Normal Epidermal Keratinocytes Research Articles

Cigarette smoke condensate induces cytochromes P450 and aldo-keto reductases in oral cancer cells

Our objective is to identify molecular factors which contribute to the increased risk of smokers for oral squamous cell carcinoma (OSCC). In the present study, we investigated the effects of cigarette smoke condensate (CSC) on gene expression profiles in different human oral cell phenotypes: normal epidermal keratinocytes (NHEK), oral dysplasia cell lines (Leuk1 and Leuk2), and a primary oral carcinoma cell line (101A). We determined differential gene expression patterns in CSC-exposed versus non-exposed cells using high-density microarray RNA expression profiling and validation by quantitative real-time RT-PCR. A set of 35 genes was specifically up- or down-regulated following CSC treatment (25 μg/ml for 24 h) by at least 2-fold in any one cell type. Notably, five genes of the cytochrome P450 (CYP1A1, CYP1B1) and aldo-keto reductase (AKR1C1, AKR1C3, AKR1B10) families were highly increased in expression, some of them 15- to 30-fold. The timing and extent of induction for these genes differed among the four cell phenotypes. A potential biological interaction network for the CSC response in oral cells was derived from these data, proposing novel putative response pathways. These CSC-responsive genes presumably participate in the prevention or repair of carcinogen-induced DNA damage in tobacco-related oral carcinogenesis, and may potentially be exploited for determining the severity of exposure and for correcting mutagenic damage in exposed tissues of the oral cavity.

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer

Harlequin ichthyosis (HI) is a devastating skin disorder with an unknown underlying cause. Abnormal keratinocyte lamellar granules (LGs) are a hallmark of HI skin. ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily are known to have closely related functions as lipid transporters. ABCA3 is involved in lipid secretion via LGs from alveolar type II cells, and missense mutations in ABCA12 have been reported to cause lamellar ichthyosis type 2, a milder form of ichthyosis. Therefore, we hypothesized that HI might be caused by mutations that lead to serious ABCA12 defects. We identify 5 distinct ABCA12 mutations, either in a compound heterozygous or homozygous state, in patients from 4 HI families. All the mutations resulted in truncation or deletion of highly conserved regions of ABCA12. Immunoelectron microscopy revealed that ABCA12 localized to LGs in normal epidermal keratinocytes. We confirmed that ABCA12 defects cause congested lipid secretion in cultured HI keratinocytes and succeeded in obtaining the recovery of LG lipid secretion after corrective gene transfer of ABCA12. We concluded that ABCA12 works as an epidermal keratinocyte lipid transporter and that defective ABCA12 results in a loss of the skin lipid barrier, leading to HI. Our findings not only allow DNA-based early prenatal diagnosis but also suggest the possibility of gene therapy for HI.

CRH stimulates POMC activity and corticosterone production in dermal fibroblasts

It has been previously documented that human skin cells including epidermal keratinocytes and dermal fibroblasts produce and process proopiomelanocortin (POMC), corticotropin releasing hormone (CRH), and express functional CRH receptors type-1 (CRH-R1). The skin also has corticosteroidogenic activity, suggesting a functional connection between these elements. In the current study, we found that human dermal fibroblasts (but not normal epidermal keratinocytes) respond to CRH with stimulation of cAMP, with POMC gene and protein expression, and ACTH production and release. Furthermore, CRH and ACTH stimulate production of corticosterone in fibroblasts, with ACTH being more potent. Although cortisol-immunoreactivity accumulation/production in fibroblasts has been detected by ELISA, it appears to be constitutive (not affected by CRH or ACTH). These effects are absent in keratinocytes. Therefore, we propose that fibroblasts but not keratinocytes display a functional CRH–POMC–corticosteroid axis organized similarly to the hypothalamus–pituitary–adrenal (HPA) axis. However, it diverges from the HPA organization in its distal step, where CRH and ACTH stimulate production of corticosterone, instead of cortisol.

Phospholipase Cγ1 Is Required for Activation of Store-Operated Channels in Human Keratinocytes

Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R.

