Targeted inhibition of YAP/TAZ alters the biological behaviours of keloid fibroblasts.

Abnormal activation of fibroblasts plays a crucial role in keloid development. However, the mechanism of fibroblast activation remains to be determined. YAP/TAZ are key molecules in the Hippo signalling pathway that promote cell proliferation and inhibit apoptosis. Here, we show that keloid fibroblasts have higher levels of YAP/TAZ mRNA and proteins on primary culture. Targeted knockdown of endogenous YAP or TAZ significantly inhibited cell proliferation, reduced cell migration, induced cell apoptosis and down-regulated collagen1a1 production by keloid fibroblasts. Moreover, we demonstrate that verteporfin, an inhibitor of YAP/TAZ, has similar but stronger inhibitory effects on fibroblasts compared to YAP/TAZ knockdown. Our study provides evidence that YAP/TAZ may be involved in the pathogenesis of keloids. Targeted inhibition of YAP/TAZ could change the biological behaviours of fibroblasts and can potentially be used as therapy for keloids.

Lipidomics profiling of skin surface lipids in senile pruritus.

BACKGROUND: Senile pruritus is common, yet its etiology remains unknown. Aging-associated skin barrier defects and skin surface lipid (SSL) alterations have been postulated to play important roles in its occurrence. In the present study, the lipidomic profiles of SSLs in elderly patients were examined to better understand the potential causes of senile pruritus. METHODS: Transepidermal water loss (TEWL) was evaluated to assess the skin barrier function. The Ameliorated Kawashima Itch Scale score was used to measure the pruritus severity. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and multivariate data analysis were employed to investigate SSL alterations. RESULTS: The results showed that senile pruritus patients had higher TEWL values than control subjects (13.13 ± 4.28 versus 6.71 ± 2.45, p < 0.01). LC-MS/MS revealed significant differences in the lipidomic profiles and identified 81 species of SSLs that differed between the two groups. Compared with control subjects, senile pruritus patients had increased levels of ceramides (Cers), diacylglycerols, fatty acids, phosphatidylcholines, phosphatidylethanolamines, phytosphingosines, sphingosines, diacylceryl-3-O-carboxyhydroxymethylcholine, diacylglyceryl trimethylhomoserine, and unsaturated free fatty acids, but decreased levels of triacylglycerol. Cer-EOS, Cer-NDS, and Cer-NS were positively correlated with TEWL value (p < 0.05). Pruritus severity score was positively correlated with sphingomyelin, Cer-NP, Cer-AS, Cer-NDS, and Cer-NS, but negatively correlated with Cer-BS, Cer-EODS, Cer-EOS, and Cer-AP. CONCLUSIONS: The present study indicated that patients with senile pruritus have impaired skin barrier function and altered SSL composition. Certain SSL species identified in this study may be potential targets for future studies on the pathogenesis of senile pruritus. TRIAL REGISTRATION: Peking University International Hospital (Number: YN2018QN04 ; date: January 2019).

Circ-FOXM1 contributes to cell proliferation, invasion, and glycolysis and represses apoptosis in melanoma by regulating miR-143-3p/FLOT2 axis.

BACKGROUND: Numerous literatures have demonstrated that circular RNAs (circRNAs) are involved in multiple types of tumors. However, the effects of circRNAs in melanoma are not very clear. In this study, we aimed to investigate the roles and mechanisms of circ-FOXM1 in melanoma. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of circ-FOXM1, microRNA-143-3p (miR-143-3p), and Flotillin 2 (FLOT2) mRNA. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometry analysis, and transwell assay were employed to test cell proliferation, apoptosis, and invasion, respectively. The glucose consumption and lactate production were examined by specific kits. Western blot assay was utilized for the detection of hexokinase2 (HK2), pyruvate kinase isozyme type M2 (PKM2), and FLOT2. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the targeting association between miR-143-3p and circ-FOXM1 or FLOT2. A murine xenograft model was established to explore the effect of circ-FOXM1 in vivo. RESULTS: Circ-FOXM1 was elevated and miR-143-3p was reduced in melanoma tissues and cells. Circ-FOXM1 deficiency impeded cell proliferation, invasion, and glycolysis and facilitated cell apoptosis in melanoma in vitro and tumorigenesis in vivo. Circ-FOXM1 acted as a sponge of miR-143-3p and the impacts of circ-FOXM1 silencing on cell proliferation, apoptosis, invasion, and glycolysis were overturned by miR-143-3p deletion. Moreover, FLOT2 was a target gene of miR-143-3p and FLOT2 overexpression rescued the inhibitory effect of miR-143-3p on melanoma progression. CONCLUSION: Circ-FOXM1 facilitated the development of melanoma by upregulating FLOT2 through miR-143-3p.

