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1.  Epidermal α6β4 integrin stimulates the influx of immunosuppressive cells during skin tumor promotion 
Journal of Dermatological Science  2012;66(2):108-118.
Induction of α6β4 integrin in the differentiated epidermal cell layers in skin is a hallmark of human cutaneous SCC pathogenesis and stimulates chemically induced SCC formation in Invα6β4 transgenic mice, which exhibit persistent expression of α6β4 in the suprabasal epidermal layers. However, the molecular basis for the support of SCC development by suprabasal α6β4 is not fully understood.
We examined the relevance for suprabasal α6β4 expression in the epidermis for the recruitment of immunosuppressive leukocytes during the early stages of tumor promotion.
In this study, we made use of the Invα6β4 transgenic mouse model, which exhibits expression of α6β4 integrin in the suprabasal layers of the epidermis driven by the involucrin promoter. First, we examined protein lysates from Invα6β4 transgenic skin using a pro-inflammatory cytokine array panel. Next, we immunofluorescence labeling of murine skin sections was employed to immunophenotype tumor promoter-treated Invα6β4 transgenic skin. Finally, a M-CSF neutralizing antibody strategy was administered to resolve Invα6β4 transgenic skin inflammation.
Employing the Invα6β4 transgenic mouse model, we show that suprabasal α6β4 integrin expression selectively alters the profile of secreted pro-inflammatory molecules by epidermal cells, in particular CXCL5 and M-CSF, in response to acute tumor promoter treatment. The induction of CXCL5 and M-CSF in Invα6β4 transgenic epidermis was shortly followed by an exacerbated influx of CD200R+ myeloid-derived suppressor cells (MDSCs), which co-expressed the M-CSF receptor, and FoxP3+ Treg cells compared to wild-type mice. As a result, the levels of activated CD4+ T lymphocytes were dramatically diminished in Invα6β4 transgenic compared to wild-type skin, whereas similar levels of lymphocyte activation were observed in the peripheral blood. Finally, TPA-induced CD200R+ infiltrative cells and epidermal proliferation were suppressed in Invα6β4 mice treated with M-CSF neutralizing antibodies.
We conclude that aberrant expression of α6β4 integrin in post-mitotic epidermal keratinocytes stimulates a pro-tumorigenic skin microenvironment by augmenting the influx of immunosuppressive granular cells during tumor promotion.
PMCID: PMC3328604  PMID: 22464766
skin carcinogenesis; myeloid-derived suppressor cell; microenvironment; squamous cell carcinoma; keratinocyte
2.  A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis 
Journal of Clinical Investigation  2001;108(4):527-536.
In normal epidermis, β1 integrin expression is confined to the basal layer, whereas in hyperproliferative epidermis, integrins are also expressed in the suprabasal layers. Transgenic mice in which integrins are expressed suprabasally via the involucrin promoter have a sporadic psoriatic phenotype; however, the mechanism by which integrins contribute to the pathogenesis of psoriasis is unknown. We observed activation of mitogen-activated protein kinase (MAPK) in basal and suprabasal keratinocytes of human and transgenic mouse psoriatic lesions and healing mouse skin wounds, correlating in each case with suprabasal integrin expression. Phenotypically normal human and transgenic mouse epidermis did not contain activated MAPK. Transgene-positive keratinocytes produced more IL-1α than controls did, and keratinocyte MAPK could be activated by ligation of suprabasal integrins or treatment with IL-1α. Constitutive activation of MAPK increased the growth rate of human keratinocytes and delayed the onset of terminal differentiation, recreating many of the histological features of psoriatic epidermis. We propose that activation of MAPK by integrins, either directly or through increased IL-1α production, is responsible for epidermal hyperproliferation in psoriasis and wound healing, and that the sporadic phenotype of the transgenic mice may reflect the complex mechanisms by which IL-1 release and responsiveness are controlled in skin.
PMCID: PMC209397  PMID: 11518726
3.  L-3-Phosphoserine Phosphatase (PSPH) Regulates Cutaneous Squamous Cell Carcinoma Proliferation Independent of L-serine Biosynthesis 
Journal of dermatological science  2011;63(3):164-172.
L-3-phosphoserine phosphatase (Psph) is a highly conserved and widely expressed member of the haloacid dehalogenase superfamily and the rate-limiting enzyme in L-serine biosynthesis. We previously found Psph expression to be uniquely upregulated in a α6β4 integrin transgenic mouse model that is predisposed to epidermal hyperproliferation and squamous cell carcinoma (SCC) formation implicating a role for Psph in epidermal homeostasis.
