Notch Signaling May Be Involved in the Abnormal Differentiation of Epidermal Keratinocytes in Psoriasis

Localization of each keratin isoform differs among epidermal layers. Proliferating basal cells synthesize keratin 14 (K14) and suprabasal cells express keratin 10 (K10) in normal skin. Notch signaling is essential for keratinocyte differentiation. Notch1 is expressed in all epidermal layers, Notch2 in the basal cell layer and Notch3 in basal cell and spinous cell layers in normal epidermis. It has been poorly elucidated how localization and expression levels of Notch molecules are related to epidermal molecular markers K10 and K14 in psoriatic skin with abnormal differentiation of epidermal tissue. This study aimed to investigate the relationship between abnormal differentiation of epidermal cells in psoriatic skin and expression of Notch molecules. We investigated keratins (K14 and K10) and Notches (1, 2, 3 and 4) using immunohistochemistry in psoriatic skin (n=30) and normal skin (n=10). In normal skin, K14 and K10 were discretely observed in the basal cell layer and suprabasal layer, respectively. In psoriatic skin, K14 was expressed in the pan epidermal layer while it and K10 were co-expressed in some middle suprabasal layer cells. Notch1, 2, 3, and 4 localized in all epidermal layers in normal skin. In psoriatic skin, Notch1, 2, and 4 mainly localized in suprabasilar layers and Notch3 is lacalized in pan epidermal, suprabasilar, and basilar layers. Protein and mRNA of Notch1, 2, and 3 isoforms decreased in psoriatic epidermis compared with normal epidermis. These data suggest that decrements in these Notch molecules might cause aberrant expression of K10 and K14 leading to anomalous differentiation of the epidermis in psoriatic lesions.     

Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development, and differentiation. Normalization of the nu/nu follicular differentiation defect and amelioration of chemotherapy-induced alopecia.

The growth and development of hair follicles is influenced by a number of different growth factors and cytokines, particularly members of the fibroblast growth factor (FGF) family. Keratinocyte growth factor (KGF or FGF-7) is a recently identified 28-kd member of the FGF family that induces proliferation of a wide variety of epithelial cells, including keratinocytes within the epidermis and dermal adnexa. Because KGF induces marked proliferation of keratinocytes, and both KGF and KGF receptor (KGFR) mRNA are expressed at high levels in skin, we sought to localize KGF and KGFR in skin by in situ hybridization. KGFR mRNA was relatively strongly expressed by keratinocytes in the basilar epidermis as well as throughout developing hair follicles of rat embryos and neonates. KGF mRNA was expressed at lower levels than was KGFR but could be localized to follicular dermal papillae in rat embryos and neonates. These results prompted us to investigate the effects of KGF on hair follicles in two distinct murine models of alopecia. In the first model, recombinant KGF (rKGF) induced dose-dependent hair growth over most of the body in nu/nu athymic nude mice when administered intraperitoneally or subcutaneously over 17 to 18 days. When administered subcutaneously, rKGF induced the most extensive hair growth at the sites of injection. Histologically, rKGF induced marked follicular and sebaceous gland hypertrophy, a normalization of the nu/nu follicular keratinization defect, and an increase in follicular keratinocyte proliferation as assessed by bromodeoxyuridine labeling. In the second model, a neonatal rat model of cytosine arabinoside chemotherapy-induced alopecia in which interleukin-1, epidermal growth factor, and acidic FGF have all demonstrated some degree of alopecia cytoprotection, rKGF induced a dose-dependent cytoprotective effect, abrogating as much as 50% of the alopecia in this model when administered beginning 1 day before the onset of chemotherapy. Taken together, these data suggest that KGF is an important endogenous mediator of normal hair follicle growth, development, and differentiation.     

