occurs. In this study, we investigated the molecular mechanism of the autoinduction of amphiregulin (AREG), a member of the EGF family. We found that ultraviolet B (UVB) exposure increased the AREG mRNA level by stabilization of its mRNA in a human immortalized keratinocyte cell line, HaCaT. The 3' UTR of AREG mRNA was responsible for binding to an mRNA-binding protein, human antigen R (HuR), and the interaction between AREG mRNA and HuR was enhanced by UVB. Inducible knockdown of HuR expression significantly decreased AREG mRNA stability. Interestingly, treatment of HaCaT cells with an EGFR inhibitor, an EGFR neutralizing antibody, or an ADAM inhibitor destabilized AREG mRNA. In the case of ADAM inhibition, administration of soluble AREG restored the mRNA level, indicating that the stabilization occurs in a shedding-dependent manner of EGFR ligands. The HuR dependence of AREG mRNA and protein expression was also confirmed in human primary keratinocytes. Taken together, we propose a novel mechanism by which HuR regulates the stability of AREG mRNA in keratinocytes after UVB exposure and suggest that targeting of HuR functions might be crucial for understanding skin cancers caused by aberrant EGF family member-EGFR signaling.

Second harmonic generation reveals collagen fibril remodeling in fibroblast-populated collagen gels.

Remodeling of collagen fibrils is involved in a variety of physiological and pathological processes including development, tissue repair, and metastasis. Fibroblast-populated collagen gel contraction has been employed as a model system to investigate the collagen fibril remodeling within three-dimensional collagen matrices. Research on collagen gel contraction is also important for understanding the mechanism underlying connective tissue repair, and for design considerations for engineered tissues in regenerative medicine. Second harmonic generation (SHG) is a non-linier optical effect by which well-ordered protein assemblies, including collagen fibrils, can be visualized without any labeling, and used for a noninvasive imaging of collagen fibrils in the skin. Here we demonstrate that the remodeling of collagen fibrils in the fibroblast-populated collagen gel can be analyzed by SHG imaging with a multiphoton microscope. Two models of collagen gel contraction (freely versus restrained contraction) were prepared, and orientation of fibroblasts, density, diameter, and distribution of collagen fibrils were examined by multiphoton fluorescent and SHG microscopy. Three-dimensional construction images revealed vertical and horizontal orientation of fibroblasts in freely and restrained gel contraction, respectively. Quantitative analysis indicated that collagen fibrils were accumulated within the gel and assembled into the thicker bundles in freely but not restrained collagen gel contraction. We also found that actomyosin contractility was involved in collagen fibril remodeling. This study elucidates how collagen fibrils are remodeled by fibroblasts in collagen gel contraction, and also proves that SHG microscopy can be used for the investigation of the fibroblast-populated collagen gel.

Genome-wide association study identifies HLA-A*3101 allele as a genetic risk factor for carbamazepine-induced cutaneous adverse drug reactions in Japanese population.

An anticonvulsant, carbamazepine (CBZ), is known to show incidences of cutaneous adverse drug reactions (cADRs) including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) and drug-induced hypersensitivity syndrome (DIHS). To identify a gene(s) susceptible to CBZ-induced cADRs, we conducted a genome-wide association study (GWAS) in 53 subjects with the CBZ-induced cADRs, including SJS, TEN and DIHS, and 882 subjects of a general population in Japan. Among the single nucleotide polymorphisms (SNPs) analyzed in the GWAS, 12 SNPs showed significant association with CBZ-induced cADRs, and rs1633021 showed the smallest P-value for association with CBZ-induced cADRs (P = 1.18 × 10⁻¹³). These SNPs were located within a 430 kb linkage disequilibrium block on chromosome 6p21.33, including the HLA-A locus. Thus, we genotyped the individual HLA-A alleles in 61 cases and 376 patients who showed no cADRs by administration of CBZ (CBZ-tolerant controls) and found that HLA-A*3101 was present in 60.7% (37/61) of the patients with CBZ-induced cADRs, but in only 12.5% (47/376) of the CBZ-tolerant controls (odds ratio = 10.8, 95% confidence interval 5.9-19.6, P = 3.64 × 10⁻¹5), implying that this allele has the 60.7% sensitivity and 87.5% specificity when we apply HLA-A*3101 as a risk predictor for CBZ-induced cADRs. Although DIHS is clinically distinguished from SJS and TEN, our data presented here have indicated that they share a common genetic factor as well as a common pathophysiological mechanism. Our findings should provide useful information for making a decision of individualized medication of anticonvulsants.