We examined the status of PSPH in normal skin epidermis and skin tumors along with its sub-cellular localization in epidermal keratinocytes and its requirement for squamous cell carcinoma (SCC) proliferation.
First, an immunohistochemical study was performed for PSPH in normal skin and skin cancer specimens and in cultured keratinocytes. Next, biochemical analyses were performed to confirm localization of PSPH and to identify candidate binding proteins. Finally, proliferation and apoptosis studies were performed in human SCC and normal keratinocytes, respectively, transduced with vectors encoding small hairpin RNAs targeting PSPH or overexpressing a phosphatase-deficient PSPH mutant.
PSPH is expressed throughout the proliferative layer of the epidermis and hair follicles in rodent and human skin and is highly induced in SCC. In keratinocytes, PSPH is a cytoplasmic protein that primarily localizes to endosomes and is present primarily as a homodimer. Knock down of PSPH dramatically diminished SCC cell proliferation and cyclin D1 levels in the presence of exogenous of L-serine production suggesting a non-canonical role for PSPH in epithelial carcinogenesis.
Psph is highly induced in proliferative normal keratinocytes and in skin tumors. PSPH appears to be critical for the proliferation of SCC cells; however, this phenomenon may not involve the phosphoserine metabolic pathway.
PMCID: PMC3152677  PMID: 21726982
PSPH; epidermis; squamous cell carcinoma; keratinocyte; serine metabolism
4.  Induction of GITRL Expression in Human Keratinocytes by TH2 cytokines and TNFα: Implications for Atopic Dermatitis 
Clinical and Experimental Allergy  2012;42(4):550-559.
Glucocorticoid-induced TNF receptor-related protein ligand (GITRL), a ligand for the T cell co-stimulatory molecule GITR, is expressed by keratinocytes and involved in chemokine production. The expression of GITRL in skin inflammation is unknown.
This study investigated cytokine regulation of keratinocyte GITRL expression.
GITRL expression was evaluated in cytokine treated human epidermal keratinocytes (HEK)s, murine PAM 212 cell line, murine and human skin explants by real time PCR, flow cytometry and immunostaining. Functional responses to GITR fusion protein were examined by real time PCR and ELISA. GITRL expression in AD and psoriasis was studied by immunohistochemistry.
Skin biopsies from STAT6VT transgenic mice, which develop spontaneous atopic skin inflammation, were found by immunofluoresence, to have increased keratinocyte GITRL expression. Exposure to Th2 cytokines augmented GITRL mRNA expression in the murine PAM 212 keratinocytic cell line and murine skin explants. In contrast, GITRL mRNA and protein expression was only increased in HEKs and human skin explants in the presence of the combination of TNFα and Th2 cytokines. A synergistic effect of Th2 cytokines and GITR fusion protein on production of CCL17, the Th2 chemokine, by murine keratinocytes was demonstrated. Immunohistochemical staining showed that acute AD lesions have increased expression of GITRL compared with normal skin, chronic AD lesions and psoriatic plaques.
Conclusions and Clinical Relevance
Our studies demonstrate that GITRL expression is augmented by Th2 cytokines and TNFα in keratinocytes. Increased GITRL expession in acute AD skin lesions is shown. This data suggests a link between cytokine regulated keratinocyte GITRL expression and its role in inflammatory responses in AD.
PMCID: PMC3306062  PMID: 22417213
Skin; Th2 cytokines; atopic dermatitis
5.  Systemic FasL and TRAIL Neutralisation Reduce Leishmaniasis Induced Skin Ulceration 
Cutaneous leishmaniasis (CL) is caused by Leishmania infection of dermal macrophages and is associated with chronic inflammation of the skin. L. aethiopica infection displays two clinical manifestations, firstly ulcerative disease, correlated to a relatively low parasite load in the skin, and secondly non-ulcerative disease in which massive parasite infiltration of the dermis occurs in the absence of ulceration of epidermis. Skin ulceration is linked to a vigorous local inflammatory response within the skin towards infected macrophages. Fas ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressing cells are present in dermis in ulcerative CL and both death ligands cause apoptosis of keratinocytes in the context of Leishmania infection. In the present report we show a differential expression of FasL and TRAIL in ulcerative and non-ulcerative disease caused by L. aethiopica. In vitro experiments confirmed direct FasL- and TRAIL-induced killing of human keratinocytes in the context of Leishmania-induced inflammatory microenvironment. Systemic neutralisation of FasL and TRAIL reduced ulceration in a model of murine Leishmania infection with no effect on parasitic loads or dissemination. Interestingly, FasL neutralisation reduced neutrophil infiltration into the skin during established infection, suggesting an additional proinflammatory role of FasL in addition to direct keratinocyte killing in the context of parasite-induced skin inflammation. FasL signalling resulting in recruitment of activated neutrophils into dermis may lead to destruction of the basal membrane and thus allow direct FasL mediated killing of exposed keratinocytes in vivo. Based on our results we suggest that therapeutic inhibition of FasL and TRAIL could limit skin pathology during CL.