Apoptosis mediated by cytolytic molecules might be responsible for maintenance of psoriatic plaques

Psoriasis is a chronic hyperproliferative skin disease characterized by keratinocyte hyperproliferation and inflammation. It is generally considered as an autoimmune disease mediated by T cells. The precise mechanism of triggering keratinocyte hyperproliferation is as yet unknown. Apoptosis seems to be important in the maintenance of skin cell homeostasis as well as in the pathogenesis of some skin diseases. We hypothesize how apoptosis mediated by cytolytic mechanisms could be involved in initiating and maintenance of psoriatic plaque. Increased keratinocyte hyperproliferation might develop as a consequence of failure to remove self-reactive T cells by apoptosis that in other way cause significant keratinocyte damage. Apoptotic keratinocytes might trigger an injury response program causing regenerative hyperplasia of epidermal keratinocytes. Another possibility is that the failure to eliminate these abnormal keratinocytes could result in the persistence of chronic inflammatory conditions constantly recruiting specific T cells. Increased epidermal thickness in psoriasis could be also explained by imbalance between the expression of pro- and anti-apoptotic proteins. Epidermal keratinocytes have the ability to produce cytolytic molecules, thus they might also have the potential to protect the epidermis from T cell-mediated damage. In conclusion, hyperproliferation of psoriatic keratinocytes might be partly due to changes in the keratinocyte expression of pro- and anti-apoptotic genes, partly to the damaged keratinocytes triggering an inappropriate wound repair response and partly by the failure to eliminate these abnormal keratinocytes resulting in the persistence of chronic inflammation. Each of the proposed mechanisms might be a possible therapeutic target mainly by new immunomodulatory agents.     

Keratinocytes express functional CARD18, a negative regulator of inflammasome activation,and its altered expression in psoriasis may contribute to disease pathogenesis

Caspase recruitment domain family member 18 (CARD18, Iceberg) is known as a negative regulatory molecule that inhibits inflammatory events by terminating inflammasome activation due to a direct interaction with pro-caspase-1.During the investigation of molecular mechanisms in keratinocytes that contribute to the pathogenesis of psoriasis, we found that CARD18 expression differs in healthy and psoriatic skin; moreover, CARD18 demonstrated altered response under inflammatory conditions in healthy and psoriatic skin. In healthy skin, low basal CARD18 expression was detected, which showed significant elevation in response to inflammatory stimuli (lymphokine treatment or mechanical injury). In contrast, higher basal expression was observed in psoriatic non-involved skin, but no further induction could be detected.We demonstrated that keratinocytes express CARD18 both at mRNA and protein levels and the expression increased in parallel with differentiation. The investigation of cellular inflammatory processes revealed that psoriasis-associated danger signals triggered the expression of inflammasome components (AIM2, Caspase-1) and CARD18 as well as IL-1β production of keratinocytes. Furthermore, gene-specific silencing of CARD18 in cells treated with cytosolic DNA (poly(dA:dT)) resulted in increased IL-1β secretion, suggesting a negative regulatory role for CARD18 in keratinocyte inflammatory signaling.The differential regulation of CARD18 in healthy and psoriatic uninvolved epidermis may contribute to the susceptibility of psoriasis. Furthermore, our in vitro results indicate that CARD18 may contribute to the fine tuning of keratinocyte innate immune processes.     

A study on altered expression of serine palmitoyltransferase and ceramidase in psoriatic skin lesion

Ceramides are the main lipid component maintaining the lamellae structure of stratum corneum, as well as lipid second messengers for the regulation of cellular proliferation and/or apoptosis. In our previous study, psoriatic skin lesions showed marked decreased levels of ceramides and signaling molecules, specially protein kinase C-alpha (PKC-alpha) and c-jun N-terminal kinase (JNK) in proportion to the psoriasis area and severity index (PASI) scores, which suggested that the depletion of ceramide is responsible for epidermal hyperproliferation of psoriasis via downregulation of proapoptotic signal cascade such as PKC-alpha and JNK. In this study, we investigated the protein expression of serine palmitoyltransferase (SPT) and ceramidase, two major ceramide metabolizing enzymes, in both psoriatic epidermis and non-lesional epidermis. The expression of SPT, the ceramide generating enzyme in the de novo synthesis in psoriatic epidermis, was significantly less than that of the non-lesional epidermis, which was inversely correlated with PASI score. However, the expression of ceramidase, the degradative enzyme of ceramides, showed no significant difference between the lesional epidermis and the non-lesional epidermis of psoriatic patients. This might suggest that decreased expression of SPT protein is one of the important causative factors for decreased ceramide levels in psoriasis.     

Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor.

