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.

CCL27 is downregulated by interferon gamma via epidermal growth factor receptor in normal human epidermal keratinocytes.

The cutaneous T cell-attracting chemokine (CTACK)/CCL27 is indispensable in skin inflammation. CTACK/CCL27 is exclusively produced by epidermal keratinocytes to attract CCR10-expressing T lymphocytes to the skin. We investigated the mechanism of CTACK/CCL27 production from normal human epidermal keratinocytes (NHEKs) by the proinflammatory cytokines TNFα and IFNγ. CTACK/CCL27 production was induced by TNFα via ERK, JNK, p38, and NFκB. The induction of CTACK/CCL27 by TNFα was suppressed by IFNγ via a pathway dependent on JAK, STAT1, and STAT3. Our results also demonstrated that IFNγ and TNFα induced the phosphorylation of EGFR and the following phosphorylation of ERK, which is partly responsible for the suppressive effect of IFNγ on TNFα-induced production of CTACK/CCL27. Peri-lesional skin of psoriasis demonstrates early inflammatory changes as we have previously reported. CTACK/CCL27 expression was diffuse in the peri-lesional epidermis, while it was restricted to basal layer in lesional epidermis, suggesting that CTACK/CCL27 expression was induced in the early stage of psoriatic plaque formation, and IFNγ could participate in the suppression of CTACK/CCL27 expression in the lesional epidermis, reflecting the later stage of psoriatic plaque formation. Our study suggests that CTACK/CCL27 may have a pivotal role in the early stage of psoriasis plaque formation, but should be downregulated in the later stage to induce inflammation characteristic for chronic psoriasis plaques.

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.

Desmosomes and disease: pemphigus and bullous impetigo.

Desmosomal cadherins are the pathophysiologic targets of autoimmune or toxin-mediated disruption in the human diseases pemphigus and bullous impetigo (including its generalized form, called staphylococcal scalded skin syndrome). Experiments exploiting the production of both pathogenic and nonpathogenic antidesmoglein antibodies in pemphigus patients' sera have afforded data that make an invaluable contribution towards identifying the functional domains of the desmogleins involved in intercellular adhesion. Conformational epitopes of antidesmoglein autoantibodies in pemphigus patients' sera and the specific cleavage site of desmoglein 1 by exfoliative toxin have been identified, implicating the N-terminal extracellular domains of the desmogleins as critical regions for controlling intercellular adhesion. Furthermore, the development of active autoimmune mouse models for pemphigus allows in vivo characterization of the disease and its pathogenesis. These studies offer new insight into the potential mechanisms of acantholysis in pemphigus and staphylococcal-associated blistering disease, with implications for the role of desmogleins in desmosomal structure and function.

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.

Ramified microglial cells promote astrogliogenesis and maintenance of neural stem cells through activation of Stat3 function.

The differentiation and proliferation of neural stem cells (NSCs) are regulated by a combination of their intrinsic properties (e.g., transcription factors, epigenetic factors, and microRNA regulation) and cell-extrinsic properties from the microenvironment around NSC (e.g., cytokines, growth factors, and cell-cell contact). Recently, there has been a great interest in clarifying the mechanism of the influence of the microenvironment on NSCs, especially cell-cell contact between NSCs and other types of cells nearby. In this study, we investigated whether microglial (Mi) cells influence the fate of NSCs. Coculture study showed that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs through their paracrine effects. This microglia-induced astrogliogenesis was inhibited by AG490 and by overexpression of the dominant-negative form of Stat3 and SOCS3. Promoter assay revealed transactivation of Stat3 function in NSCs by Mi cells. Gene expression study revealed that mRNA of Notch family members (notch1-3) and sox9 in NSCs was significantly upregulated by Mi cells, and this up-regulation was inhibited by AG490. These results demonstrated that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs by activating Stat3 function and via notch and sox9 signaling pathways.

Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.

Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.

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.

Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome.