Author Summary
Cutaneous leishmaniases are associated with parasite-induced inflammatory lesions of the skin. The degree of clinical pathology is not associated with parasitic burden; on the contrary, ulcerative lesions are associated with low infectious load, and non-ulcerative lesions are associated with an abundant parasite infiltration. Leishmania are intracellular parasites in mammalian hosts and reside in macrophages in the deep layers of the skin, the dermis. The exact mechanism of ulceration in CL is not known and Leishmania parasites do not directly induce destruction of keratinocytes in the most superficial layer of the skin, the epidermis. In this study we investigated if ulcerated lesions were associated with higher expression of FasL- and TRAIL-induced cell-death of keratinocytes. We found a higher expression of FasL and TRAIL in human skin samples from ulcerative as compared to non-ulcerative leishmaniasis. In a mouse model of ulcerative leishmaniasis neutralisation of FasL and TRAIL reduced ulceration. We suggest that FasL and TRAIL participate in the ulcer formation during leishmaniasis both as a chemoattractant of activated neutrophils leading to tissue destruction and through direct killing of keratinocytes. Possible approaches to use this concept in therapeutical interventions with the aim to reduce immunopathology associated with leishmaniasis are discussed.
PMCID: PMC2953481  PMID: 20967287
6.  Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation 
The Journal of Experimental Medicine  2007;204(11):2603-2614.
The interleukin (IL)-1 family members IL-1α, -1β, and -18 are potent inflammatory cytokines whose activities are dependent on heterodimeric receptors of the IL-1R superfamily, and which are regulated by soluble antagonists. Recently, several new IL-1 family members have been identified. To determine the role of one of these family members in the skin, transgenic mice expressing IL1F6 in basal keratinocytes were generated. IL1F6 transgenic mice exhibit skin abnormalities that are dependent on IL-1Rrp2 and IL-1RAcP, which are two members of the IL-1R family. The skin phenotype is characterized by acanthosis, hyperkeratosis, the presence of a mixed inflammatory cell infiltrate, and increased cytokine and chemokine expression. Strikingly, the combination of the IL-1F6 transgene with an IL1F5 deficiency results in exacerbation of the skin phenotype, demonstrating that IL-1F5 has antagonistic activity in vivo. Skin from IL1F6 transgenic, IL1F5−/− pups contains intracorneal and intraepithelial pustules, nucleated corneocytes, and dilated superficial dermal blood vessels. Additionally, expression of IL1RL2, -1F5, and -1F6 is increased in human psoriatic skin. In summary, dysregulated expression of novel agonistic and antagonistic IL-1 family member ligands can promote cutaneous inflammation, revealing potential novel targets for the treatment of inflammatory skin disorders.
PMCID: PMC2118475  PMID: 17908936
7.  Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes Triggers Atopic Dermatitis-Like Skin Inflammatory Responses in Adult Mice 
PLoS ONE  2012;7(12):e51262.
Ctip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice.
Methodology/Principal Findings
To study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2ep−/− mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT-qPCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes.
Our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in barrier functions. Our results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism enforced by Ctip2.
PMCID: PMC3527437  PMID: 23284675
8.  Halting angiogenesis by non-viral somatic gene therapy alleviates psoriasis and murine psoriasiform skin lesions 
Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice with epidermal expression of human TGF-β1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein–coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue. High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders.
PMCID: PMC3007133  PMID: 21135506
9.  CXCR2 ligands and G-CSF mediate PKCα-induced intraepidermal inflammation 
Journal of Clinical Investigation  2006;116(10):2757-2766.