The distribution of TNF-alpha, p55 TNF receptor (TNF-R) and p75 TNF-R in normal skin and uninvolved and lesional skin from psoriasis patients has been investigated, using specific mono- and polyclonal antibodies. In normal skin, and uninvolved and lesional skin from psoriasis patients, p55 TNF-R is associated with epidermal keratinocytes and a network of upper dermal dendritic cells. This suggests that the actions of TNF-alpha on epidermal cells in vivo are mediated by binding to the p55 TNF-R. In lesional psoriasis skin, there was staining of the parakeratotic stratum corneum and increased expression of p55 TNF-R in association with upper dermal blood vessels. Staining for p75 TNF-R in normal skin was restricted to eccrine sweat ducts and dermal dendritic cells, and was absent from the epidermis. In lesional psoriasis skin, there was staining for p75 TNF-R in association with upper dermal blood vessels and perivascular infiltrating cells. TNF-alpha in normal skin was predominantly localized to the basal cell layers of the epidermis, and was seen in association with eccrine ducts and sebaceous glands. In lesional psoriasis skin, and to a lesser extent in uninvolved psoriasis skin, TNF-alpha was distributed throughout the epidermis, and was also specifically localized to upper dermal blood vessels. Up-regulation of TNF-alpha, p55 TNF-R and p75 TNF-R on dermal blood vessels in psoriasis may play an important role in the pathogenesis of this condition by promoting cutaneous recruitment of inflammatory cells.     

Cellular localization of interleukin-8 and its inducer, tumor necrosis factor-alpha in psoriasis.

The importance of immunologic mechanisms in psoriasis has been deduced from the ability of immunosuppressive therapies to ameliorate this common and chronic skin disease. Certainly the histology of psoriatic lesions suggests a dialogue between the hyperplastic keratinocytes and infiltrating T lymphocytes and macrophages. To begin dissecting the cytokine network involved in the pathophysiology of psoriasis, the location, in both epidermal and dermal compartments, of tumor necrosis factor-alpha, interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha at the protein and/or mRNA levels were identified. Tumor necrosis factor-alpha was selected as a potentially key regulatory cytokine, first because it induces cultured keratinocyte interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha production, and second because intercellular adhesion molecule-1 expression by keratinocytes in psoriatic epidermis had been identified previously. Using immunohistochemical localization, tumor necrosis factor-alpha was identified in 12 psoriatic lesions as intense and diffuse expression by dermal dendrocytes (macrophages) in the papillary dermis (without significant staining of endothelial cells, mast cells, or dermal Langerhans cells), and focally by keratinocytes and intraepidermal Langerhans cells. Functional interaction between the dermal dendrocytes and keratinocytes was suggested by the presence of interleukin-8 expression of suprabasal keratinocytes immediately above the tumor necrosis factor-alpha-positive dermal dendrocytes. Interleukin-8 mRNA and transforming growth factor-alpha mRNA were detectable in the epidermal roof of psoriatic lesions, but neither was detectable at the protein or mRNA levels in any normal skin specimens. Treatment of cultured human keratinocytes with phorbol ester (which experimentally produces psoriasiform changes on mouse skin) or tumor necrosis factor-alpha also increased interleukin-8 and transforming growth factor-alpha mRNAs. Further elucidation of the cellular and molecular basis for the genesis and evolution of psoriasis will provide the framework for a better evaluation of the cause and treatment of this skin disease.     

Influence of infliximab on keratinocyte apoptosis in psoriasis patients

Biological drugs targeting tumor necrosis factor-α, such as infliximab, are highly effective in psoriasis. The interference with keratinocyte apoptosis has been included among the possible effects of infliximab in psoriasis, although the available data are still controversial. The purpose of our study was to verify the action of infliximab on psoriatic keratinocytes. Keratinocyte apoptosis was evaluated in the lesional psoriatic skin of 11 patients at baseline and a different time point during treatment with infliximab. Infliximab (5?mg/kg) was given intravenously at weeks 0, 2, and 6, followed by maintenance infusions every 8 weeks. Pretreatment with intravenous hydrocortisone was performed prior to each infusion. Keratinocytes with apoptotic features were histologically identified according to the following changes: chromatin condensation at the periphery of the nucleus, cytoplasmic vesiculation, nuclear fragmentation, nuclear pyknosis. Immunohistochemical assessment of p53 and caspase-3 expression was also performed. At baseline, prior to treatment with infliximab, lesional epidermis showed 1.2–3.2% p53-positive apoptotic keratinocytes in the basal zone. The number of p53-positive apoptotic keratinocytes increased after treatment with infliximab, already at day 1–2 after the first infusion, and such cells were localized at basal and suprabasal layers or were through all layers. There was no immunoreactivity for caspase-3 at any time point examined. Our results suggest that induction of p53-related keratinocyte apoptosis might be one of the mechanisms of infliximab action in psoriasis.     

