Carlotta Castagnoli

New p63 targets in keratinocytes identified by a genome‐wide approach

p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The ΔNp63α isoform is thought to play a ‘master’ role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing ΔNp63α; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.

An integrated humoral and cellular response is elicited in pancreatic cancer by α-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen†

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5‐year survival rate. α‐Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to α‐enolase. The present work was intended to assess the ability of α‐enolase to induce antigen‐specific T cell responses. We show that α‐enolase‐pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN‐γ and lyse PDAC cells but not normal cells. In vivo, α‐enolase‐specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to α‐enolase, the existence of α‐enolase‐specific T cells was also demonstrated. Taken as a whole, these results indicate that α‐enolase elicits a PDAC‐specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer.

Characterization of T-cell subsets infiltrating post-burn hypertrophic scar tissues

In this study, skin-infiltrating cells were characterized in both the active and remission phases of post-burn hypertrophic scar biopsies. Immunohistochemistry examination of active phase samples showed an abundant presence of Langerhans cells, T cells, macrophages, a low presence of natural killer cells and the lack of B lymphocytes. In active hypertrophic scars T lymphocytes infiltrate deep into the superficial dermis and are also observed in the epidermis: CD3+ cells were present at about 222 +/- 107 per 0.25 mm2. In particular the analysis of lymphocyte subpopulations showed that CD4+ T cells predominate in the dermis as well as in the epidermis of active hypertrophic scars whereas CD8+ cells were less well represented (CD4/CD8 ratio is 2.06). This distribution was also shown in remission phase samples and in normotrophic scar specimens, although the lymphocyte number was significantly lower. Approximately 70 per cent of T lymphocytes present in the tissue involved in active phase hypertrophic scar samples were activated (positive with anti-HLA-DR and IL-2 receptor antibodies) which is significantly higher than remission phase hypertrophic and normotrophic scars, in which positivity was 40 and 38 per cent, respectively. Upon activation, the lesional lymphocytes release several cytokines, locally and transiently, that interact with specific receptors in response to different stimulation. Central to the immune hypothesis of hypertrophic scars is that some of the T-cell lymphokines act on keratinocytes, fibroblasts and other cell types to induce changes characteristic of these scars. The presence and close proximity of activated T lymphocytes and antigen-presenting cells of various phenotypes in both the epidermis and dermis of hypertrophic tissues provides strong circumstantial evidence of a local immune response. However, the manner in which T cells achieve and maintain their activated state in hypertrophic tissues is not yet known, and both antigen-dependent and independent mechanisms may contribute.

Preparation and Characterization of a Novel Skin Substitute

Autologous epidermal cell cultures (CEA) represent a possibility to treat extensive burn lesions, since they allow a significative surface expansion which cannot be achieved with other surgical techniques based on autologous grafting. Moreover currently available CEA preparations are difficult to handle and their take rate is unpredictable. This study aimed at producing and evaluating a new cutaneous biosubstitute made up of alloplastic acellular glycerolized dermis (AAGD) and CEA to overcome these difficulties. A procedure that maintained an intact basement membrane was developed, so as to promote adhesion and growth of CEA on AAGD. Keratinocytes were seeded onto AAGD and cultured up to 21 days. Viability tests and immunohistochemical analysis with specific markers were carried out at 7, 14, and 21 days, to evaluate keratinocyte adhesion, growth, and maturation. Our results support the hypothesis that this newly formed skin substitute could allow its permanent engraftment in clinical application.

Imbalance between activin A and follistatin drives postburn hypertrophic scar formation in human skin

Abstract:  Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF‐β, play an important role in this process. Activin A, a member of the TGF‐β family, shares the same intra‐cellular Smad signalling pathway with TGF‐β, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of α‐SMA+ myofibroblasts and DC‐SIGN+ dendritic cells coexpressing activin A. Ex‐vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF‐βs stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF‐βs. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced α‐SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin‐induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.

Identification of new p63 targets in human keratinocytes.

p63 is a transcription factor involved in the development of ectodermal tissues, including limb, skin and, in general, multilayered epithelia. We identified both activated and repressed genes in human keratinocytes via gene expression profiling of p63-depleted cells and validated 21 new primary targets by RT-PCR and ChIP location analysis. The p63 isoforms differentially activate or repress selected promoters. ChIPs in primary keratinocytes indicate that p63 targets are generally shared with p53, but some are p63-specific. Several growth suppressors are among repressed genes. The newly identified genes belong to pathways of growth and differentiation and are regulated in HaCaT differentiation and in stratification of human skin.

Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFβ. Ιn vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFβ. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFβ family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFβ. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFβ, responsible for LC differentiation during inflammatory/autoimmune conditions.

Production and function of activin A in human dendritic cells

Activin A, a member of the transforming growth factor-beta superfamily, has a role in tissue repair and inflammation. In our previous studies, we identified by immunohistochemistry DC-SIGN(+) dendritic cells as a source of activin A in vivo. The present study was aimed at investigating activin A production by dendritic cells (DC) in vitro and its function. Here we demonstrate that monocyte-derived DC (Mo-DC) released abundant levels of activin A during the maturation process induced by TLR agonists, bacteria (B. henselae, S. thyphimurium), TNF and CD40L. Activin A was also induced in monocyte-derived Langerhans cells (LC) and in blood myeloid DC by LPS and/or CD40L stimulation, but not in blood plasmacytoid DC following stimulation with influenza virus. Activin A production by DC was selectively down-regulated by anti-inflammatory molecules such as dexamethasone or IL-10. Neutralization of endogenous activin A using its inhibitor follistatin, or the addition of exogenous activin A during LPS maturation did not affect Mo-DC maturation marker expression, cytokine release or allostimulatory function. However, Mo-DC matured with LPS in the presence of exogenous activin A displayed a higher FITC-dextran uptake, similar to that of immature DC. Moreover, activin A promoted monocyte differentiation to DC and reversed the inhibitory effects of IL-6 on DC differentiation of monocytes. These findings demonstrate that different subsets of DC release activin A, a cytokine that promotes DC generation, and affects the ability of mature DC to take up antigens (Ags).

p63 regulates the caspase-8-FLIP apoptotic pathway in epidermis.

The transcription factor p63, member of the p53 family, is crucial for epithelial development. An RNAi screening identified the apoptotic gene Procaspase-8 as a target activated by p63. The caspase-8 inhibitor FLIP is also under p63 control. We analysed and detailed the direct transactivation through the use of RNAi, reporter assays, ChIPs, western blots, confocal studies in HaCat, as well as in primary human keratinocytes. The direct ΔNp63 regulation of these targets was confirmed in vivo using transgenic ΔNp63 mice under the K5 promoter, as compared with p63 knockout mice, and in vitro in normal human primary keratinocytes following UV irradiation. Lowering the steady state of p63 protein levels changes the relative ratio of FLIP isoforms, causing the activation of the expressed, inactive Procaspase-8, into the active isoform thus triggering the proapoptotic cascade. Therefore, p63 fine-tunes the Procaspase-8-FLIP pro- and antiapoptotic pathway in keratinocytes.

Reciprocal regulation of p63 by C/EBP delta in human keratinocytes

BackgroundGenetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis.ResultsC/EBPδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPδ as a primary target of ΔNp63α by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the ΔNp63 promoter. Overexpression of C/EBPδ leads to alteration in the normal profile of p63 isoforms, with the emergence of ΔNp63β and γ, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPδ leads to gene expression modifications, in part due to the concomitant repression of ΔNp63α. Finally, C/EBPδ is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct.ConclusionOur data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets.