Basal Epidermal Keratinocytes Research Articles

O19 Improvement of a junctional epidermolysis bullosa mouse model

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is the most severe of the epidermolysis bullosa (EB) disorders and is caused by mutations in genes encoding the skin basement membrane proteins laminin 332, type XVII collagen or integrin α6β4. Affected individuals experience blistering from birth leading to scarring, granulation tissue and susceptibility to infection. In JEB, generalized severe, there is 50% mortality in the first 2 years of life, often owing to failure to thrive or overwhelming infection. A number of animal models for JEB exist; however, because of severe blistering on the entire surface of the skin, these mice die soon after birth, reducing their experimental utility. In 2017, the first adult inducible knockout mouse model of JEB was published. In this mouse model, the disruption of the Lama3 gene is specifically induced in epidermal basal keratinocytes after birth. These mice remain viable, which allows us to study the longer-term consequences of the loss of laminin α3. However, the mice did not develop the severe phenotype observed in patients with JEB. Methods We have optimized this previously published methodology of a conditional knockout JEB mouse model (Lama3flx/flxK14CreERT) to now include more regular tamoxifen injections, which Results in a more controlled and severe phenotype of JEB. Results Widespread skin blistering develops after 12 weeks of tamoxifen treatment with an impaired skin barrier (transepidermal water loss) and a loss of epidermal lipids. Immunofluorescent staining was conducted to assess skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane proteins (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Conclusions These mice recapitulate all key features of human JEB with loss of laminin α3 at the basement membrane, epidermal thickening and visible blisters. This allows us to delve deeper into the functional and downstream pathways affected by the loss of laminin α3 and discover why the prognosis of patients with JEB is so severe.

Zinc supplements may prevent and treat cutaneous toxicities induced by epidermal growth factor receptor inhibitors.

Inhibitors of Epidermal Growth Factor Receptor (EGFRi) widely used in the therapy of cancer are associated with cutaneous adverse reactions (CARs) in >90% of continuously treated patients, including papulo-pustular or acneiform eruptions, xerosis, pruritus, paronychia and alopecia1 and, very occasionally, necrolytic migratory erythema-like (NME) reactions.2, 3 These CAR are usually associated with better treatment response, but may severely impact patient's quality of life and are a frequent reason for treatment withdrawal or dose reduction. The mechanisms underlying these CAR are not completely understood, but have been explained mostly by the pharmacologic activity of the drugs. EGF is involved in the survival, migration, proliferation and maturation of basal epidermal keratinocytes but is essential also in the follicles, nail apparatus, sebaceous and eccrine glands. Therefore, EGF receptor inhibition induces cell growth arrest, premature differentiation and keratinocyte apoptosis, causing thinner skin with epidermal barrier disruption, oxidative stress, inflammation with a Th2 pattern, reduction of skin innate immunity, and disturbance of sebaceous lipids.4, 5 This may explain xerosis, dermatitis and increased lesional Staphylococcus aureus colonization, often observed in these patients. However, based on the report of low-zinc levels in a case of erlotinib-induced NME-like2 and 25 cases of EGFRi-induced xerotic dermatitis with low- or marginal serum zinc levels who improved after zinc supplementation in more than 75% of the cases,6 Chun-Wei Lu and the team of Wen-Hung Chung from Taiwan decided to study the relationship between zinc levels and CAR from EGFRi, as presented in this issue of JEADV.3 They studied 101 patients treated with different EGFR inhibitors targeting the Tyrosine kinase (EGFR-TKI) (gefitinib, erlotinib, afatinib, osimertinib, and zorifertinib) who developed papulo-pustular reactions, dry skin and eczema, paronychia, NME-like eruption and/or dysgeusia. They had a significantly lower plasma zinc level when compared with patients with cancer without these therapies or out-patients with no cancer and no ECGFR therapy. Low-zinc levels occurred in all the phenotypes of CAR and zinc reduction was correlated with CAR severity. Moreover, in 55 patients who received supplementation with zinc gluconate skin lesions improved significantly with no need to reduce the dose or stop the EGFR-TKI.3 To further demonstrate the relation between zinc and EGFRi, the authors have shown that zinc levels decreased significantly in animals treated with erlotinib for 2 weeks, and these rats developed periorificial lesions simulating NME or acrodermatitis enteropathica-like lesions with histopathology similar to the patients who had developed this phenotype of CAR.3 This data supports that zinc deficiency may be one of the drivers of EGFRi-induced CAR, although there is no proven explanation for this effect. Some EGFRi, like erlotinib, may have zinc-binding activity or the EGFRi may interfere with zinc transport during absorption in the duodenum or with keratinocyte zinc transporters (ZnT2).3, 6 Zinc is abundant in the epidermis, controlling multiple metalloenzymes and transcription factors that regulate protein, lipid and DNA synthesis and degradation, and is essential for controlling several epidermal functions (keratinocyte differentiation, proliferation and apoptosis, skin inflammation and wound repair). EGFRi-induced CAR has many similarities with skin manifestations of zinc deficiency, namely NME or acrodermatitis enteropathica, paronychia, alopecia, dysgeusia and Staphylococcal skin colonization. Acneiform or papulo-pustular reactions, as observed in EGFRi-induced CAR, are not reported in zinc deficiency, but even though plasma zinc levels measured in acne are conflicting, topical or systemics treatments with zinc have been proposed for the treatment of acne.7 If zinc deficiency is further confirmed in all these phenotypes of CAR induced by EGFRi, both monoclonal antibodies anti-EGFR and oral ECGR-TKI, it might be important to perform systematic zinc supplementation in association with these therapies, instead of using prolonged treatments with tetracyclines, retinoids or topical steroids which may have other adverse effects. Has participated in advisory boards or performed paid lectures for Abbvie, Astra-Zeneca, Leo, Lilly, Novartis, Pfizer, Sanofi, Takeda. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