Role of the aryl hydrocarbon receptor in tobacco smoke extract-induced matrix metalloproteinase-1 expression.

Findings from large epidemiologic studies indicate that there is a link between smoking and extrinsic skin ageing. We previously reported that matrix metalloproteinases (MMPs) mediate connective tissue damage in skin exposed to tobacco smoke extracts. Tobacco smoke contains more than 3800 constituents, including numerous water-insoluble polycyclic aromatic hydrocarbons (PAHs) that trigger aryl hydrocarbon receptor (AhR) signalling pathways. To analyse the molecular mechanisms involved in tobacco smoke-induced skin ageing, we exposed primary human fibroblasts and keratinocytes to tobacco smoke extracts. Hexane- and water-soluble tobacco smoke extracts significantly induced MMP-1 mRNA in both human cultured fibroblasts and keratinocytes in a dose-dependent manner. To clarify the involvement of the AhR pathway, we used a stable AhR-knockdown HaCaT cell line. AhR knockdown abolished the increased transcription of the AhR-dependent genes CYP1A1/CYP1B1 and MMP-1 induced by either of the tobacco smoke extracts. Furthermore, the tobacco smoke extracts induced 7-ethoxyresorufin-O-deethylase activity, which was almost completely abolished by AhR knockdown. Likewise, treating fibroblasts with AhR pathway inhibitors, that is, the flavonoids 3-methoxy-4-nitroflavone and α-naphthoflavone, blocked the expression of CYP1B1 and MMP-1. These findings suggest that the tobacco smoke extracts induce MMP-1 expression in human fibroblasts and keratinocytes via activation of the AhR pathway. Thus, the AhR pathway may be pathogenetically involved in extrinsic skin ageing.

Hair-inducing ability of human dermal papilla cells cultured under Wnt/ß-catenin signalling activation.

It is well known that dermal papilla cells (DPCs) play crucial roles in hair follicle induction. In this study, we examined whether Wnt/ß-catenin activation results in maintenance of the hair-inducing ability of human DPCs. Expression of DPC marker genes was maintained under Wnt/ß-catenin signalling stimulation by GSK-3ß inhibition. Furthermore, human DPCs showed constant hair induction when transplanted with murine epidermal cell fraction. Alu-positive human DPCs were essentially detected adjacent to the reconstructing epidermal structure positive for P-cadherin immunoreactivity. The transplanted human DPCs were abundant in the surrounding dermal sheath portion of the fully regenerated hair follicles. These results support the importance of Wnt/ß-catenin signalling in hair follicle induction. This study may provide valuable information to establish a culture method of human DPCs for cell-based therapy.

Mite allergen is a danger signal for the skin via activation of inflammasome in keratinocytes.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disorder caused by multiple factors. Among them, house dust mite (HDM) allergens are important in the development of AD. In airway allergy, HDM allergens activate innate immunity. However, information regarding the activation of innate immunity by HDM allergens in the skin is limited. OBJECTIVES: The inflammasome is a key regulator of pathogen recognition and inflammation. We investigated whether HDM allergens activate the inflammasome in epidermal keratinocytes. METHODS: Keratinocytes were stimulated with Dermatophagoides pteronyssinus, and the activation of caspase-1 and secretion of IL-1ß and IL-18 were examined. Formation of the inflammasome was studied by analyzing the subcellular distributions of inflammasome proteins. The importance of specific inflammasome proteins was studied by knocking down their expression through transfection of keratinocytes with lentiviral particles carrying short hairpin RNAs (shRNAs). RESULTS: D pteronyssinus activated caspase-1 and induced caspase-1-dependent release of IL-1ß and IL-18 from keratinocytes. Moreover, D pteronyssinus stimulated assembly of the inflammasome by recruiting apoptosis-associated specklike protein containing a caspase-recruitment domain (ASC), caspase-1, and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin-domain containing 3 (NLRP3) to the perinuclear region. Finally, infection with lentiviral particles carrying ASC, caspase-1, or NLRP3 shRNAs suppressed the release of IL-1ß and IL-18 from the keratinocytes. Activation of the NLRP3 inflammasome by D pteronyssinus was dependent on cysteine protease activity. CONCLUSION: House dust mite allergens are danger signals for the skin. In addition, HDM-induced activation of the NLRP3 inflammasome may play a pivotal role in the pathogenesis of AD.