Transgenic mice overexpressing PKCα in the epidermis (K5-PKCα mice) exhibit an inducible severe intraepidermal neutrophilic inflammation and systemic neutrophilia when PKCα is activated by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This inducible model of cutaneous inflammation was used to define mediators of skin inflammation that may have clinical relevance. Activation of cutaneous PKCα increased the production of the chemotactic factors cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein 2 (MIP-2) in murine plasma. TPA treatment of cultured K5-PKCα keratinocytes also released KC and MIP-2 into culture supernatants through an NF-κB–dependent pathway. MIP-2 and KC mediated the infiltration of neutrophils into the epidermis, since this was prevented by ablating CXCR2 in K5-PKCα mice or administering neutralizing antibodies against KC or MIP-2. The neutrophilia resulted from PKCα-mediated upregulation of cutaneous G-CSF released into the plasma independent of CXCR2. These responses could be inhibited by topical treatment with a PKCα-selective inhibitor. Inhibiting PKCα also reduced the basal and TNF-α– or TPA-induced expression of CXCL8 in cultured psoriatic keratinocytes, suggesting that PKCα activity may contribute to psoriatic inflammation. Thus, skin can be the source of circulating factors that have both local and systemic consequences, and these factors, their receptors, and possibly PKCα could be therapeutic targets for inhibition of cutaneous inflammation.
PMCID: PMC1560349  PMID: 16964312
10.  CD8+ T cells Mediate RAS-induced Psoriasis-like Skin Inflammation Through IFN-γ 
The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RASV12G in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses and increased Th1/Th17 and Tc1/Tc17 skin infiltration. The majority of skin infiltrating CD8+ T cells co-expressed IFN-γ and IL-17A. When RAS was expressed on a Rag1−/− background, microabscess formation, iNOS expression and keratinocyte hyperproliferation were suppressed. Depletion of CD8+ but not CD4+ T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17+ γΔ T cells, and epidermal hyperproliferation to levels similar to a Rag1−/− background. Reconstitution of Rag1−/− inducible RAS mice with purified CD8+ T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ but not IL-17A in CD8+ T cell reconstituted Rag1−/− mice expressing RAS blocked CD8-mediated skin inflammation, iNOS expression and keratinocyte hyperproliferation. These results show for that CD8+ T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ.
PMCID: PMC3577939  PMID: 23151849
CD8+ T cells; RAS; psoriasis model; inflammation
11.  GATA3 Expression Is Decreased in Psoriasis and during Epidermal Regeneration; Induction by Narrow-Band UVB and IL-4 
PLoS ONE  2011;6(5):e19806.
Psoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serves as a key switch in both epidermal and T helper cell differentiation, we investigated its function in psoriasis. Because psoriatic skin inflammation shares many characteristics of epidermal regeneration during wound healing, we also studied GATA3 expression under such conditions.
Psoriatic lesional skin showed decreased GATA3 mRNA and protein expression compared to non-lesional skin. GATA3 expression was also markedly decreased in inflamed skin of mice with a psoriasiform dermatitis induced with imiquimod. Tape-stripping of non-lesional skin of patients with psoriasis, a standardized psoriasis-triggering and skin regeneration-inducing technique, reduced the expression of GATA3. In wounded skin of mice, low GATA3 mRNA and protein expression was detected. Taken together, GATA3 expression is downregulated under regenerative and inflammatory hyperproliferative skin conditions. GATA3 expression could be re-induced by successful narrow-band UVB treatment of both human psoriasis and imiquimod-induced psoriasiform dermatitis in mice. The prototypic Th2 cytokine IL-4 was the only cytokine capable of inducing GATA3 in skin explants from healthy donors. Based on these findings we argue that GATA3 serves as a key regulator in psoriatic inflammation, keratinocyte hyperproliferation and skin barrier dysfunction.
PMCID: PMC3096641  PMID: 21611195
12.  Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis 
The Journal of Cell Biology  2010;188(6):935-952.
Loss of FGFRs results in skin abnormalities due to activation of keratinocytes and epidermal T cells.
Fibroblast growth factors (FGFs) are master regulators of organogenesis and tissue homeostasis. In this study, we used different combinations of FGF receptor (FGFR)-deficient mice to unravel their functions in the skin. Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. We identified loss of FGF-induced expression of tight junction components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier causes activation of keratinocytes and epidermal γδ T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiate an inflammatory response and induce a double paracrine loop through production of keratinocyte mitogens by dermal cells. Our results identify essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlight the importance of stromal–epithelial interactions in skin homeostasis and disease.
PMCID: PMC2845079  PMID: 20308431
13.  Development of Transgenic Minipigs with Expression of Antimorphic Human Cryptochrome 1 
PLoS ONE  2013;8(10):e76098.
Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3–20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1AP). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.
PMCID: PMC3797822  PMID: 24146819
14.  Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice 
The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.