Bullous impetigo due to Staphylococcus aureus is one of the most common bacterial infections of man, and its generalized form, staphylococcal scalded skin syndrome (SSSS), is a frequent manifestation of staphylococcal epidemics in neonatal nurseries. Both diseases are mediated by exfoliative toxins (ETs), which show exquisite pathologic specificity in blistering only the superficial epidermis. We show that these toxins act as serine proteases with extremely focused molecular specificity to cleave mouse and human desmoglein 1 (Dsg1) once after glutamic acid residue 381 between extracellular domains 3 and 4. Mutation of the predicted catalytically active serine to alanine completely inhibits cleavage. The mutated ETs bind specifically to Dsg1 by immunofluorescence colocalization and by coimmunoprecipitation. Thus, ETs, through specific recognition and proteolytic cleavage of one structurally critical peptide bond in an adhesion molecule, cause its dysfunction and allow S. aureus to spread under the stratum corneum, the main barrier of the skin, explaining how, although they circulate through the entire body in SSSS, they cause pathology only in the superficial epidermis.

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.

SOCS1-negative feedback of STAT1 activation is a key pathway in the dsRNA-induced innate immune response of human keratinocytes.

Toll-like receptor (TLR)3 is a receptor for virus-associated double-stranded RNA, and triggers antiviral immune responses during viral infection. Epidermal keratinocytes express TLR3 and provide an innate immune defense against viral infection. Since the intracellular regulatory mechanism is unknown, we hypothesized that the signal transducers and activators of transcription (STAT)-suppressors of cytokine signaling (SOCS) system regulates the innate immune response of keratinocytes. Treatment with polyinosinic-polycytidylic acid (poly(I:C)) resulted in the rapid translocation of IFN regulatory factor (IRF)-3 into the nucleus, followed by phosphorylation of STAT1 and STAT3. The activation of STATs by poly(I:C) probably occurs in an indirect fashion, through poly(I:C)-induced IFN. We infected cells with the dominant-negative forms of STAT1 (STAT1F), STAT3 (STAT3F), and SOCS1 using adenovirus vectors. Infection with STAT1F suppressed the induction of macrophage inflammatory protein (MIP)-1alpha by poly(I:C), whereas STAT3F had a minimal effect, which indicates that STAT1 mediates MIP-1alpha induction. SOCS1, which is a negative feedback regulator of STAT1 signaling, was induced by treatment with poly(I:C). SOCS1 infection inhibited the phosphorylation of STAT1 and significantly reduced poly(I:C)-induced MIP-1alpha production. Furthermore, STAT1-SOCS1 regulated poly(I:C)-induced TLR3 and IRF-7 expression. However, SOCS1 did not affect NF-kappaB signaling. Thus, the STAT1-SOCS1 pathway regulates the innate immune response via TLR3 signaling in epidermal keratinocytes.

Bone morphogenetic protein-2 modulates Wnt and frizzled expression and enhances the canonical pathway of Wnt signaling in normal keratinocytes.

BACKGROUND: Bone morphogenetic protein-2 (BMP-2) and Wnt are involved in the normal development and tumorigenesis of several organs, and in the development of skin and skin appendages as a morphogen. However, the crosstalk between BMP-2 and the Wnt/beta-catenin signaling pathway is not clear. OBJECTIVE: We examined BMP-2-dependent expression of Wnt and its receptor frizzled in normal human keratinocytes. METHODS: The mRNA expression of the Wnt and frizzled families was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or ribonuclease protection assay. beta-Catenin expression was measured using RT-PCR and Western blotting. T-cell factor/lymphoid enhancing factor activity was analyzed using the luciferase reporter assay. RESULTS: We detected the expression of Wnt-2b/13, -4, -5a, -5b, -7a, -7b, and -10a, frizzled-1, -4, -5, -6, -8, -9, and -10, MFRP, and SFRP-1/SARP-2 in keratinocytes. BMP-2 increased Wnt-2b/13, -5b, and -7b, and frizzled-6, -8, and -10. Conversely, BMP-2 suppressed Wnt-10a and SFRP-1/SARP-2. Although Wnt-4 expression was not affected by BMP-2 in confluent conditioned keratinocytes, BMP-2 suppressed cell density-dependent Wnt-4 induction. The transcriptional activity of TCF/LEF, which is a target of the canonical Wnt pathway, was upregulated by BMP-2 in both time- and dose-dependent manners. However, BMP-2-dependent differentiation of keratinocytes suppressed TCF/LEF transcriptional activity. CONCLUSION: These results suggest that BMP-2 modulates the expression of molecules involved in Wnt signaling, and activates the canonical Wnt pathway in normal human keratinocytes. Moreover, Wnt signaling may be influenced by the fate of keratinocytes, such as proliferation, migration, and differentiation.