Blue light differentially alters cellular redox properties

Blue light (lambda = 380-500 nm) historically has been used to initiate polymerization of biomaterials and recently has been proposed as a therapeutic agent. New evidence suggests that cell-type-specific responses result from redox changes induced by exposure to blue light. Cultured cells were exposed to defined doses of blue light, equivalent to exposure times of 10 s and 2 min, to achieve energies of 5 J/cm2 and 60 J/cm2, respectively, after which (a) viable cell number, (b) cellular protein profiles, (c) mitochondrial succinate dehydrogenase (SDH) activity, (d) total reactive oxygen species (ROS), and (e) induction of apoptosis were compared to that of nonexposed control cultures. Results showed that blue-light exposure arrested monocyte cell growth and increased levels of peroxiredoxins. SDH activity of normal epidermal keratinocytes (NHEK) was slightly enhanced by blue light, whereas identical treatment of OSC2 oral tumor cells resulted in significant suppression of SDH activity. Blue-light exposure generally induced higher levels of total ROS in OSC2 cells than in NHEK. Finally, only OSC2 cells exhibited signs of apoptosis via Annexin V staining following exposure to blue light. These data support the central hypothesis that blue light induces an oxidative stress response in cultured cells resulting in cell-type-specific survival outcomes. The identification of oxidative stress as a mediator of the effects of blue light is a critical first step in defining its biological risks and therapeutic opportunities.

Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes.

Beneficial effects attributed to green tea, such as its anticancer and antioxidant properties, may be mediated by (-)-epigallocatechin-3-gallate (EGCG). In this study, the effects of EGCG on cell proliferation and UV-induced apoptosis were investigated in normal epidermal keratinocytes. When topically applied to aged human skin, EGCG stimulated the proliferation of epidermal keratinocytes, which increased the epidermal thickness. In addition, this topical application also inhibited the UV-induced apoptosis of epidermal keratinocytes. EGCG was found to increase the phosphorylation of Bad protein at the Ser112 and Ser136. Moreover, EGCG-induced Erk phosphorylation was found to be critical for the phosphorylation of Ser112 in Bad protein, and the EGCG-induced activation of the Akt pathway was found to be involved in the phosphorylation of Ser136. Furthermore, EGCG increased Bcl-2 expression but decreased Bax expression, causing an increase in the Bcl-2-to-Bax ratio. In addition, we demonstrate the differential growth inhibitory effects of EGCG on cancer cells. In conclusion, this study demonstrates that EGCG promotes keratinocyte survival and inhibits the UV-induced apoptosis via two mechanisms: by phosphorylating Ser112 and Ser136 of Bad protein through Erk and Akt pathways, respectively, and by increasing the Bcl-2-to-Bax ratio. Moreover, these two proposed mechanisms of EGCG-induced cell proliferation may differ kinetically to promote keratinocyte survival.

Isolation and clonal analysis of human epidermal keratinocyte stem cells in long-term culture.

We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence. Clonal analysis revealed the presence of holoclones, meroclones, and paraclones. Only emerging colonies with high proliferative potentials and extensive capacities for division (holoclones and meroclones) were subcultured, favoring the expansion of stem cells and progenitors capable of prolonged self-maintenance when subcloned, thus accounting for the prevailing long-term proliferation of the original culture. We found that meroclones included bipotent progenitors capable of generating both keratinocytes and mucin-producing cells. The numbers of these cells were greater after confluence, suggesting that commitment for their differentiation occurred late in the life of a single clone. On a three-dimensional gelatin matrix and on a collagen layer containing the fibroblast feeder, cells isolated from the expansion of holoclones and meroclones formed stratified cohesive layers of keratinocytes that were able to further differentiate, as in normal skin. These results indicate that our procedure will serve as a valuable tool to study expansion of epidermal stem cells as well as the growth mechanisms and cell products associated with their growth and differentiation.

Platelet Derived Growth Factor (PDGF) Responsive Epidermis Formed from Human Keratinocytes Transduced with the PDGFβ Receptor Gene