PMCID: PMC2662566  PMID: 19307725
15.  Inhibition of Keratinocyte Differentiation by the Synergistic Effect of IL-17A, IL-22, IL-1α, TNFα and Oncostatin M 
PLoS ONE  2014;9(7):e101937.
Keratinocyte differentiation program leading to an organized epidermis plays a key role in maintaining the first line of defense of the skin. Epidermal integrity is regulated by a tight communication between keratinocytes and leucocytes, particularly under cytokine control. Imbalance of the cytokine network leads to inflammatory diseases such as psoriasis. Our attempt to model skin inflammation showed that the combination of IL-17A, IL-22, IL-1α, OSM and TNFα (Mix M5) synergistically increases chemokine and antimicrobial-peptide expression, recapitulating some features of psoriasis. Other characteristics of psoriasis are acanthosis and down-regulation of keratinocyte differentiation markers. Our aim was to characterize the specific roles of these cytokines on keratinocyte differentiation, and to compare with psoriatic lesion features. All cytokines decrease keratinocyte differentiation markers, but IL-22 and OSM were the most powerful, and the M5 strongly synergized the effects. In addition, IL-22 and OSM induced epidermal hyperplasia in vitro and M5 induced epidermal thickening and decreased differentiation marker expression in a mouse model, as observed in human psoriatic skin lesions. This study highlights the precise role of cytokines in the skin inflammatory response. IL-22 and OSM more specifically drive epidermal hyperplasia and differentiation loss while IL-1α, IL-17A and TNFα were more involved in the activation of innate immunity.
PMCID: PMC4092099  PMID: 25010647
16.  Luteolin Inhibits Human Keratinocyte Activation and Decreases NF-κB Induction That Is Increased in Psoriatic Skin 
PLoS ONE  2014;9(2):e90739.
Psoriasis (Ps) is an autoimmune disease characterized by keratinocyte hyperproliferation and chronic inflammation, with increased expression of tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Anti-TNF biologic agents are effective in treating Ps, but are associated with increased risk of infections and blood malignancies. Moreover, keratinocyte hyperproliferation and activation have yet to be addressed. Flavonoids, such as luteolin, are natural compounds with potent anti-inflammatory properties, but their actions on keratinocytes remain unknown. We show that TNF (50 ng/mL) triggers significant production of inflammatory mediators interleukin-6, interleukin-8 and VEGF from both human HaCaT and primary keratinocytes. Pretreatment with the flavonoid luteolin (10–100 µM) significantly inhibits mRNA expression and release of all three mediators in a concentration-dependent manner. More importantly, luteolin decreases TNF-induced phosphorylation, nuclear translocation and DNA binding of the nuclear factor-kappa B (NF-κB) typically involved in inflammatory mediator transcription. We also report that luteolin reduces TNF-induced mRNA expression of two genes (NFKB1 and RELA) encoding two NF-κB subunits (NF-κB p50 and NF-κB p65, respectively). Interestingly, we show that gene expression of RELA is increased in human psoriatic skin. Keratinocyte proliferation, which is a characteristic feature of psoriatic skin, is effectively reduced by luteolin in HaCaT cells, but not in primary keratinocytes. Finally, luteolin does not affect intracellular ATP production or viability. Appropriate formulations of luteolin and related flavones may be promising candidates to be developed into local and systemic treatments for Ps and other inflammatory skin diseases.
PMCID: PMC3938790  PMID: 24587411
17.  Keratinocyte Expression of CGRPβ: Implications for Neuropathic and Inflammatory Pain Mechanisms 
Pain  2011;152(9):2036-2051.
Calcitonin Gene-Related Peptide (CGRP) is a vasodilatory peptide that has been detected at high levels in the skin, blood, and cerebral spinal fluid under a variety of inflammatory and chronic pain conditions, presumably derived from peptidergic C and Aδ innervation. Herein, CGRP immunolabeling (IL) was detected in epidermal keratinocytes at levels that were especially high and widespread in the skin of humans from locations afflicted with postherpetic neuralgia (PHN) and complex region pain syndrome type 1 (CRPS), of monkeys infected with simian immunodeficiency virus, and of rats subjected to L5/L6 spinal nerve ligation, sciatic nerve chronic constriction, and subcutaneous injection of Complete Freund’s Adjuvant. Increased CGRP-IL was also detected in epidermal keratinocytes of transgenic mice with keratin-14 promoter driven overexpression of noggin, an antagonist to BMP-4 signaling. Transcriptome microarray, qPCR, and Western blot analyses using laser captured mouse epidermis from transgenics, monolayer cultures of human and mouse keratinocytes, and multilayer human keratinocyte organotypic cultures, revealed that keratinocytes express predominantly the beta isoform of CGRP. Cutaneous peptidergic innervation has been shown to express predominantly the alpha isoform of CGRP. Keratinocytes also express the cognate CGRP receptor components, CRLR, RAMP1, and RCP, consistent with known observations that CGRP promotes several functional changes in keratinocytes, including proliferation and cytokine production. Our results indicate that keratinocyte derived CGRPβ may modulate epidermal homeostasis through autocrine/paracrine signaling and may contribute to chronic pain under pathological conditions.