Paraneoplastic pemphigus associated with fatal bronchiolitis obliterans and intractable mucosal erosions: Treatment with cyclosporin in addition to steroid, rituximab and intravenous immunoglobulin.

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that presents as severe mucosal erosions and variable cutaneous lesions and is primarily associated with hematologically malignant or benign diseases. A 59-year-old Japanese woman presented with oral, ocular and vaginal mucosal erosions and erythema as well as blistering on her trunk and limbs. She developed bronchiolitis obliterans; lymphadenopathy in the cervical, subclavian, para-aortic and intraperitoneal regions; and splenomegaly. PNP with B-cell lymphoma was diagnosed. She was treated with two courses of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) for B-cell lymphoma, rituximab once every 3 weeks for five cycles, steroid pulse therapy, oral prednisolone, cyclosporin and high-dose i.v. immunoglobulin. The B-cell lymphoma was in remission after two courses of R-CHOP treatment. Although her skin erythema and blistering were also improved, the mucosal erosions and bronchiolitis obliterans gradually worsened. The patient died of bronchiolitis obliterans after 6 months of hospitalization. Because a cellular immune response is thought to be involved in the pathogenesis of PNP, cyclosporin therapy is expected to aid in suppressing the cellular response. In this case, however, the patient's mucosal lesions and bronchiolitis obliterans were not improved by regular administration of cyclosporin therapy.

Mechanism of thymus- and activation-regulated chemokine (TARC)/CCL17 production and its modulation by roxithromycin.

Stimulation with tumor necrosis factor (TNF)alpha and interferon (IFN)gamma synergistically induced thymus- and activation-regulated chemokine (TARC)/CCL17 production from HaCaT keratinocytes (KC). Inhibitors for nuclear factor kappa B (NFkappaB), parthenolide, and Bay 11-7085, and an inhibitor of p38, SB202190, inhibited TNFalpha- and IFNgamma-induced production of CCL17 by HaCaT KC. Surprisingly, an inhibitor of epidermal growth factor receptor tyrosine kinase, PD153035, enhanced the production of CCL17 in HaCaT KC. Roxithromycin (RXM), a 14-membered ring macrolide, suppressed CCL17 production by HaCaT KC induced by IFNgamma and TNFalpha. RXM partially suppressed p38 phosphorylation and NFkappaB-driven luciferase activity induced by TNFalpha and IFNgamma. Degradation of inhibitor of nuclear factor kappa B (IkappaB) alpha upon stimulation with IFNgamma and TNFalpha was not affected by the addition of RXM. Through elucidating the mechanism of CCL17 production, our study indicates that RXM suppresses the production through the inhibition of p38 and NFkappaB, independent of the inhibition of IkappaB degradation.

Microbubble-enhanced ultrasound for gene transfer into living skin equivalents.

