Seborrheic keratoses (SKs) are common, benign epithelial tumors of the skin that do not, or very rarely, progress into malignancy, for reasons that are not understood. We investigated this by gene expression profiling of human SKs and cutaneous squamous cell carcinomas (SCCs) and found that several genes previously connected with keratinocyte tumor development were similarly modulated in SKs and SCCs, whereas the expression of others differed by only a few fold. In contrast, the tyrosine kinase receptor FGF receptor–3 (FGFR3) and the transcription factor forkhead box N1 (FOXN1) were highly expressed in SKs, and close to undetectable in SCCs. We also showed that increased FGFR3 activity was sufficient to induce FOXN1 expression, counteract the inhibitory effect of EGFR signaling on FOXN1 expression and differentiation, and induce differentiation in a FOXN1-dependent manner. Knockdown of FOXN1 expression in primary human keratinocytes cooperated with oncogenic RAS in the induction of SCC-like tumors, whereas increased FOXN1 expression triggered the SCC cells to shift to a benign SK-like tumor phenotype, which included increased FGFR3 expression. Thus, we have uncovered a positive regulatory loop between FGFR3 and FOXN1 that underlies a benign versus malignant skin tumor phenotype.
Anna Mandinova, Vihren Kolev, Victor Neel, Bing Hu, Wesley Stonely, Jocelyn Lieb, Xunwei Wu, Claudia Colli, Rong Han, Mike Pazin, Paola Ostano, Reinhard Dummer, Janice L. Brissette, G. Paolo Dotto
The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin α5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.
Sophie Paquet-Fifield, Holger Schlüter, Amy Li, Tara Aitken, Pradnya Gangatirkar, Daniel Blashki, Rachel Koelmeyer, Normand Pouliot, Manuela Palatsides, Sarah Ellis, Nathalie Brouard, Andrew Zannettino, Nick Saunders, Natalie Thompson, Jason Li, Pritinder Kaur
Hemangiomas are the most common type of tumor in infants. As they are endothelial cell–derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T–transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/–, and Ang2–/– mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/– cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/– cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.
Sulochana S. Bhandarkar, Marisa Jaconi, Levi E. Fried, Michael Y. Bonner, Benjamin Lefkove, Baskaran Govindarajan, Betsy N. Perry, Ravi Parhar, Jamie Mackelfresh, Allie Sohn, Michael Stouffs, Ulla Knaus, George Yancopoulos, Yvonne Reiss, Andrew V. Benest, Hellmut G. Augustin, Jack L. Arbiser
Psoriasis is a common immune-mediated chronic inflammatory skin disorder, but the mechanisms of pathogenesis are still poorly understood. IL-23 is expressed in psoriatic skin, and IL-23 injection produces IL-22–dependent psoriasiform changes in mouse skin. Th17 cells produce IL-22 and display CCR6, the CCL20 receptor; CCR6+ T cells and CCL20 are abundant in psoriatic skin. We investigated a possible role for CCR6 in recruiting Th17 cells and producing psoriasiform pathology by injecting IL-23 into the skin of WT and Ccr6–/– mice. Unlike for WT mice, IL-23–injected ears of Ccr6–/– mice showed neither substantial epidermal/dermal changes nor increased Il22 mRNA expression. However, injection of IL-22 yielded equivalent psoriasiform changes in WT and Ccr6–/– mice. Surprisingly, IL-23–injected ears of WT and Ccr6–/– mice contained similar numbers of Th cells able to make IL-17A and/or IL-22. Furthermore, in ears of Rag1–/– mice, IL-23 initially induced skin changes and levels of Il22 mRNA that were indistinguishable from WT mice, revealing at least one non–T cell source for IL-22. We conclude that CCR6 is essential in a model of IL-23–induced, IL-22–mediated dermatitis, which develops in sequential T cell–independent and T cell–dependent phases. These findings reveal an expanded role for CCR6 in IL-23–related responses and identify CCR6 as a potential therapeutic target in psoriasis.
Michael N. Hedrick, Anke S. Lonsdorf, Aiko-Konno Shirakawa, Chyi-Chia Richard Lee, Fang Liao, Satya P. Singh, Hongwei H. Zhang, Alexander Grinberg, Paul E. Love, Sam T. Hwang, Joshua M. Farber
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.