Platelet-derived growth factor is a major proliferative and migratory stimulus for connective tissue cells during the initiation of skin repair processes. In response to injury, locally produced platelet-derived growth factor is secreted by a diversity of cutaneous cell types whereas target activity is confined to cells of mesenchymal origin, e.g. dermal fibroblasts and smooth muscle cells. Although epidermal cells contribute to cutaneous platelet-derived growth factor activity by their ample capacity to secrete platelet-derived growth factor ligand, normal epidermal keratinocytes are not known to express any member of the platelet-derived growth factor receptor family. In order to study if epidermis may be genetically transformed to a platelet-derived growth factor sensitive compartment we aimed to introduce the gene encoding human platelet-derived growth factor receptor beta (PDGF beta R) into epidermal keratinocytes using a retrovirus-derived vector. Successful gene transfer to primary cells was confirmed by immunofluorescence staining, southern blotting, and ligand-induced receptor autophosphorylation. By culturing a mixture of PDGF beta R-transduced and unmodified keratinocytes at the air-liquid interface on devitalized dermis, we were able to establish a multilayered epithelium showing histologic similarities to that evolved from native keratinocytes or keratinocytes transduced with the reporter gene encoding enhanced green fluorescent protein. Receptor-modified epidermal tissue cultured for 6 days and examined by immunofluorescence microscopy was shown to contain PDGF beta R-expressing keratinocytes distributed in all layers of living epidermis. By continued tissue culture in serum-containing medium, the epidermis became increasingly cornified although receptor-positive cells were still observed within the viable basal compartment. Stimulation of PDGF beta R-transduced epidermis with recombinant platelet-derived growth factor BB had a mitogenic effect as reflected by an increased frequency of Ki-67 positive keratinocytes. The study demonstrates that transgene expression of human PDGF beta R can be achieved in epidermal keratinocytes by retroviral transduction, and that ligand activation of such gene-modified skin equivalent enhances cell proliferation. In perspective, viral PDGF beta R gene transfer to keratinocytes may be a useful approach in studies of receptor tyrosine kinase mediated skin repair and epithelialization.

Dynamic Characterization of the Molecular Events During In Vitro Epidermal Wound Healing

The aim of this study was to characterize some of the molecular events stimulated in vitro in response to injury within a confluent culture of normal epidermal keratinocytes as a model to understand the mechanisms of wound healing. To this end, an original device was developed specifically designed to perform calibrated injuries of great lengths within mono-stratified or pluri-stratified keratinocyte cultures. The experiments performed in this study validate this device as an appropriate tool for studying epidermal wound healing; this is because it performs mechanical injuries that stimulate the expression of multiple healing markers also known to be upregulated during wound healing in vivo (growth factors, cytokines, proteinases, extracellular matrix proteins). Using this device, it was demonstrated in human keratinocytes: mechanical injuries (i) immediately stimulate the tyrosine phosphorylation of numerous cellular proteins; (ii) induce molecular cascades leading to the activation of p21ras, mitogen-activated protein kinases, extracellular signal-regulated kinases 1/2, c-Jun NH2 terminal kinase, and p38 mitogen-activated protein kinase; and (iii) increase the phosphorylation of their respective substrates, c-jun and activator transcription factor 1. Wounding of these cells also results in increases in the DNA binding activities of several jun/fos activator protein-1 transcription factor complexes. It is important to note that the development of an appropriate wounding system was essential for performing this study, as use of a classical wounding procedure did not enable the detection of the biologic parameters reported above. In conclusion, these data indicate that using the appropriate system, it is possible to identify the signaling pathways activated in normal human keratinocyte cells after injury. In this study, it was shown that the mitogen-activated protein kinase pathways and activator protein-1 are stimulated in response to physical injury, and may be involved in regulating the expression of healing markers.

Protective effect of vitamin E on ultraviolet B light-induced damage in keratinocytes.

Ultraviolet (UV) B radiation is the most common environmental factor in the pathogenesis of skin cancer. Exposure of human skin to UVB radiation leads to the depletion of cutaneous antioxidants, the activation of nuclear factor kappa B (NF-kappaB), and programmed cell death (apoptosis). Although antioxidant supplementation has been shown to prevent UVB-induced photooxidative damage, its effect on components of cell signaling pathways leading to gene expression has not been clearly established. In the present study, the effect of the antioxidant vitamin, alpha-tocopherol (alpha-T), and its acetate analog, alpha-tocopherol acetate (alpha-TAc), on UVB-induced damage in primary and neoplastic mouse keratinocytes was investigated. The ability of both vitamins to modulate UVB-induced apoptosis and activation of the transcription factor NF-kappaB were studied. Treatment of normal and neoplastic mouse epidermal keratinocytes (308 cells) with 30-60 mJ/cm(2) UVB markedly decreased viable cell number and was accompanied by DNA fragmentation. When both vitamins were applied to cells at times before and after UVB radiation, a significant increase in the percentage of viable cells and concomitant decrease in the number of apoptotic cells was noted, with vitamin pretreatment providing a better protection than posttreatment. Simultaneous posttreatment of irradiated cells with alpha-TAc abolished the cytotoxic effects of UVB and restored cell viability to control levels. In addition, simultaneous posttreatment of irradiated cells with alpha-T reduced the number of apoptotic cells by half, indicating a synergistic effect of two such treatments compared with any single one. Flow cytometry analysis indicated that vitamin treatment suppressed both an increase in pre-G0 cells and a decrease in cycling cells by UVB exposure. In addition, NF-kappaB activation was detected 2 h after UV exposure and was maintained for up to 8 h. Pretreatment with vitamins significantly inhibited NF-kappaB activation at 4 and 8 h. These results indicate that vitamin E and its acetate analog can modulate the cellular response to UVB partly through their action on NF-kappaB activation. Thus, these antioxidant vitamins are potential drugs for the protection from or the reduction of UVB-associated epidermal damage.