580 Vers la compréhension du lien entre le profil des cytokines et les phénotypes pathologiques associés à l’épidermolyse bulleuse simple à l’aide de cellules souches pluripotentes induites

Epidermolysis bullosa simplex (EBS) is a skin disorder caused by dominant missense mutations in KERATIN5 (KRT5) or KERATIN14 (KRT14) gene, which encode intermediate filament (IF) proteins mainly expressed in epidermal basal keratinocytes. Incorporation of mutant keratins in network of IF makes the network sensitive to stress inducing inflammation and skin blistering formation. Currently, no therapeutic approach is known. Significant cytokine imbalance in blister fluid samples from different patients was demonstrated, suggesting that EB could be considered as inflammatory disorder rather than skin disorder.

Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis.

Irradiation‐induced alopecia and dermatitis (IRIAD) are two of the most visually recognized complications of radiotherapy, of which the molecular and cellular basis remains largely unclear. By combining scRNA‐seq analysis of whole skin‐derived irradiated cells with genetic ablation and molecular inhibition studies, we show that senescence‐associated IL‐6 and IL‐1 signaling, together with IL‐17 upregulation and CCR6+‐mediated immune cell migration, are crucial drivers of IRIAD. Bioinformatics analysis colocalized irradiation‐induced IL‐6 signaling with senescence pathway upregulation largely within epidermal hair follicles, basal keratinocytes, and dermal fibroblasts. Loss of cytokine signaling by genetic ablation in IL‐6−/− or IL‐1R−/− mice, or by molecular blockade, strongly ameliorated IRIAD, as did deficiency of CCL20/CCR6‐mediated immune cell migration in CCR6−/− mice. Moreover, IL‐6 deficiency strongly reduced IL‐17, IL‐22, CCL20, and CCR6 upregulation, whereas CCR6 deficiency reciprocally diminished IL‐6, IL‐17, CCL3, and MHC upregulation, suggesting that proximity‐dependent cellular cross talk promotes IRIAD. Therapeutically, topical application of Janus kinase blockers or inhibition of T‐cell activation by cyclosporine effectively reduced IRIAD, suggesting the potential of targeted approaches for the treatment of dermal side effects in radiotherapy patients.