Heidi Koegel, Lukas von Tobel, Matthias Schäfer, Siegfried Alberti, Elisabeth Kremmer, Cornelia Mauch, Daniel Hohl, Xiao-Jing Wang, Hans-Dietmar Beer, Wilhelm Bloch, Alfred Nordheim, Sabine Werner
Glycogen synthase kinase–3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by 2 genes, GSK3A and GSK3B. GSK-3 is thought to be involved in tissue repair and fibrogenesis, but its role in these processes is currently unknown. To investigate the function of GSK-3β in fibroblasts, we generated mice harboring a fibroblast-specific deletion of Gsk3b and evaluated their wound-healing and fibrogenic responses. We have shown that Gsk3b-conditional-KO mice (Gsk3b-CKO mice) exhibited accelerated wound closure, increased fibrogenesis, and excessive scarring compared with control mice. In addition, Gsk3b-CKO mice showed elevated collagen production, decreased cell apoptosis, elevated levels of profibrotic α-SMA, and increased myofibroblast formation during wound healing. In cultured Gsk3b-CKO fibroblasts, adhesion, spreading, migration, and contraction were enhanced. Both Gsk3b-CKO mice and fibroblasts showed elevated expression and production of endothelin-1 (ET-1) compared with control mice and cells. Antagonizing ET-1 reversed the phenotype of Gsk3b-CKO fibroblasts and mice. Thus, GSK-3β appears to control the progression of wound healing and fibrosis by modulating ET-1 levels. These results suggest that targeting the GSK-3β pathway or ET-1 may be of benefit in controlling tissue repair and fibrogenic responses in vivo.
Mohit Kapoor, Shangxi Liu, Xu Shi-wen, Kun Huh, Matthew McCann, Christopher P. Denton, James R. Woodgett, David J. Abraham, Andrew Leask
Mohit Kapoor, Shangxi Liu, Xu Shi-wen, Kun Huh, Matthew McCann, Christopher P. Denton, James R. Woodgett, David J. Abraham, Andrew Leask
PKC isoforms t, α, and β play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.
Hans Skvara, Markus Dawid, Elise Kleyn, Barbara Wolff, Josef G. Meingassner, Hilary Knight, Thomas Dumortier, Tamara Kopp, Nasanin Fallahi, Georg Stary, Christoph Burkhart, Olivier Grenet, Juergen Wagner, Youssef Hijazi, Randall E. Morris, Claire McGeown, Christiane Rordorf, Christopher E.M. Griffiths, Georg Stingl, Thomas Jung
Dysfunctional Tregs have been identified in individuals with psoriasis. However, their role in the pathogenesis of the disease remains unclear. Here we explored the effect of diminished CD18 (β2 integrin) expression on the function of CD4+CD25+CD127– Tregs using the Cd18 hypomorphic (Cd18hypo) PL/J mouse model of psoriasis that closely resembles the human disease. We found that reduced CD18 expression impaired cell-cell contact between Tregs and DCs. This led to dysfunctional Tregs, which both failed to suppress the pathogenic T cells and promoted the onset and severity of the disease. This failure was TGF-β–dependent, as Tregs derived from Cd18hypo PL/J mice had diminished TGF-β1 expression. Adoptive transfer of Tregs expressing wild-type levels of CD18 into affected Cd18hypo PL/J mice resulted in a substantial improvement of the psoriasiform skin disease, which did not occur upon coinjection of the cells with TGF-β–specific neutralizing antibody. Our data indicate a primary dysfunction of Cd18hypo Tregs, allowing subsequent hyperproliferation of pathogenic T cells in the Cd18hypo PL/J mouse model of psoriasis. This study may provide a step forward in our understanding of the unique role of CD18 expression levels in avoiding autoimmunity.
Honglin Wang, Thorsten Peters, Anca Sindrilaru, Daniel Kess, Tsvetelina Oreshkova, Xue-Zhong Yu, Anne Maria Seier, Heike Schreiber, Meinhard Wlaschek, Robert Blakytny, Jan Röhrbein, Guido Schulz, Johannes M. Weiss, Karin Scharffetter-Kochanek
Dystrophic epidermolysis bullosa (DEB) is a severe skin fragility disorder associated with trauma-induced blistering, progressive soft tissue scarring, and increased risk of skin cancer. DEB is caused by mutations in type VII collagen. In this study, we describe the generation of a collagen VII hypomorphic mouse that serves as an immunocompetent animal model for DEB. These mice expressed collagen VII at about 10% of normal levels, and their phenotype closely resembled characteristics of severe human DEB, including mucocutaneous blistering, nail dystrophy, and mitten deformities of the extremities. The oral blistering experienced by these mice resulted in growth retardation, and repeated blistering led to excessive induction of tissue repair, causing TGF-β1–mediated contractile fibrosis generated by myofibroblasts and pseudosyndactyly in the extremities. Intradermal injection of WT fibroblasts resulted in neodeposition of collagen VII and functional restoration of the dermal-epidermal junction. Treated areas were also resistant to induced frictional stress. In contrast, untreated areas of the same mouse showed dermal-epidermal separation following induced stress. These data demonstrate that fibroblast-based treatment can be used to treat DEB in a mouse model and suggest that this approach may be effective in the development of clinical therapeutic regimens for patients with DEB.
Anja Fritsch, Stefan Loeckermann, Johannes S. Kern, Attila Braun, Michael R. Bösl, Thorsten A. Bley, Hauke Schumann, Dominik von Elverfeldt, Dominik Paul, Miriam Erlacher, Dirk Berens von Rautenfeld, Ingrid Hausser, Reinhard Fässler, Leena Bruckner-Tuderman