Altered claudin expression is a feature of chronic plaque psoriasis

Epithelial tight junctions play a central role in cell-cell adhesion and are necessary for the selective paracellular movement of ions. Claudins are key components of tight junctions and their expression is altered in gut epithelia in a variety of inflammatory enteropathies, including ulcerative colitis and Crohn's disease. Psoriasis is a chronic inflammatory skin disease affecting approximately 2% of the western population, with significantly increased occurrence in individuals with Crohn's disease. Initial studies investigated the expression of claudins in skin of healthy volunteers and patients with chronic plaque psoriasis. We report here that claudins-1 and -3 are the major protein species present in the epidermis of healthy skin; they are expressed on the surface of epidermal keratinocytes, consistent with their localization to tight junctions. In plaques of psoriasis, claudin-1 was not identifiable in the epidermis, although typical staining patterns were observed in clinically normal, uninvolved skin of patients with psoriasis. Claudin-3 was present in the epidermal granular cell layer in normal skin, but was only identified within the cytosol of epidermal keratinocytes in both involved and uninvolved skin of psoriasis patients. We examined further whether exposure of keratinocytes in vitro to pro-inflammatory cytokines mimicked the observed changes in claudin expression seen in chronic plaque psoriasis; lipopolysaccharide, interferon-gamma and tumour necrosis factor-alpha had no effect on claudin protein expression or distribution. Addition of interleukin-1beta, however, resulted in down-regulation of claudins-1 and -3. Tumour necrosis factor-alpha and interleukin-1beta were further used in an in vivo model of skin inflammation; interleukin-1beta alone modulated claudin protein expression in this system. These data demonstrate that epidermal claudin expression is altered in chronic plaque psoriasis and that expression is in part modulated by interleukin-1beta.     

Hyaluronan participates in the epidermal response to disruption of the permeability barrier in vivo

Hyaluronan (hyaluronic acid, HA) is a glycosaminoglycan in the extracellular matrix of tissues that plays a role in cellular migration, proliferation and differentiation. Injury to the stratum corneum elicits an epidermal hyperproliferative response, a pathogenic feature in many cutaneous diseases including eczema and psoriasis. Because HA is abundant in the matrix between keratinocytes, we asked whether the presence of HA is required for epidermal hyperplasia to occur in response to barrier injury. Disruption of the stratum corneum, by acetone application on the skin of hairless mice, led to a marked accumulation of HA in the matrix between epidermal basal and spinous keratinocytes, and also within keratinocytes of the upper epidermis. To test whether HA may have a functional role in epidermal hyperplasia, we used Streptomyces hyaluronidase (StrepH), delivered topically, to degrade epidermal HA and blunt the accumulation of epidermal HA after acetone. StrepH signficantly reduced epidermal HA levels, and also significantly inhibited the development of epidermal hyperplasia. This reduction in epidermal thickness was not attributable to any decrease in keratinocyte proliferation, but rather to an apparent acceleration in terminal differentiation (ie, increased keratin 10 and filaggrin expression). Overall, the data show that HA is a significant participant in the epidermal response to barrier injury.     

Arginase 1 overexpression in psoriasis: limitation of inducible nitric oxide synthase activity as a molecular mechanism for keratinocyte hyperproliferation

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.     

Discordant expression of Bcl-x and Bcl-2 by keratinocytes in vitro and psoriatic keratinocytes in vivo.