PMCID: PMC3157543  PMID: 21641113
Sensory transduction; Cutaneous innervation; BMP-4; Noggin; Skin; Dermatology
18.  Transgene-specific host responses in cutaneous gene therapy: the role of cells expressing the transgene 
Gene therapy  2009;16(9):1138-1145.
A major issue in long-term gene therapy is host immune responses to therapeutic cells when transgene encodes a potential antigen. The nature of these responses depends on several factors including the type of cell and tissue expressing the transgene. Keratinocytes and fibroblasts, which are known to display distinct immunogenic profiles, are both potential targets for transgene expression in cutaneous gene therapy. However, whether there is an immunological advantage in targeting one cell type over the other is not known. To study the effect of cell type on transgene-specific host responses independent of antigen levels or methods of gene transfer and transplantation, we used a skin transplantation model in which transgene expression can be targeted transgene to either keratinocytes or fibroblasts. Although targeting an antigen to either cell type resulted in the induction of immune responses, these responses differed significantly. Transgenic keratinocytes were rejected acutely by a dominant Th2 response, while in the majority of grafted animals transgenic fibroblasts failed to induce acute rejection despite the induction of Th1 type inflammation in the graft. In a small number of mice, transgenic fibroblasts persisted for at least 20 weeks despite elicitation of antigen-specific responses. Therefore, fibroblasts may be an immunologically preferred target over keratinocytes for cutaneous gene therapy.
PMCID: PMC2741533  PMID: 19440226
Skin; Gene therapy; Fibroblast; keratinocytes; Immune responses
19.  Kindlin-1 controls Wnt and TGF-β availability to regulate cutaneous epithelial stem cell proliferation 
Nature medicine  2014;20(4):350-359.
Kindlin-1 is an integrin tail binding protein that controls integrin activation. Mutations in the FERMT-1 gene lead to Kindler Syndrome in man, which is characterized by skin blistering, premature skin ageing and skin cancer of unknown etiology. Here we show that loss of Kindlin-1 in mouse keratinocytes recapitulates Kindler Syndrome, and in addition produces enlarged and hyperactive stem cell compartments, which lead to hyperthickened epidermis, ectopic hair follicle development and increased skin tumor susceptibility. Mechanistically, Kindlin-1 controls keratinocyte adhesion through β1-class integrins and proliferation and differentiation of cutaneous epithelial stem cells by promoting αvβ6 integrin-mediated TGFβ activation and by inhibiting Wnt-β-catenin signaling through an integrin-independent regulation of Wnt ligand expression. Our findings assign Kindlin-1 the novel and essential task to control cutaneous epithelial stem cell homeostasis by balancing TGFβ mediated growth inhibitory and Wnt-β-catenin mediated growth-promoting signals.
PMCID: PMC3982140  PMID: 24681597
20.  Stress-Mediated Increases in Systemic and Local Epinephrine Impair Skin Wound Healing: Potential New Indication for Beta Blockers 
PLoS Medicine  2009;6(1):e1000012.
Stress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair.
Methods and Findings
Migratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithelialization.
This work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte β2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific β2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.
Rivkah Isseroff and colleagues describe how stress-induced elevation of epinephrine levels can impair the healing of burns in mice and suggest that β2 adrenergic receptor antagonists may have a role in improving skin wound repair.
Editors' Summary
Skin—the largest organ in the human body—protects the rest of the body against infection by forming an impervious layer over the whole external body surface. Consequently, if this layer is damaged by rubbing, cutting, or burning, it must be quickly and efficiently repaired. Wound repair (healing) involves several different processes. First, the clotting cascade stops bleeding at the wound site and immune system cells attracted into the site remove any bacteria or debris in the wound. Various factors are released by the immune cells and the other cells in and near the damaged area that encourage the migration of several different sorts of cells into the wound. These cells proliferate and prepare the wound for “re-epithelialization.” In this process, keratinocytes (a type of epithelial cell that makes a tough, insoluble protein called keratin; epithelial cells cover all the surfaces of the body) migrate into the wound site and form a new, intact epithelial layer. If any of these processes fail, the result can be a chronic (long-lasting) nonhealing wound. In particular, if the wound does not re-epithelialize, it remains open and susceptible to infection and loss of body fluids.