Differentiation of Human Induced Pluripotent Stem Cells into Keratinocytes.

Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create “designer” iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC‐derived keratinocytes (iPSC‐K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF™ Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell™400 6‐well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells

Development of a 3D psoriatic skin model optimized for infiltration of IL-17A producing T cells: Focus on the crosstalk between T cells and psoriatic keratinocytes

Psoriasis is a chronic inflammatory skin disease involving several cell types, including T cells, via the IL-23/IL-17 axis. IL-17A acts on the surrounding epithelial cells thus resulting in an inflammatory feedback loop. The development of immunocompetent models that correctly recapitulate the complex phenotype of psoriasis remains challenging, which also includes both the T cell isolation and activation methods. The purpose of this work was to develop an advanced in vitro 3D psoriatic skin model that enables the study of the impact of T cells on psoriatic epithelial cells. To reach that aim, healthy and psoriatic fibroblasts and keratinocytes were used to reproduce this tissue-engineered skin model in which activated T cells, isolated beforehand from human whole blood, have been incorporated. Our study showed that isolation of T cells with the EasySep procedure, followed by activation with PMA/ionomycin, mimicked the psoriatic characteristics in an optimal manner with the production of inflammatory cytokines important in the pathogenesis of psoriasis, as well as increased expression of Ki67, S100A7, elafin and involucrin. This psoriatic model enriched in activated T cells displayed enhanced production of IL-17A, IFN-ƴ, CCL2, CXCL10, IL-1ra, IL-6 and CXCL8 compared with the healthy model and whose increased secretion was maintained over time. In addition, anti-IL17A treatment restored some psoriatic features, including epidermal thickness and basal keratinocytes proliferation, as well as a downregulation of S100A7, elafin and involucrin expression. Altogether, our study demonstrated that this model reflects a proper psoriatic inflammatory environment and is effective for the investigation of epidermal and T cell interaction over time. Statement of significanceThe aim of this study was to provide an innovative 3D immunocompetent human psoriatic skin model. To our knowledge, this is the first immunocompetent model that uses skin cells from psoriatic patients to study the impact of IL-17A on pathological cells. Through the use of this model, we demonstrated that the T-cell enriched psoriatic model differs from T-cell enriched healthy model, highlighting efficient crosstalk between pathologic epithelial cells and T cells. This advanced preclinical model further mimics the original psoriatic skin and will prove relevant in predicting clinical outcomes, thereby decreasing inaccurate predictions of compound effects.

Deconstructing progressive inflammatory fibrosis in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disease, resulting from biallelic mutations in COL7A1, the gene encoding type VII collagen (C7). At mucocutaneous barriers, tissue integrity relies upon linked extracellular matrix (ECM) proteins forming a physiologic suture, connecting basal epidermal keratinocytes to the underlying dermis. C7 secreted from epidermal keratinocytes and dermal fibroblasts homotrimerizes in the upper dermis to form anchoring fibrils, a critical component of this suture. Clinical manifestations of RDEB are apparent at birth and include exquisite skin fragility, pain and itch, high metabolic demand, and complications downstream of systemic inflammation. Dermal fibrosis is a critical complication of RDEB. Repeated cycles of mechanical injury and healing trigger characteristic fibrotic changes. In addition to functional limitations from joint strictures and pseudosyndactyly formation, dermal fibrosis in RDEB is a nidus for and potential driver of aggressive squamous cell carcinoma (SCC), the leading cause of death in RDEB. A greater understanding of fibrosis in RDEB promises to inform impactful, life-prolonging clinical trials in this patient population with no proven systemic therapy or cure.