MicroRNA-31 is overexpressed in psoriasis and modulates inflammatory cytokine and chemokine production in keratinocytes via targeting serine/threonine kinase 40.

Psoriasis is characterized by a specific microRNA expression profile, distinct from that of healthy skin. MiR-31 is one of the most highly overexpressed microRNAs in psoriasis skin; however, its biological role in the disease has not been studied. In this study, we show that miR-31 is markedly overexpressed in psoriasis keratinocytes. Specific inhibition of miR-31 suppressed NF-κB-driven promoter luciferase activity and the basal and TNF-α-induced production of IL-1ß, CXCL1/growth-related oncogene-α, CXCL5/epithelial-derived neutrophil-activating peptide 78, and CXCL8/IL-8 in human primary keratinocytes. Moreover, interference with endogenous miR-31 decreased the ability of keratinocytes to activate endothelial cells and attract leukocytes. By microarray expression profiling, we identified genes regulated by miR-31 in keratinocytes. Among these genes, we identified serine/threonine kinase 40 (STK40), a negative regulator of NF-κB signaling, as a direct target for miR-31. Silencing of STK40 rescued the suppressive effect of miR-31 inhibition on cytokine/chemokine expression, indicating that miR-31 regulates cytokine/chemokine expression via targeting STK40 in keratinocytes. Finally, we demonstrated that TGF-ß1, a cytokine highly expressed in psoriasis epidermis, upregulated miR-31 expression in keratinocytes in vitro and in vivo. Collectively, our findings suggest that overexpression of miR-31 contributes to skin inflammation in psoriasis lesions by regulating the production of inflammatory mediators and leukocyte chemotaxis to the skin. Our data indicate that inhibition of miR-31 may be a potential therapeutic option in psoriasis.

Alternations in the human skin, gut and vaginal microbiomes in perimenopausal or postmenopausal Vulvar lichen sclerosus.

Vulvar lichen sclerosus (VLS) is a chronic and progressive dermatologic condition that can cause physical dysfunction, disfigurement, and impaired quality of life. However, the etiology of VLS remains unknown. The vulvar skin, intestinal and vaginal microbiomes have been postulated to play important roles in the pathogenesis of this disease. The aim of this study was to compare the compositional characteristics of the vulvar skin, vagina, and gut microbiota between perimenopausal or postmenopausal VLS patients and healthy controls. The study involved six perimenopausal or postmenopausal VLS patients which were based on characteristic clinical manifestations and histologic confirmation and five healthy controls. The pruritus severity of each patient was evaluated using the NRS scale, and the dermatology-specific health-related quality of life was assessed using the Skindex-16. Metagenomic sequencing was performed, and the results were analyzed for alpha and beta diversity. LEfSe analysis were used to investigate the microbial alterations in vulvar skin, gut and vagina. KEGG databases were used to analyze differences in functional abundance. The study found significant differences in alpha diversity between the two groups in stool and vaginal samples (P < 0.05). Patients with VLS had a higher abundance of Enterobacter cloacae, Flavobacterium_branchiophilum, Mediterranea_sp._An20, Parabacteroides_johnsoniiand Streptococcus_bovimastitidis on the vulvar skin, while Corynebacterium_sp._zg-913 was less abundant compared to the control group. The relative abundance of Sphingomonas_sp._SCN_67_18, Sphingobium_sp._Ant17, and Pontibacter_sp_BT213 was significantly higher in the gut samples of patients with VLS.Paenibacillus_popilliae,Gemella_asaccharolytica, and Coriobacteriales_bacterium_DNF00809 compared to the control group. Additionally, the vaginal samples of patients with VLS exhibited a significantly lower relative abundance of Bacteroidales_bacterium_43_8, Bacteroides_sp._CAG:20, Blautia_sp._AM28-10, Fibrobacter_sp._UWB16, Lachnospiraceae_bacterium_AM25-39, Holdemania_filiformis, Lachnospiraceae_bacterium_GAM79, and Tolumonas_sp. Additionally, the butyrate-producing bacterium SS3/4 showed a significant difference compared to the controls. The study found a negative relationship between Sphingobium_sp._Ant17 in stool and Skindex-16 (P < 0.05), while Mediterranea_sp._An20 had a positive correlation with Skindex-16 (P < 0.05) in the skin. Additionally, our functional analysis revealed alterations in Aminoacyl_tRNA_biosynthesis, Glutathione_metabolism, the pentose phosphate pathway, and Alanine__aspartate_and_glutamate_metabolism in the VLS patient group. The study suggests that perimenopausal or postmenopausal patients with VLS have a modified microbiome in the vulvar skin, gut, and vagina. This modification is linked to abnormal energy metabolism, increased oxidative stress, and abnormal amino acid metabolism.