E2 Polyubiquitin-conjugating enzyme Ubc13 in keratinocytes is essential for epidermal integrity.

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13(flox/flox)K5-Cre). At birth, the skin of the Ubc13(flox/flox)K5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13(flox/flox)K5-Cre mouse epidermis. In culture, Ubc13(flox/flox)K5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13(flox/flox) keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13(flox/flox)K5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.

Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.

IL-17 and IL-22 mediate IL-20 subfamily cytokine production in cultured keratinocytes via increased IL-22 receptor expression.

IL-20 cytokine subfamily members, including IL-19, IL-20, and IL-24, are highly expressed in psoriatic skin lesions. Here, we demonstrate that psoriasis mediators IL-17 and IL-22 synergistically induce the production of IL-20 subfamily proteins in cultured human keratinocytes. Interestingly, expression of the IL-22 receptor (IL-22R) also increased in epidermal lesions versus normal skin. IL-22R over-expression using an adenoviral vector to mimic psoriatic conditions in cultured keratinocytes significantly enhanced IL-17- and IL-22-induced production of IL-20 subfamily cytokines. Furthermore, IL-17 and IL-22 coordinately enhanced MIP-3alpha, IL-8, and heparin-binding EGF-like growth factor (HB-EGF) production, depending on the amount of IL-22R expression. Additionally, because IL-20 and IL-24 share the IL-22R with IL-22, the function of IL-20 and IL-24 was also increased. IL-20 and IL-24 have effects similar to that of IL-22; IL-24 showed more potent expression than IL-20. A combination of IL-24 and IL-17 increased the production of MIP-3alpha, IL-8, and HB-EGF, as did a combination of IL-22 and IL-17. These data indicate that increased IL-22R expression in epidermal keratinocytes contributes to the pathogenesis of psoriasis through enhancing the coordinated effects of IL-22 and IL-17, inducing the production of the IL-20 subfamily, chemokines, and growth factors.

Nodal lymphangiogenesis and metastasis: Role of tumor-induced lymphatic vessel activation in extramammary Paget's disease.

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget's disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

Alteration of TLR3 pathways by glucocorticoids may be responsible for immunosusceptibility of human corneal epithelial cells to viral infections.