Immunohistological Analysis of CD34-Positive Dermal Dendritic Cells and Microvessel Density in the Genital and Extragenital Lichen Sclerosus

BackgroundLichen sclerosus (LiS) is a chronic scleroatrophic condition that usually affects the anogenital area and occasionally the extragenital sites. CD34-positive dermal dendritic cells (DDCs) contribute to the maintenance of the dermal microarchitecture and modulation of the immune response. p53 is a tumor suppressor gene important for the regulation of the cell cycle and apoptosis. Similar to morphea (a LiS-closely related scleroatrophic condition), dermal sclerosis, alterations of DDCs, and dermal microvasculature may be important underlying pathogenetic mechanisms in LiS. ObjectivesTo examine the profile of CD34-positive DDCs, microvessel density (MVD), and p53 protein in LiS. Materials and methodsThe immunohistological profiles of DDCs, MVD, and p53 were examined in 19 cases of LiS and their age- and sex-matched normal skin (10 specimens), using antibodies against CD34 and p53. ResultsThere was a markedly decreased counts (1.7 ± 0.5/mm2) or complete loss of CD34-positive DDCs in LiS against their abundance in the normal skin (23.4 ± 2.1/mm2, p = 0.000). MVD was markedly increased in LiS lesions (20 ± 0.47) as compared to normal skin (5.50 ± 0.20, p = 0.000). Discontinuous single-cell p53 weakly positive nuclear staining was seen in the epidermal basal cell keratinocytes in normal skin and LiS lesions. ConclusionsTo the best of this author's knowledge, this is the first study analyzing DDCs, MVD, and p53 profiles together in LiS. The findings suggest that alterations of DDCs and MVD have roles in the pathogenesis of LiS.

Análisis inmunohistológico de las células dendríticas dérmicas positivas para CD34 y de la densidad de microvasos en el liquen escleroso genital y extragenital

BackgroundLichen sclerosus (LiS) is a chronic scleroatrophic condition that usually affects the anogenital area and occasionally the extragenital sites. CD34-positive dermal dendritic cells (DDCs) contribute to the maintenance of the dermal microarchitecture and modulation of the immune response. p53 is a tumor suppressor gene important for the regulation of the cell cycle and apoptosis. Similar to morphea (a LiS-closely related scleroatrophic condition), dermal sclerosis, alterations of DDCs, and dermal microvasculature may be important underlying pathogenetic mechanisms in LiS. ObjectivesTo examine the profile of CD34-positive DDCs, microvessel density (MVD), and p53 protein in LiS. Materials and methodsThe immunohistological profiles of DDCs, MVD, and p53 were examined in 19 cases of LiS and their age- and sex-matched normal skin (10 specimens), using antibodies against CD34 and p53. ResultsThere was a markedly decreased counts (1.7±0.5/mm2) or complete loss of CD34-positive DDCs in LiS against their abundance in the normal skin (23.4±2.1/mm2, p=0.000). MVD was markedly increased in LiS lesions (20±0.47) as compared to normal skin (5.50±0.20, p=0.000). Discontinuous single-cell p53 weakly positive nuclear staining was seen in the epidermal basal cell keratinocytes in normal skin and LiS lesions. ConclusionsTo the best of this author's knowledge, this is the first study analyzing DDCs, MVD, and p53 profiles together in LiS. The findings suggest that alterations of DDCs and MVD have roles in the pathogenesis of LiS.

Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3σ and YAP1.

The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14's codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.

Life before and beyond blistering: The role of collagen XVII in epidermal physiology.

Type XVII collagen (COL17) is a transmembranous protein that is mainly expressed in the epidermal basal keratinocytes. Epidermal-dermal attachment requires COL17 expression at the hemidesmosomes of the epidermal basement membrane zone because congenital COL17 deficiency leads to junctional epidermolysis bullosa and acquired autoimmunity to COL17 induces bullous pemphigoid. Recently, in addition to facilitating epidermal-dermal attachment, COL17 has been reported to serve as a niche for hair follicle stem cells, to regulate proliferation in the interfollicular epidermis and to be present along the non-hemidesmosomal plasma membrane of epidermal basal keratinocytes. This review focuses on the physiological properties of COL17 in the epidermis, its role in maintaining stem cells and its association with signalling pathways. We propose possible solutions to unanswered questions in this field.

631 Laminin-511 is a key component of epidermal basement membrane to maintain epidermal homeostasis

Daily exposure to sunlight damages the epidermal basement membrane (BM) and disrupts epidermal homeostasis. However, the role of epidermal BM components in maintaining epidermal homeostasis is unclear. Here, we investigated the effect of sun exposure on BM components and epidermal homeostasis. We found that laminin-511 in epidermal BM is less in sun-exposed skin compared to sun-protected skin. b1 integrin was also decreased. Since laminin-511 interacts with b1 integrin expressed on basal keratinocytes, we hypothesized that laminin-511 plays a key role in epidermal homeostasis by maintaining epidermal proliferation and differentiation. Through a screening study, we identified 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) as an inhibitor of laminin-511 degradation. HEI potently inhibited the activity of heparanase and matrix metalloproteinase 9 (MMP9), which degrade epidermal BM and are activated in sun-exposed skin. In the skin equivalent model of human skin, HEI promoted laminin-511 deposition at the epidermal BM, increased filaggrin and loricrin expression in granular layer in epidermis, and maintained Ki67 positive basal keratinocytes. HEI also increased laminin-511 deposition and promoted formation of continuous lamina densa and hemidesmosomes at the epidermal BM, maintained Ki67 positive basal keratinocytes, promoted filaggrin and loricrin expression, decreased transepidermal water loss (TEWL), and increased water content of stratum corneum in UVB exposed organ culture model. In a pilot study, it improved the barrier function of human facial skin. These results suggest laminin-511 is a key regulator of epidermal homeostasis by regulating proliferation and differentiation of basal keratinocytes in epidermis.

Zinc and Skin Disorders.

The skin is the third most zinc (Zn)-abundant tissue in the body. The skin consists of the epidermis, dermis, and subcutaneous tissue, and each fraction is composed of various types of cells. Firstly, we review the physiological functions of Zn and Zn transporters in these cells. Several human disorders accompanied with skin manifestations are caused by mutations or dysregulation in Zn transporters; acrodermatitis enteropathica (Zrt-, Irt-like protein (ZIP)4 in the intestinal epithelium and possibly epidermal basal keratinocytes), the spondylocheiro dysplastic form of Ehlers-Danlos syndrome (ZIP13 in the dermal fibroblasts), transient neonatal Zn deficiency (Zn transporter (ZnT)2 in the secretory vesicles of mammary glands), and epidermodysplasia verruciformis (ZnT1 in the epidermal keratinocytes). Additionally, acquired Zn deficiency is deeply involved in the development of some diseases related to nutritional deficiencies (acquired acrodermatitis enteropathica, necrolytic migratory erythema, pellagra, and biotin deficiency), alopecia, and delayed wound healing. Therefore, it is important to associate the existence of mutations or dysregulation in Zn transporters and Zn deficiency with skin manifestations.