Two Cases of Immune Drift Phenomena Caused by Biologic Agents for Treating Psoriasis and Atopic Dermatitis.

Atopic dermatitis and psoriasis are both common chronic inflammatory skin conditions that can significantly affect the quality of life for individuals affected by them. With the growing use of biologic agents, there have been remarkable clinical advancements in the treatment of these diseases. Interestingly, during biologic therapy for either condition, a phenomenon has emerged where treatment can paradoxically induce a transition to the phenotype of the other condition.We present two cases of immune drift phenomena caused by biologic agents for treating psoriasis and atopic dermatitis.The first one is a case of psoriasis lesion that developed in an old patient with AD who was treated with dupilumab for two months. The second one is a case of eczematoid lesion that developed in an adult patient with ankylosing spondylitis who was treated with Secukinumab for 1 year.

Transcriptional down-regulation of Brca1 and E-cadherin by CtBP1 in breast cancer.

Carboxyl-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor with oncogenic potential. Immunohistochemistry staining using human breast cancer tissue arrays revealed that 92% of invasive ductal breast cancer cases have CtBP1-positive staining compared to 4% CtBP1-positive in normal breast tissue. To explore the functional impact of CtBP1 in breast cancer, we examined CtBP1's transcriptional regulation of known tumor suppressors, breast cancer susceptibility gene 1 (Brca1), and E-cadherin. We found CtBP1 was recruited to the promoter regions of Brca1 and E-cadherin genes in breast cancer cells. Concomitantly, Brca1 loss was detected in 57% and E-cadherin loss was detected in 76% of human invasive ductal breast cancers, and correlated with CtBP1 nuclear staining in these lesions. Importantly, siRNA knock down of CtBP1 restored Brca1 and E-cadherin expression in breast cancer cell lines, implying CtBP1 down-regulates Brca1 and E-cadherin genes in human breast cancer. This study provides evidence that although genetic loss of Brca1 and E-cadherin are infrequent in breast cancer, they are down-regulated at the transcriptional level by CtBP1 expression. Thus, CtBP1 activation could be a potential biomarker for breast cancer development.

Temporal smad7 transgene induction in mouse epidermis accelerates skin wound healing.

The expression of Smad7, a tumor growth factor-ß (TGFß) antagonist, is increased during cutaneous wound healing. To assess this significance, we temporally induced Smad7 transgene expression in wounded skin in gene-switch-Smad7 transgenic (Smad7 tg) mice. Smad7 induction in epidermal keratinocytes caused an increase in keratinocyte proliferation with reduced Smad2 activation, indicating that Smad7 abrogated TGFß-mediated growth inhibition. Additionally, wounded skin from Smad7 tg mice exhibited accelerated re-epithelialization, with increased activation of extracellular signal-regulated kinase (Erk), and an in vitro migration assay revealed that Erk activation contributed to Smad7-mediated keratinocyte migration. Notably, epidermis-specific Smad7 transgene expression also has a profound effect on the wound stroma, resulting in reduced inflammation, angiogenesis, and production of type I collagen. Reduced Smad2 activation was observed in wounded stroma from Smad7 transgenic (Smad7 tg) mice, possibly owing to fewer infiltrated TGFß-producing leukocytes compared to those in wounds from control mice. Because Smad7 is not secreted, these effects could reflect functional changes in Smad7 tg keratinocytes. Supporting this notion, the activation of NF-κB, a nonsecreted protein complex that transcriptionally activates inflammatory cytokines, was reduced in wounded epidermis from Smad7 tg mice compared to that in wounded wild-type epidermis. In sum, epidermal Smad7 overexpression accelerated wound healing through its direct effects on keratinocyte proliferation and migration, and through indirect effects on wound stroma.

Inhibition of CtBP-Regulated Proinflammatory Gene Transcription Attenuates Psoriatic Skin Inflammation.