PURPOSE: The toll-like receptor 3 (TLR3) recognizes viral double-stranded RNA and its synthetic analog polyriboinosinic-polyribocytidylic acid (poly(I:C)), and the activation of TLR3 is known to induce the production of type I interferon (IFN) and inflammatory cytokines/chemokines. The purpose of this study was to determine the role played by innate responses to a herpes simplex virus 1 (HSV-1) infection of the corneal epithelial cells. In addition, we determined the effects of immunosuppressive drugs on the innate responses. METHODS: Cultured human corneal epithelial cells (HCECs) were exposed to poly(I:C), and the expressions of the mRNAs of the cytokines/chemokines macrophage-inflammatory protein 1 alpha (MIP1-alpha), macrophage-inflammatory protein 1 beta (MIP1-beta), interleukin-6 (IL-6), interleukin-8 (IL-8), regulated on activation, normal T cell expressed and secreted (RANTES), Interferon-beta (IFN-beta), and TLR3 were determined using real-time reverse transcription-polymerase chain reaction (RT-PCR). The effects of dexamethasone (DEX, 10(-6) or 10(-5) M) and cyclosporine A (CsA, 10(-6) or 10(-5) M) on the expression of these cytokines and TLR3 were also determined using real-time RT-PCR. Levels of MIP1-alpha, MIP1-beta, IL-6, IL-8, RANTES, and IFN-beta were measured using the enzyme-linked immunosorbent assay (ELISA). The activation of nuclear factor kappa B (NFkappaB) and interferon regulatory factor 3 (IRF3) in HCECs was assessed by immunohistochemical staining. The effects of DEX and CsA on HCECs exposed to HSV-1 (McKrae strain) were also examined. RESULTS: The expressions of MIP1-alpha, MIP1-beta, IL-6, IL-8, RANTES, IFN-beta, and TLR3 were up-regulated in HCECs exposed to poly(I:C). The poly(I:C)-induced expressions of IL-6 and IL-8 were down-regulated by both DEX and CsA, while the expressions of IFN-beta and TLR3 were suppressed by DEX alone. Similarly, the poly(I:C)-induced activation of NFkappaB was decreased by both DEX and CsA, and the activation of IRF3 was reduced by DEX alone. When HCECs were inoculated with HSV-1, DEX led to a decrease in the expression of IL6, IFN-beta, and TLR3, and an extension of plaque formation. CONCLUSION: These results indicate that DEX may increase the susceptibility of HCECs to viral infections by altering the TLR3 signaling pathways.

Diagnosis and treatment of Stevens-Johnson syndrome and toxic epidermal necrolysis with ocular complications.

PURPOSE: To present a detailed clarification of the symptoms at disease onset of Stevens-Johnson syndrome (SJS) and its more severe variant, toxic epidermal necrolysis (TEN), with ocular complications and to clarify the relationship between topical steroid use and visual prognosis. DESIGN: Cross-sectional study. PARTICIPANTS: Ninety-four patients with SJS and TEN with ocular complications. METHODS: A structured interview, examination of the patient medical records, or both addressing clinical manifestations at disease onset were conducted for 94 patients seen at Kyoto Prefectural University of Medicine. Any topical steroid use during the first week at the acute stage also was investigated. MAIN OUTCOME MEASURES: The incidence and the details of prodromal symptoms and the mucosal involvements and the relationship between topical steroid use and visual outcomes. RESULTS: Common cold-like symptoms (general malaise, fever, sore throat, etc.) preceded skin eruptions in 75 cases, and extremely high fever accompanied disease onset in 86 cases. Acute conjunctivitis and oral and nail involvements were reported in all patients who remembered the details. Acute conjunctivitis occurred before the skin eruptions in 42 patients and simultaneously in 21 patients, whereas only 1 patient reported posteruption conjunctivitis. Visual outcomes were significantly better in the group receiving topical steroids compared with those of the no-treatment group (P<0.00001). CONCLUSIONS: Acute conjunctivitis occurring before or simultaneously with skin eruptions accompanied by extremely high fever and oral and nail involvement indicate the initiation of SJS or TEN. Topical steroid treatment from disease onset seems to be important for the improvement of visual prognosis.

The NF-kappaB, p38 MAPK and STAT1 pathways differentially regulate the dsRNA-mediated innate immune responses of epidermal keratinocytes.