Novel role for the testis-enriched HSPA2 protein in regulating epidermal keratinocyte differentiation.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

Abstract B07: The loss function of BP180 (Collagen type XVII) in skin, leads to a mast cell dependent pro-inflammatory microenvironment which promotes melanoma progression

Abstract BP180, also known as type 17 collagen, is a transmembrane glycoprotein located in the hemidesmosome of epidermal basal keratinocytes. BP180 is a critical cell/matrix adhesion molecule in dermal-epidermal junction of the skin. Various clinical reports indicated that the altered expression of hemidesmosomal protein BP180 is associated with melanoma progression. However, it is unclear whether BP180 is directly involved in melanoma progression. To investigate the role of BP180 in melanoma progression, we generated novel mouse strains lacking BP180 functions in whole body (by crossing floxed mice with germline Cre), in the local skin (tamoxifen-inducible Cre), or in basal keratinocytes (K14-driven Cre). These BP180 functional knockout mice showed dermal-epidermal junction separation in the skin, indicating that the cell/cell matrix adhesion mediated by BP180 is impaired. Loss of BP180 function also lead to a proinflammatory microenvironment in skin accompanied with an influx of immune cells including mast cells, neutrophils and T cells. When tested in B16 mouse melanoma model BP180 functional deficient mice (globally, in the local skin, or in basal keratinocytes) showed significantly increased melanoma progression. These results demonstrated that BP180 deficiency limited in local skin microenvironment or basal keratinocyte is sufficient to promote melanoma progression. To determine whether increased neutrophil or mast cells are required for the accelerated melanoma progression in BP180 functional deficient mice, we injected B16 melanoma cells into mice lacking both mast cells and BP180 function. Mast cell deficiency drastically reduced the inflammation, neutrophil influx into skin and melanoma progression caused by loss of BP180 function. These findings provide the first evidence suggesting that BP180, as a hemidesmosomal cell-cell matrix adhesion protein, plays a vital role in host defense against melanoma progression through modulating basal keratinocytes, neutrophil and mast cells in local tumor microenvironment. Citation Format: Bin-Jin Hwang, Bin Peng, Yang Zhang, Jaime Marie Brozowski, Lin Lin, Scott E. Williams, Ning Li, Zhen Liu, Maureen Su, Nancy E. Thomas, Luis A. Diaz, Zhi Liu. The loss function of BP180 (Collagen type XVII) in skin, leads to a mast cell dependent pro-inflammatory microenvironment which promotes melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B07.

Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane.

ABSTRACTMatriptase and prostasin, acting as a tightly coupled proteolytic cascade, were reported to be required for epidermal barrier formation in mouse skin. Here we show that, in human skin, matriptase and prostasin are expressed with an inverse pattern over the course of differentiation. Matriptase was detected primarily in epidermal basal keratinocytes and the basaloid cells in the outer root sheath of hair follicles and the sebaceous gland, where prostasin was not detected. In contrast, prostasin was detected primarily in differentiated cells in the epidermal granular layer, the inner root sheath of hair follicles, and the sebaceous gland, where matriptase expression is negligible. While co-expressed in the middle stage of differentiation, prostasin was detected as polarized patches, and matriptase at intercellular junctions. Targeting to different subcellular localizations is also observed in HaCaT human keratinocytes, in which matriptase was detected primarily at intercellular junctions, and prostasin primarily on membrane protrusion. Furthermore, upon induction of zymogen activation, free active prostasin remains cell-associated and free active matriptase is rapidly shed into the extracellular milieu. Our data suggest that matriptase and prostasin likely function as independent entities in human skin rather than as a tightly coupled proteolytic cascade as observed in mouse skin.

CD99 Is Strongly Expressed in Basal Cells of the Normal Adult Epidermis and Some Subpopulations of Appendages: Comparison with Developing Fetal Skin.