Psoriasis is a chronic immune-mediated disease characterized by excessive proliferation of epidermal keratinocytes and increased immune cell infiltration to the skin. Although it is well-known that psoriasis pathogenesis is driven by aberrant production of proinflammatory cytokines, the mechanisms underlying the imbalance between proinflammatory and anti-inflammatory cytokine expression are incompletely understood. In this study, we report that the transcriptional coregulators CtBP1 and 2 can transactivate a common set of proinflammatory genes both in the skin of imiquimod-induced mouse psoriasis model and in human keratinocytes and macrophages stimulated by imiquimod. We find that mice overexpressing CtBP1 in epidermal keratinocytes display severe skin inflammation phenotypes with increased expression of T helper type 1 and T helper type 17 cytokines. We also find that the expression of CtBPs and CtBP-target genes is elevated both in human psoriatic lesions and in the mouse imiquimod psoriasis model. Moreover, we were able to show that topical treatment with a peptidic inhibitor of CtBP effectively suppresses the CtBP-regulated proinflammatory gene expression and thus attenuates psoriatic inflammation in the imiquimod mouse model. Together, our findings suggest to our knowledge previously unreported strategies for therapeutic modulation of the immune response in inflammatory skin diseases.

Smad7-induced beta-catenin degradation alters epidermal appendage development.

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.

Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression.

TGF-beta and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4-/- mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3-/- mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT mice. Consistent with the loss of Smad2 in poorly differentiated human SCCs, Smad2-/- tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2-/- mouse epidermis and occurred more frequently in Smad2-negative human SCCs than in Smad2-positive SCCs. Knocking down Snail abrogated Smad2 loss-associated EMT, suggesting that Snail upregulation is a major mediator of Smad2 loss-associated EMT. Furthermore, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss-associated Snail overexpression. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis.

Epidermal Smad4 deletion results in aberrant wound healing.

In the present study, we assessed the role of Smad4, a component of the transforming growth factor-beta signaling pathway, in cutaneous wound repair. Interestingly, when Smad4 was deleted in the epidermis, several defects in wound healing were observed in non-keratinocyte compartments. In comparison with wounded wild-type mouse skin, Smad4-deficient wounds had delayed wound closure and remodeling. Increased angiogenesis and inflammation were found in Smad4-deficient skin; these effects were exacerbated throughout the entire wound healing process. In addition, increased numbers of myofibroblasts but reduced collagen levels were found in Smad4-deficient wounds in comparison with wild-type wounds. Since Smad4 is not a secreted protein, we assessed if the above non-cell autonomous alterations were the result of molecular alterations in Smad4-deficient keratinocytes, which exert paracrine effects on wound stroma. Smad4-deficient skin and wounds had elevated levels of transforming growth factor-beta1, which have been shown to induce similar phenotypes, as well as of several transforming growth factor-beta1 target genes, such as matrix metalloproteinases, vascular endothelial growth factor-A, and chemokine (C-C motif) ligand 5. Furthermore, the above pathological and molecular alterations were exacerbated in skin cancer lesions that spontaneously developed from Smad4-deficient skin. Therefore, loss of Smad4 in the epidermis appears to significantly affect the microenvironment during wound healing and carcinogenesis.

IKKalpha is a critical coregulator of a Smad4-independent TGFbeta-Smad2/3 signaling pathway that controls keratinocyte differentiation.

Cell-cycle exit and differentiation of suprabasal epidermal keratinocytes require nuclear IkappaB kinase alpha (IKKalpha), but not its protein kinase activity. IKKalpha also is a suppressor of squamous cell carcinoma (SCC), but its mode of action remains elusive. Postulating that IKKalpha may serve as a transcriptional regulator in keratinocytes, we searched for cell-cycle-related genes that could illuminate this function. IKKalpha was found to control several Myc antagonists, including Mad1, Mad2, and Ovol1, through the association with TGFbeta-regulated Smad2/3 transcription factors and is required for Smad3 recruitment to at least one of these targets. Surprisingly, Smad2/3-dependent Mad1 induction and keratinocyte differentiation are independent of Smad4, the almost universal coregulator of canonical TGFbeta signaling. IKKalpha also is needed for nuclear accumulation of activated Smad2/3 in the epidermis, and Smad2/3 are required for epidermal differentiation. We suggest that a TGFbeta-Smad2/3-IKKalpha axis is a critical Smad4-independent regulator of keratinocyte proliferation and differentiation.

PARP Inhibition Enhances Radiotherapy of SMAD4-Deficient Human Head and Neck Squamous Cell Carcinomas in Experimental Models.

PURPOSE: SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). EXPERIMENTAL DESIGN: We analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed. RESULTS: SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. CONCLUSIONS: SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.

Smad4-dependent desmoglein-4 expression contributes to hair follicle integrity.

We have previously shown that keratinocyte-specific deletion of Smad4, a TGFbeta/Activin/BMP signaling mediator, results in a progressive alopecia. To further assess the molecular mechanisms of Smad4 loss-mediated alopecia, we examined expression levels of key molecules associated with hair follicle differentiation in Smad4-deleted skin. Among them, Desmoglein 4 (Dsg4) was down-regulated in Smad4-deleted skin prior to the onset of hair follicle abnormalities with gradual depletion coinciding with hair follicle degeneration. Chromatin immunoprecipitation (ChIP) assay showed that Smad4, together with the BMP mediators Smad1 and Smad5, but not the TGFbeta/Activin mediators Smad2 or Smad3, bound to the Smad Binding Element (SBE) of the Dsg4 promoter. A Dsg4 reporter assay revealed that Smad4 was required for the maximal transactivation of Dsg4 in cooperation with Smad1 and Smad5. Mutating the SBE of the Dsg4 promoter abrogated Smad4 transactivation of Dsg4. Furthermore, BMP ligands, but not ligands of TGFbeta and Activin, induced endogenous Dsg4 expression. Our data demonstrate that in the presence of Smad4, BMP signaling participated in transcriptional regulation of Dsg4. Thus, Smad4 loss-associated Dsg4 depletion contributed, at least in part, to hair follicles degeneration in Smad4 deficient skin.

Smad7 Ameliorates TGF-ß-Mediated Skin Inflammation and Associated Wound Healing Defects but Not Susceptibility to Experimental Skin Carcinogenesis.

We assessed the roles of Smad7 in skin inflammation and wound healing using genetic and pharmacological approaches. In K5.TGFß1/K5.Smad7 bigenic (double transgenic) mice, Smad7 transgene expression reversed transforming growth factor (TGF)-ß1 transgene-induced inflammation, fibrosis, and subsequent epidermal hyperplasia and molecularly abolished TGF-ß and NF-κB activation. Next, we produced recombinant human Smad7 protein with a Tat-tag (Tat-Smad7) that rapidly enters cells. Subcutaneous injection of Tat-Smad7 attenuated infiltration of F4/80+ and CD11b+ leukocytes and α-smooth muscle actin+ fibroblasts before attenuating epidermal hyperplasia in K5.TGFß1 skin. Furthermore, topically applied Tat-Smad7 on K5.TGFß1 skin wounds accelerated wound closure, with improved re-epithelialization and reductions in inflammation and fibrotic response. A short treatment with Tat-Smad7 was also sufficient to reduce TGF-ß and NF-κB signaling in K5.TGFß1 skin and wounds. Relevant to the clinic, we found that human diabetic wounds had elevated TGF-ß and NF-κB signaling compared with normal skin. To assess the oncogenic risk of a potential Smad7-based therapy, we exposed K5.Smad7 skin to chemical carcinogenesis and found reduced myeloid leukocyte infiltration in tumors but not accelerated carcinogenesis compared with wild-type littermates. Our study suggests the feasibility of using exogenous Smad7 below an oncogenic level to alleviate skin inflammation and wound healing defects associated with excessive activation of TGF-ß and NF-κB.

Distinct mechanisms of TGF-beta1-mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis.