BACKGROUND: Gene transfer to skin is an attractive therapeutic approach because of the accessibility of the skin and the high rate of cure for many cutaneous diseases. However, safety concerns over viral vectors and the low efficiency of most non-viral gene transfer techniques have encumbered their clinical application for gene transfer. By contrast, efficient gene transfers into various cell types using microbubble-enhanced ultrasound has been reported. OBJECTIVES: The purpose of this study was to investigate whether ultrasound with microbubble enhancement allowed effective transfer of foreign genes into living skin equivalents (LSEs). METHODS: Microbubbles and plasmid DNA encoding green fluorescent protein (GFP) were added to the dermal-epidermal junctions of LSEs, which were then exposed to ultrasound. The LSEs were harvested at different time points to investigate transgene expression using confocal laser microscopy. Transfected LSEs were also transplanted onto nude mice, and the in vivo transgene expression was observed. RESULTS: From days 2 to 7 after transfection, most GFP-positive cells continued to migrate upward from the basal layer, while other GFP-positive cells lagged behind or remained in the basal layer on days 5 and 7. Transfection resulted in 20-30% GFP-positive cells. Multiple transfections further increased the percentage of transfected cells and resulted in multi-layer transgene expression. Grafts from the transfected LSEs survived on nude mice and continued to express GFP up to 2 weeks post-transplantation. CONCLUSION: Gene transfer into LSE using ultrasound with microbubble enhancement is an effective alternative to viral and non-viral methods.

Inhibition of STAT3 prevents neointima formation by inhibiting proliferation and promoting apoptosis of neointimal smooth muscle cells.

In cultured vascular smooth muscle cells (SMCs), STAT3 mediates proliferation signal by directly activating transcription of early growth response genes. Recently, we have found that balloon injury transiently induces JAK2 and STAT3 expressions and activations with a peak at day 7 in rat carotid artery. However, the specific role of STAT3 in neointima formation remains unknown. Adenoviral vector encoding a dominant negative STAT3 (AxCAdnSTAT3) or beta-galactosidase (control) was overexpressed in a balloon-injured artery to inhibit endogenous STAT3 activation selectively. In controls, neointima became evident after day 4, and reached a maximum at day 14. The number of bromodeoxyuridine (BrdU)-positive proliferating or TUNEL-positive apoptotic neointimal SMCs peaked at day 7, decreasing to lower levels by day 14. AxCAdnSTAT3 not only abrogated STAT3 phosphorylation but also decreased BrdU labeling index by 60% and increased TUNEL index by 35% at day 7 versus controls, resulting in the 40% reduction in the intima/media area ratio at day 14. At day 7, in controls, vascular injury upregulated antiapoptotic mediator Mcl-i and Bcl-xL expression by 8-fold to 5-fold, respectively, versus sham, whereas proapoptotic Bax slightly increased by 1.5-fold versus sham. AxCAdnSTAT3 reversed the upregulated Mcl-1 and Bcl-xL levels by 70% and 37%,respectively, while having no affect on Bax expression. In conclusion, the STAT3-mediated pathway plays an important role in neointima formation through enhanced vascular SMC accumulation by promoting cell proliferation and survival.

Expression of desmoglein 1 compensates for genetic loss of desmoglein 3 in keratinocyte adhesion.

The desmoglein compensation hypothesis, namely that one desmoglein can compensate for loss of function of another, has been proposed to explain the tissue specificity of the autoantibody-induced loss of cell adhesion in pemphigus. To validate this hypothesis genetically, we used desmoglein-3 knockout mice (DSG3-/-) that lose their telogen hair prematurely due to loss of adhesion between keratinocytes of the telogen hair club and the outer root sheath, where the only desmoglein expressed in normal mice is desmoglein-3. To determine if desmoglein-1 could substitute for the function of desmoglein-3 in telogen hair, we produced transgenic mice that express desmoglein-1 driven off the keratin 14 promoter, and then bred the transgene (TG) into DSG3-/- mice. Immunoblotting showed transgene expression in skin, and immunofluorescence showed desmoglein-1 in the telogen club of DSG3-/-TG+ but not DSG3-/-TG- mice. DSG3-/-TG- mice lost telogen hair with each wave of telogen, whereas DSG3-/-TG+ mice had markedly delayed and decreased hair loss. DSG3-/- mice also show low weights due to blisters in the oral mucosa. Surprisingly, DSG3-/-TG+ mice showed similar low weights, because the transgene, although expressed in skin, was not well expressed in oral mucous membranes. These studies show that desmoglein-1 can compensate for loss of desmoglein-3-mediated adhesion, and provide genetic evidence confirming the desmoglein compensation hypothesis.