The epidermis is the primary boundary between the body and the environment, and it serves as the first line of defense against microbial pathogens. Production of chemokines and cytokines is an important step in the initiation of innate immune responses to viral infections. Epidermal keratinocytes produce IFN-alpha, -beta and macrophage inflammatory protein (MIP)-1alpha in response to double-stranded RNA (dsRNA) or viral infections. We showed that human keratinocytes produced cytokines [tumor necrosis factor (TNF)-alpha, IL-1beta and IL-15] and chemokines [MIP-1beta, RANTES and liver and activation-regulated chemokine (LARC)] in response to dsRNA, with activation of the nuclear factor kappaB (NF-kappaB), p38 mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription 1 (STAT1) pathways. To study the roles of these pathways in their production, we transfected keratinocytes with adenoviral vectors (Ax) carrying a dominant-negative form of inhibitor kappaB alpha (IkappaBalpha) (IkappaBalphaM), a dominant-negative mutant form of STAT1 (STAT1F) or suppressors of cytokine signaling 1 (SOCS1). Transfection with AxIkappaBalphaM or addition of a p38 inhibitor (SB203580) significantly decreased the dsRNA-mediated production of TNF-alpha, IL-1beta and MIP-1alpha, but not of IFN-beta, IL-15, MIP-1beta, RANTES or LARC. Transfection with AxSTAT1F or AxSOCS1 inhibited the dsRNA-mediated production of TNF-alpha, IL-15, MIP-1alpha, MIP-1beta, RANTES and LARC, but not IFN-beta or IL-1beta. In conclusion, the NF-kappaB, p38 MAPK and STAT1 pathways differentially regulate dsRNA-mediated innate immune responses in epidermal keratinocytes.

An intermediary role of proHB-EGF shedding in growth factor-induced c-Myc gene expression.

Activation of growth factor receptors by ligand binding leads to an increased expression of c-Myc, a transcriptional regulator for cell proliferation. The activation of transcriptional factors via the activated receptors is thought to be the main role of c-Myc gene expression. We demonstrate here that epidermal growth factor receptor (EGFR)- and fibroblast growth factor receptor (FGFR)-mediated c-Myc induction and cell cycle progression in primary cultured mouse embryonic fibroblasts (MEFs) are abrogated by knockout of the heparin-binding EGF-like growth factor (Hb-egf) gene, or by a metalloproteinase inhibitor, although molecules downstream of the receptors are activated. Induction of c-Myc expression by EGF or basic FGF is recovered in Hb-egf-depleted MEFs by overexpression of wild-type proHB-EGF, but no recovery was observed with an uncleavable mutant of proHB-EGF. The uncleavable mutant also inhibited EGF-induced acetylation of histone H3 at the mouse c-Myc first intron region, which could negatively affect transcriptional activation. We conclude that signal transduction initiated by generation of the carboxyl-terminal fragment of proHB-EGF (HB-EGF-CTF) in the shedding event plays an important intermediary role between growth factor receptor activation and c-Myc gene induction.

PPAR gamma is an important transcription factor in 1 alpha,25-dihydroxyvitamin D3-induced involucrin expression.

BACKGROUND: 1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D, suppresses keratinocyte proliferation, promotes keratinocyte differentiation, and induces involucrin expression. Peroxisome proliferation-activated receptors (PPARs) are ligand-activated transcription factors. It has been reported that PPARs stimulate keratinocyte differentiation and regulate the expression of differentiation molecules. OBJECTIVE: Keratinocytes treated with 1 alpha,25(OH)2D3 induced PPAR gamma, which was followed by increased involucrin expression. In this study, we investigated whether PPAR gamma is involved in the 1 alpha,25(OH)2D3-induced involucrin expression in human keratinocytes. METHODS: Subconfluent keratinocytes were treated with 10(-7)M 1 alpha,25(OH)2D3 for the indicated times, and PPAR and involucrin mRNA expression were determined by real-time RT-PCR. The levels of PPARs, involucrin, p38, and phospho-p38 proteins were assayed by Western blotting, and the DNA binding activities of PPAR gamma and AP-1 were investigated by electrophoretic mobility shift assays (EMSA). To examine the role of PPAR gamma in 1 alpha,25(OH)2D3 responses, recombinant adenovirus carrying a dominant-negative form of PPAR gamma (Axdn-PPAR gamma) was constructed and transfected into keratinocytes. The p38 inhibitor SB203580 was added to the cultures to evaluate the involvement of p38 in involucrin expression. RESULTS: 1 alpha,25(OH)2D3 induced PPAR gamma expression and stimulated PPAR gamma activity. The introduction of dn-PPAR gamma inhibited the expression of involucrin mRNA and protein induced by 1 alpha,25(OH)2D3, and suppressed AP-1 DNA binding activity. 1 alpha,25(OH)2D3 also triggered the phosphorylation of p38, which contributes to involucrin induction. Moreover, dn-PPAR gamma prevented the 1 alpha,25(OH)2D3-induced phosphorylation of p38. CONCLUSIONS: These results suggest that PPAR gamma regulates involucrin expression by controlling the AP-1 signal and p38 activation in 1 alpha,25(OH)2D3-induced keratinocyte differentiation.