In the present study, we demonstrated that human skin cancers frequently overexpress TGF-beta1 but exhibit decreased expression of the TGF-beta type II receptor (TGF-(beta)RII). To understand how this combination affects cancer prognosis, we generated a transgenic mouse model that allowed inducible expression of TGF-beta(1) in keratinocytes expressing a dominant negative TGF-(beta)RII (Delta(beta)RII) in the epidermis. Without Delta(beta)RII expression, TGF-beta1 transgene induction in late-stage, chemically induced papillomas failed to inhibit tumor growth but increased metastasis and epithelial-to-mesenchymal transition (EMT), i.e., formation of spindle cell carcinomas. Interestingly, Delta(beta)RII expression abrogated TGF-beta1-mediated EMT and was accompanied by restoration of membrane-associated E-cadherin/catenin complex in TGF-beta1/Delta(beta)RII compound tumors. Furthermore, expression of molecules thought to mediate TGF-beta1-induced EMT was attenuated in TGF-beta1/Delta(beta)RII-transgenic tumors. However, TGF-beta1/Delta(beta)RII-transgenic tumors progressed to metastasis without losing expression of the membrane-associated E-cadherin/catenin complex and at a rate higher than those observed in nontransgenic, TGF-beta1-transgenic, or Delta(beta)RII-transgenic mice. Abrogation of Smad activation by Delta(beta)RII correlated with the blockade of EMT. However, Delta(beta)RII did not alter TGF-beta1-mediated expression of RhoA/Rac and MAPK, which contributed to increased metastasis. Our study provides evidence that TGF-beta1 induces EMT and invasion via distinct mechanisms. TGF-beta1-mediated EMT requires functional TGF-(beta)RII, whereas TGF-beta1-mediated tumor invasion cooperates with reduced TGF-(beta)RII signaling in tumor epithelia.

Topical Application of Tat-Rac1 Promotes Cutaneous Wound Healing in Normal and Diabetic Mice.

The endogenous small GTPase, Rac1, plays a critical role during normal skin wound healing. It remains to be determined whether endogenous Rac1 can be appropriately activated in chronic wounds; if not, whether exogenous Rac1 has therapeutic effects on wound healing. Here we show that Rac1 protein levels were lower in wounds of db/db diabetic mice than wounds in wild type mice during the healing process. To assess the therapeutic potential of exogenous Rac1 in wound healing, we produced a Tat-Rac1 fusion protein that enters into cells through protein transduction. Tat-Rac1 increased proliferation and migration of keratinocytes and dermal fibroblasts in vitro. Topical application of Tat-Rac1 accelerated cutaneous wound closure in vivo in db/db mice as well as wild type mice. Further analyses revealed that Tat-Rac1 had faster re-epithelialization, higher keratinocyte proliferation and migration without an earlier onset of myofibroblast activation than vehicle treated wounds. Tat-Rac1 also reduced inflammation in wounds. Our findings revealed the failure of diabetic wounds to elevate Rac1 expression and suggested a therapeutic strategy utilizing a Rac1-based biologic to compensate for this defect thereby promoting wound healing.

Effects of Photodynamic Therapy Using Yellow LED-light with Concomitant Hypocrellin B on Apoptotic Signaling in Keloid Fibroblasts.

Keloid is a common and refractory disease characterized by abnormal fibroblast proliferation and excessive deposition of extracellular matrix components. Hypocrellin B (HB) is a natural perylene quinone photosensitizer. In this experiment, we studied the effects of photodynamic therapy (PDT) using yellow light from light-emitting diode (LED) combined with HB on keloid fibroblasts (KFB) in vitro. Our results showed that HB-LED PDT treatment induced significant KFB apoptosis and decreased KFB cell viability. HB-LED PDT treatment lead to significant BAX upregulation and BCL-2 downregulation in KFB cells, which led to elevation of intracellular free Ca2+ and activation of caspase-3. Our data provides preliminary evidence for the potential of HB-LED PDT as a therapeutic strategy for keloid.

The role of Smad7 in oral mucositis.

Oral mucositis, a severe oral ulceration, is a common toxic effect of radio- or chemoradio-therapy and a limiting factor to using the maximum dose of radiation for effective cancer treatment. Among cancer patients, at least 40% and up to 70%, of individuals treated with standard chemotherapy regimens or upper-body radiation, develop oral mucositis. To date, there is no FDA approved drug to treat oral mucositis in cancer patients. The key challenges for oral mucositis treatment are to repair and protect ulcerated oral mucosa without promoting cancer cell growth. Oral mucositis is the result of complex, multifaceted pathobiology, involving a series of signaling pathways and a chain of interactions between the epithelium and submucosa. Among those pathways and interactions, the activation of nuclear factor-kappa B (NF-κB) is critical to the inflammation process of oral mucositis. We recently found that activation of TGFß (transforming growth factor ß) signaling is associated with the development of oral mucositis. Smad7, the negative regulator of TGFß signaling, inhibits both NF-κB and TGFß activation and thus plays a pivotal role in the prevention and treatment of oral mucositis by attenuating growth inhibition, apoptosis, and inflammation while promoting epithelial migration. The major objective of this review is to evaluate the known functions of Smad7, with a particular focus on its molecular mechanisms and its function in blocking multiple pathological processes in oral mucositis.