Why Was This Study Done?
One factor that impairs the repair of skin wounds is stress. In stressful situations (including situations in which wounds are likely to occur), the human body releases several chemicals that prepare the body for “fight or flight,” including cortisol and epinephrine (also called adrenaline). Most scientists ascribe the effects of stress on wound healing to stress-induced increases in cortisol, but might stress-induced epinephrine also affect wound healing? In this study, the researchers test whether epinephrine impairs keratinocyte migration and re-epithelialization of burn wounds (keratinocytes have a receptor for epinephrine called the β2 adrenergic receptor [β2AR] on their cell surface that allows them to respond to epinephrine). They chose to study burn wounds for two reasons. First, major burns cause a massive release of stress chemicals into the bloodstream that raises blood levels (systemic levels) of cortisol and epinephrine for days or weeks after the initial trauma. Second, despite recent therapeutic advances, many people still die from major burns (4,000 every year in the USA alone) so there is a pressing need for better ways to treat this type of wound.
What Did the Researchers Do and Find?
The researchers investigated the effects of epinephrine on wound healing in three types of experiments. First, they looked at the effect of epinephrine on keratinocytes growing in dishes (in vitro experiments). Levels of epinephrine similar to those in the blood of stressed individuals greatly inhibited the motility and migration of human keratinocytes (isolated from the foreskin of newborn babies) and of mouse keratinocytes. It also inhibited the repair of scratch wounds made in monolayers of keratinocytes growing on dishes. Treatment of the cultures with a β2AR antagonist (a chemical that prevents epinephrine activating the β2AR) reversed the effects of epinephrine. In addition, the migration of mouse keratinocytes that had been genetically altered so that they did not express β2AR was not inhibited by epinephrine. Next, the researchers investigated the healing of burn wounds made in small pieces of human skin growing in dishes (ex vivo experiments). Burn injuries rapidly increased the amount of epinephrine in these tissue explants, they report, and treatment of the explants with a βAR antagonist (an inhibitor of all types of βARs) greatly increased wound re-epithelialization. Finally, the researchers report that the re-epithelialization of burn wounds in living mice was improved when the mice were treated with a β2AR antagonist.
What Do These Findings Mean?
These findings reveal a second pathway by which stress can impair wound healing. They show that stress-induced increases in systemic and local epinephrine activate β2ARs on keratinocytes and that this activation inhibits keratinocyte motility and wound re-epithelialization. Although results obtained in animals do not always reflect what happens in people, the finding that the treatment of mice with β2AR antagonists improves the rate of burn wound re-epithelialization, suggests that beta blockers—drugs that inhibit all βARs and that are widely used to treat high blood pressure and to prevent heart disease—or specific β2AR antagonists might provide a new therapeutic approach to the treatment of burns and, perhaps, chronic nonhealing wounds.
Additional Information.
Please access these Web sites via the online version of this summary at
Wikipedia has pages on wound healing, burn injuries, and epinephrine (Note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The MedlinePlus Encyclopedia has a page on burns (in English and Spanish)
MedlinePlus provides links to other information on burns (in English and Spanish)
PMCID: PMC2621262  PMID: 19143471
21.  Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes. 
Journal of Clinical Investigation  1995;95(1):317-327.
Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes.
PMCID: PMC295434  PMID: 7529261
22.  Loss of syndecan-1 is associated with malignant conversion in skin carcinogenesis 
Molecular carcinogenesis  2010;49(4):363-373.