BackgroundCD99 is a cell surface transmembrane glycoprotein expressed in various tissues. CD99 is differentially expressed between subpopulations of each tissue and is highly expressed in certain hematopoietic and precursor cells. However, there has been no comprehensive study of CD99 expression in normal skin. We evaluated CD99 expression in normal human skin and developing fetal skin. MethodsSeventy-five adult skin samples containing normal skin and eight fetal skin samples of different gestational ages were collected. CD99 immunohistochemical staining was performed to evaluate expression pattern in adult and fetal skin samples. CD99 and CD34 expression were compared by double immunofluorescence. ResultsIn normal adult skin, CD99 was strongly expressed in the membrane of epidermal basal keratinocytes, hair follicle bulges and outer root sheaths, and inner secretory cells of eccrine sweat glands. In fetal skin, CD99 was not expressed on the periderm at 16 weeks of gestation but was expressed in basal cells of fetal skin at around 19 weeks of gestation. CD99 expression became comparable to that of the adult skin after 20 weeks of gestation. CD99 and CD34 were co-expressed in hair follicle outer root sheaths, as seen by double immunofluorescence study. ConclusionsThis is the first study examining CD99 expression pattern in normal adult and fetal skin. CD99 tends to be expressed in the basal/precursor cells of epidermis and in hair follicles. These results provide a basis for future investigation on functions of CD99 in the skin and provide a novel potential target for the treatment of dermatologic lesions.

Expression and Function of Group IIE Phospholipase A2 in Mouse Skin

Recent studies using knock-out mice for various secreted phospholipase A2 (sPLA2) isoforms have revealed their non-redundant roles in diverse biological events. In the skin, group IIF sPLA2 (sPLA2-IIF), an "epidermal sPLA2" expressed in the suprabasal keratinocytes, plays a fundamental role in epidermal-hyperplasic diseases such as psoriasis and skin cancer. In this study, we found that group IIE sPLA2 (sPLA2-IIE) was expressed abundantly in hair follicles and to a lesser extent in basal epidermal keratinocytes in mouse skin. Mice lacking sPLA2-IIE exhibited skin abnormalities distinct from those in mice lacking sPLA2-IIF, with perturbation of hair follicle ultrastructure, modest changes in the steady-state expression of a subset of skin genes, and no changes in the features of psoriasis or contact dermatitis. Lipidomics analysis revealed that sPLA2-IIE and -IIF were coupled with distinct lipid pathways in the skin. Overall, two skin sPLA2s, hair follicular sPLA2-IIE and epidermal sPLA2-IIF, play non-redundant roles in distinct compartments of mouse skin, underscoring the functional diversity of multiple sPLA2s in the coordinated regulation of skin homeostasis and diseases.

Hemidesmosomes: how much plakins do they need?

Hemidesmosomes: how much plakins do they need?

Paclitaxel-induced epithelial damage and ectopic MMP-13 expression promotes neurotoxicity in zebrafish

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in cancer treatment and in a number of curative and palliative regimens. Despite its beneficial effects on cancer, paclitaxel also damages healthy tissues, most prominently the peripheral sensory nervous system. The mechanisms leading to paclitaxel-induced peripheral neuropathy remain elusive, and therapies that prevent or alleviate this condition are not available. We established a zebrafish in vivo model to study the underlying mechanisms and to identify pharmacological agents that may be developed into therapeutics. Both adult and larval zebrafish displayed signs of paclitaxel neurotoxicity, including sensory axon degeneration and the loss of touch response in the distal caudal fin. Intriguingly, studies in zebrafish larvae showed that paclitaxel rapidly promotes epithelial damage and decreased mechanical stress resistance of the skin before induction of axon degeneration. Moreover, injured paclitaxel-treated zebrafish skin and scratch-wounded human keratinocytes (HEK001) display reduced healing capacity. Epithelial damage correlated with rapid accumulation of fluorescein-conjugated paclitaxel in epidermal basal keratinocytes, but not axons, and up-regulation of matrix-metalloproteinase 13 (MMP-13, collagenase 3) in the skin. Pharmacological inhibition of MMP-13, in contrast, largely rescued paclitaxel-induced epithelial damage and neurotoxicity, whereas MMP-13 overexpression in zebrafish embryos rendered the skin vulnerable to injury under mechanical stress conditions. Thus, our studies provide evidence that the epidermis plays a critical role in this condition, and we provide a previously unidentified candidate for therapeutic interventions.