Apoptosis is a required event in maintaining kinetic homeostasis within continually renewing tissues such as skin. However, no systematic study of the apoptotic process in epidermal keratinocytes of the skin has been performed. In this report, we examined the expression of proteins associated with promoting (Fas) or preventing (Bcl-2, Bcl-x, CD40) apoptosis in the normal, psoriatic, and malignant keratinocyte. Immunohistochemical staining and flow cytometry analysis revealed that normal cultured keratinocytes express low levels of Fas, CD40, and Bcl-x that was enhanced by cytokines including gamma-interferon (IFN-gamma) and a phorbol ester tumor promoter, TPA. Only faint Bcl-2 staining was detected in cultured keratinocytes exposed to IFN-gamma and TPA compared with the prominent expression of Bcl-x. Biopsies of normal skin, psoriatic plaques, and basal cell carcinomas were examined to extend the in vitro observations. Immunohistochemical staining revealed that while keratinocytes in normal epithelium express low to absent levels of Fas and Bcl-x, psoriatic keratinocytes expressed significantly higher levels of Fas and Bcl-x. In contrast, malignant keratinocytes in basal cell carcinomas expressed high levels of Bcl-2, but minimal Bcl-x, and no Fas. Immunoblot analysis revealed that the long form of Bcl-x (Bcl-xI), which prevents apoptosis in lymphocytes, is expressed by cultured keratinocytes and psoriatic plaque keratinocytes. We conclude that normal cytokine-activated keratinocytes can express an apoptotic (Fas) and an anti-apoptotic protein (Bcl-x). The overexpression of Bcl-x in psoriasis, or Bcl-2 in basal cell carcinomas, may contribute to the longevity of these cells by blocking the normal apoptotic process involved in the terminal differentiation program of epidermal keratinocytes.     

The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice

The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.     

Gene from a psoriasis susceptibility locus primes the skin for inflammation

Psoriasis is a common complex genetic disease characterized by hyperplasia and inflammation in the skin; however, the relative contributions of epidermal cells and the immune system to disease pathogenesis remain unclear. Linkage studies have defined a psoriasis susceptibility locus (PSORS4) on 1q21, the epidermal differentiation complex, which includes genes for small S100 calcium-binding proteins. These proteins are involved in extracellular and intracellular signaling during epithelial host defense, linking innate and adaptive immunity. Inflammation-prone psoriatic skin constitutively expresses elevated concentrations of S100A7 (psoriasin) and S100A15 (koebnerisin) in the epidermis. Here, we report that genetically modified mice expressing elevated amounts of doxycycline-regulated mS100a7a15 in skin keratinocytes demonstrated an exaggerated inflammatory response when challenged by exogenous stimuli such as abrasion (Koebner phenomenon). This immune response was characterized by immune cell infiltration and elevated concentrations of T helper 1 (T(H)1) and T(H)17 proinflammatory cytokines, which have been linked to the pathogenesis of psoriasis and were further amplified upon challenge. Both inflammation priming and amplification required mS100a7a15 binding to the receptor of advanced glycation end products (RAGE). mS100a7a15 potentiated inflammation by acting directly as a chemoattractant for leukocytes, further increasing the number of inflammatory cells infiltrating the skin. This study provides a pathogenetic psoriasis model using a psoriasis candidate gene to link the epidermis and innate immune system in inflammation priming, highlighting the S100A7A15-RAGE axis as a potential therapeutic target.     

Inhibitory effect of Paeonia lactiflora Pallas extract (PE) on poly (I:C)-induced immune response of epidermal keratinocytes

Epidermal keratinocytes provide protective role against external stimuli by barrier formation. In addition, kertinocytes exerts their role as the defense cells via activation of innate immunity. Disturbance of keratinocyte functions is related with skin disorders. Psoriasis is a common skin disease related with inflammatory reaction in epidermal cells. We attempted to find therapeutics for psoriasis, and found that Paeonia lactiflora Pallas extract (PE) has an inhibitory potential on poly (I:C)-induced inflammation of keratinocytes. PE significantly inhibited poly (I:C)-induced expression of crucial psoriatic cytokines, such as IL-6, IL-8, CCL20 and TNF-α, via down-regulation of NF-κB signaling pathway in human keratinocytes. In addition, PE significantly inhibited poly (I:C)-induced inflammasome activation, in terms of IL-1β and caspase-1 secretion. Finally, PE markedly inhibited poly (I:C)-increased NLRP3, an important component of inflammasome. These results indicate that PE has an inhibitory effect on poly (I:C)-induced inflammatory reaction of keratinocytes, suggesting that PE can be developed for the treatment of psoriasis.     

Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis.

Psoriasis is a common inherited skin disease that is characterized by hyperproliferation of epidermal keratinocytes and excessive dermal angiogenesis. A growing body of evidence supports a key pathogenetic role for activated keratinocytes in the angiogenic response that accompanies psoriasis. We investigated the role of psoriatic epidermis in the aberrant expression of angiogenesis by examining the ability of pure populations of multipassaged keratinocytes obtained from the skin of normal individuals and psoriatic patients to induce angiogenesis in vivo in the rat corneal bioassay and endothelial cell chemotaxis in vitro. Media conditioned by keratinocytes from psoriatic patients, including both symptomless skin and psoriatic plaques, induced vigorous angiogenic responses in over 90% of corneas tested and potently stimulated directional migration of capillary endothelial cells in vitro. In contrast, conditioned medium from normal keratinocyte cultures was weakly positive in less than 10% of corneas assayed and failed to stimulate endothelial cell chemotaxis. Furthermore, keratinocytes from psoriatic skin exhibited a 10- to 20-fold increase in interleukin-8 production and a seven-fold reduction in thrombospondin-1 production. The angiogenic activity present in keratinocyte-conditioned media from psoriatic patients was suppressed by adding either highly purified thrombospondin-1 (125 ng) or following the addition of either normal keratinocyte-conditioned media or neutralizing interleukin-8 antibody. We conclude that psoriatic keratinocytes are phenotypically different from normal keratinocytes with respect to their angiogenic capacity and that this aberrant phenotype is attributable to a defect in the overproduction of interleukin-8 and a deficiency in the production of the angiogenesis inhibitor thrombospondin-1.     

Possible involvement of keratinocyte growth factor and its receptor in enhanced epithelial-cell proliferation and acquired recurrence of middle-ear cholesteatoma

Middle-ear cholesteatoma is characterized by enhanced proliferation of epithelial cells and granular tissue formation. However, the molecular mechanism underlying these pathological changes is largely unknown. Keratinocyte growth factor (KGF) is a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation. In the present study, we investigated the possible involvement of KGF and its receptor, KGFR, in the pathogenesis of cholesteatoma using in situ hybridization and immunohistochemistry, respectively. We examined 56 cholesteatoma specimens, and 8 normal skin areas as control. KGF and KGFR expression was examined by immunohistochemistry using rabbit anti-human KGF and anti-human KGFR polyclonal antisera raised in our laboratories against synthetic peptides corresponding to parts of human KGF and KGFR, respectively. KGF protein and mRNA were detected exclusively in stromal fibroblasts and infiltrating T lymphocytes in 80% of cholesteatoma cases, whereas KGFR protein and mRNA were localized in the epithelium in 72% of cases. Assessment of the proliferative activity of cholesteatoma using the labeling index for Ki-67 showed a significantly higher Ki-67 labeling index (66%) in KGF+/KGFR+ cases than other cases. There was a significant correlation between KGF+/KGFR+ expression and recurrence. Our results indicate the possible involvement of both KGF and KGFR in enhanced epithelial cell proliferative activity and recurrence of cholesteatoma.     

Peroxiredoxin 6 is a potent cytoprotective enzyme in the epidermis

Peroxiredoxin 6 is an enzyme that detoxifies hydrogen peroxide and various organic peroxides. In previous studies we found strongly increased expression of peroxiredoxin 6 in the hyperproliferative epidermis of wounded and psoriatic skin, suggesting a role of this enzyme in epidermal homeostasis. To address this question, we generated transgenic mice overexpressing peroxiredoxin 6 in the epidermis. Cultured keratinocytes from transgenic mice showed enhanced resistance to the toxicity of various agents that induce oxidative stress. However, overexpression of peroxiredoxin 6 did not affect skin morphogenesis or homeostasis. On skin injury, enhancement of wound closure was observed in aged animals. Most importantly, peroxiredoxin 6 overexpression strongly reduced the number of apoptotic cells after UVA or UVB irradiation. These findings demonstrate that peroxiredoxin 6 protects keratinocytes from cell death induced by reactive oxygen species in vitro and in vivo, suggesting that activation of this enzyme could be a novel strategy for skin protection under stress conditions.