Syndecan-1 (sdc-1) is a cell surface proteoglycan that mediates the interaction of cells with their matrix, influencing attachment, migration and response to growth factors. In keratinocytes, loss of sdc-1 delays wound healing, reduces migration, and increases TGFβ1 expression. In this study we show that sdc-1 expression is significantly reduced in basal cell, squamous cell, and metastatic human skin cancers compared to normal human skin. In experimental mouse skin tumor induction, compared to wildtype (wt) BALB/c mice, papilloma formation in sdc-1 null mice was reduced by 50% and the percent of papillomas converting to squamous cell carcinoma (SCC) was enhanced. Sdc-1 expression on wildtype mouse papillomas decreased as they converted to SCC. Furthermore, papillomas forming on sdc-1 null mice expressed suprabasal α3 and β4 integrins; suprabasal β4 integrin is a marker of a high risk for progression. While the proliferative response to TPA did not differ among the genotypes, sdc-1 null mice had an enhanced inflammatory response and retained higher levels of total TGFβ1 within their skin after TPA treatment. Sdc-1 null keratinocytes, transduced in vitro by oncogenic rasHa, expressed higher levels of β4 integrin and had enhanced pSmad2 signaling and reduced senescence when compared to wildtype rasHa transduced keratinocytes. When rasHa transduced cells of both genotypes were grafted onto nude mice, null tumors converted to SCC with higher frequency confirming the skin painting experiments. These data indicate that sdc-1 is important both early in the development of skin tumors and in progression of skin cancers suggesting that reduced expression of sdc-1 could be a useful marker for progression in neoplastic skin lesions.
PMCID: PMC3653623  PMID: 20082322
skin carcinogenesis; keratinocytes; syndecan-1; integrin; laminin 332; TGFβ1; ras oncogene
23.  Human keratinocytes' response to injury upregulates CCL20 and other genes linking innate and adaptive immunity 
In the early stages of wound healing, keratinocytes become “activated” and release inflammatory molecules such as interleukin-1 and interleukin-8 that are linked to innate immune responses and neutrophil recruitment. It is unclear, however, whether keratinocytes release molecules linked to adaptive immune responses, e.g. CCL20, in their early state of activation without signals from infiltrating T cells. This study aims to isolate the immediate alterations in protective and inflammatory gene expression that occur in epidermal keratinocytes, with a particular focus on molecules associated with cell-mediated immunity. We used dispase-separated epidermis, followed by intercellular disassociation by trypsinization, as a model for epidermal injury. We obtained a pure population of keratinocytes using flow cytometry. As a control for uninjured epidermis, we performed laser capture microdissection on normal human skin. Sorted keratinocytes had an early burst of upregulated gene expression, which included CCL20, IL-15, IL-23A, IFN-κ, and several antimicrobial peptides. Our results provide insight into the potential role of keratinocytes as contributors to cell-mediated inflammation, and expand knowledge about gene modulation that occurs during early wound healing. Our findings may be relevant to cutaneous diseases such as psoriasis, where micro-injury can trigger the formation of psoriatic plaques at the site of trauma.
PMCID: PMC3235229  PMID: 21881590
wound healing; keratinocytes; skin
24.  Vascular endothelium-specific overexpression of human catalase in cloned pigs 
Transgenic Research  2010;20(5):989-1001.
The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H2O2), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H2O2 would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT–PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H2O2 in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H2O2 in vasodilation and in the mechanisms underlying vascular health and disease.
PMCID: PMC3286124  PMID: 21170678
Endothelium; Catalase; Hydrogen peroxide; Swine; Somatic cell nuclear transfer; Vascular
25.  Integrin A6 Cleavage in Mouse Skin Tumors 
We have previously identified a structural variant of the α6 integrin (Laminin receptor) called α6p. The α6p variant is a 70 kDa form of the full-length α6 integrin (140 kDa) that remains paired with either the β1 or β4 subunit on the cell surface. α6p is produced by urokinase-type plasminogen activator (uPA), which removes the extracellular β-barrel domain while the receptor is on the cell surface. The α6p integrin was present in human prostate cancer tissue but not in normal tissue and the cleavage of the α6 integrin extracellular domain promotes tumor cell invasion and migration on laminin. The objective of the present study was to determine whether the α6p integrin is observed in other models of carcinogenesis. Our results indicate detectable low levels of α6p in normal mouse skin, and comparatively elevated levels in mouse papillomas and squamous cell carcinomas induced by DMBA, TPA and MNNG treatments. Furthermore, we have found that α6p was present at high levels in skin melanomas of transgenic mice that over express activated Ha-ras under the control of the tyrosinase promoter. Finally, subcutaneous injection into athymic nude mice of a malignant mouse keratinocyte derived cell line (6M90) that is α6p negative, results in the development of tumors that contain α6p integrin. The latter results indicate that α6p is induced in vivo suggesting that the tumor microenvironment plays a major role in the production of α6p. Taken together, these data suggest that the cell surface cleavage of the α6 integrin may be a novel mechanism of integrin regulation and might be an important step during skin tissue remodeling and during carcinogenesis.
PMCID: PMC2906811  PMID: 20664806

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