Human corneal epithelial cell proliferation by epiregulin and its cross-induction by other EGF family members.

PURPOSE: To investigate the effects of epiregulin, a newly identified member of the epidermal growth factor (EGF) family, on the proliferation of human corneal epithelial cells (HCECs). METHODS: The proliferation of HCECs was determined by cell counting and BrdU incorporation assays at specific times after exposure to different concentrations of human recombinant epiregulin (0 to 20 ng/ml). Immunohistochemical staining was used to localize epiregulin in cadaveric corneas. RT-PCR and real-time PCR were used to determine the expression levels of epiregulin in cultured and cadaveric HCECs. To examine the interaction between epiregulin and epidermal growth factor receptors (EGFRs), the phosphorylation of ErbB1 and ERK1/ERK2 (ERK1/2) was estimated by western blot analysis in the presence or absence of AG1478, a specific inhibitor of EGFR kinase activity. To search for cross-induction of epiregulin by other EGF family members, the expressions of EGF, heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR), and transforming growth factor-alpha (TGF-alpha) mRNA were determined by real-time PCR in the presence of 10 ng/ml of epiregulin. Conversely, the expression of epiregulin was also determined following the incubation of HCECs with 10 nM of either of EGF, HB-EGF, TGF-alpha, or AR. RESULTS: The mRNA of epiregulin was expressed in cultured HCECs and HCECs obtained from cadaveric eyes. Epiregulin was strongly detected in the limbal epithelium and basal epithelium of the peripheral cornea, but it was weakly detected in the central corneal epithelium. HCECs proliferated in the presence of epiregulin in a dose-dependent manner as detected by an increase in cell numbers or in BrdU incorporation. When HCECs were incubated with exogenous epiregulin, the expression of the mRNA of epiregulin was upregulated as detected by real-time PCR, and the phosphorylation of ErbB1 and ERK1/2 was upregulated in a dose-dependent manner as shown by western blot analysis. These upregulations were inhibited by AG1478, a specific inhibitor of EGFR kinase activity. Epiregulin increased the expression of HB-EGF and AR, while TGF-alpha, HB-EGF, AR, and EGF increased the expression of epiregulin in HCECs. CONCLUSIONS: These findings indicate that epiregulin played an autocrine role in the proliferation of HCECs presumably through cross-induction with other EGF family members.

Suppressor of cytokine signaling 3 negative regulation of signal transducer and activator of transcription 3 in platelet-derived growth factor-induced fibroblast migration.

Platelet-derived growth factor (PDGF) is involved in wound healing, but PDGF-induced fibroblast migration and the intracellular signaling mechanisms of fibroblast migration are poorly understood. Signal transducer and activator of transcription 3 (STAT3) is involved in migration and is negatively regulated by the suppressor of cytokine signaling 3 (SOCS3). We studied the PDGF induction of fibroblast migration in vitro and the involvement of STAT3 and SOCS3. We found that PDGF activated STAT3 and strongly induced fibroblast migration. Transfection with a dominant-negative mutant of STAT3 almost completely abolished PDGF-induced fibroblast migration and STAT3 phosphorylation. Next, we studied the mechanisms that regulate fibroblast migration. PDGF enhanced the expression of SOCS3 by 2.8-fold at 1 h. Transfection with SOCS3 almost completely abolished PDGF-induced STAT3 phosphorylation and reduced fibroblast migration to 47% of control, indicating that SOCS3 acts as a negative regulator of PDGF-induced fibroblast migration. In conclusion, PDGF induces fibroblast migration under the control of STAT3-SOCS3.