Localization of Calcineurin/NFAT in Human Skin and Psoriasis and Inhibition of Calcineurin/NFAT Activation in Human Keratinocytes by Cyclosporin A

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.

Laminin-gamma2 overexpression in head-and-neck squamous cell carcinoma.

To identify molecular markers for the progression of head-and-neck squamous cell carcinoma (HNSCC), we used RNA arbitrarily primed (RAP) PCR to determine the qualitative and quantitative differences in gene expression between normal epithelial cells, those derived from dysplastic oral mucosa and invasive and metastatic HNSCC. Three differentially expressed DNA fragments (RAP20, RAP21, RAP26) that were upregulated in a tumor cell line (T45) were identified as being regions of the gamma2 subunit of human laminin-5. Northern blot analysis of total cellular RNA revealed overexpression of these transcripts in 6 of 7 HNSCC cell lines compared with normal epidermal keratinocytes. In contrast, no differences were observed in HeLa (cervical carcinoma) or HCT116 (colon carcinoma) cells. Immunostaining of HNSCC cells derived from primary (HN4) and metastatic (HN12) tumors indicated elevated levels of endogenous laminin-gamma2 protein. Furthermore, HNSCC tissues demonstrated strong laminin-gamma2 staining, particularly in the peripheral basaloid cells of tumor islands at the invasion front. In contrast, only minimal staining of laminin-gamma2 was detected in basal cells of the normal epithelium. The data indicate that laminin-gamma2 is frequently overexpressed in HNSCCs and derivative cell lines and that its overexpression is likely to be useful as a marker of head-and-neck squamous malignancy.

Regulation of Keratin Expression by Ultraviolet Radiation: Differential and Specific Effects of Ultraviolet B and Ultraviolet A Exposure

Skin, the most superficial tissue of our body, is the first target of environmental stimuli, among which is solar ultraviolet radiation. Very little is known about the regulation of keratin gene expression by ultraviolet radiation, however, although (i) it is well established that ultraviolet exposure is involved in skin cancers and photoaging and (ii) keratins represent the major epidermal proteins. The aim of this study was to analyze the regulation of human keratin gene expression under ultraviolet B (290-320 nm) or ultraviolet A (320-400 nm) irradiation using a panel of constructs comprising different human keratin promoters cloned upstream of a chloramphenicol acetyl transferase reporter gene and transfected into normal epidermal keratinocytes. By this approach, we demonstrated that ultraviolet B upregulated the transcription of keratin 19 gene and to a lesser extent the keratin 6, keratin 5, and keratin 14 genes. The DNA sequence responsible for keratin 19 induction was localized between -130 and +1. In contrast to ultraviolet B, ultraviolet A irradiation induced only an increase in keratin 17, showing a differential gene regulation between these two ultraviolet ranges. The induction of keratin 19 was confirmed by studying the endogenous protein in keratinocytes in classical cultures as well as in skin reconstructed in vitro and normal human skin. These data show for the first time that keratin gene expression is regulated by ultraviolet radiation at the transcriptional level with a specificity regarding the ultraviolet domain of solar light.

Characterization of Retinoic Acid Receptor-deficient Keratinocytes

Retinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARalpha, -beta, and -gamma) are transducers of the retinoid signal. The epidermis expresses RARgamma and RARalpha, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARalpha, RARgamma, and RARalphagamma null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARgamma is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl)-retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARalpha and RARgamma differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes.

Pathogenesis of Paget's Disease: Epidermal Heregulin- , Motility Factor, and the HER Receptor Family

In Paget's disease of the breast, the epidermis of the nipple is infiltrated by large neoplastic cells of glandular origin. It has been hypothesized that the spread of Paget cells through the nipple epidermis is induced by a motility factor that acts via the HER2/NEU receptor. To test this hypothesis, we characterized and purified a motility factor released by keratinocytes and identified its target receptors in specimens from patients with Paget's disease and in SK-BR-3 breast adenocarcinoma cells, which overexpress HER2/NEU. We isolated the motility factor from keratinocyte-conditioned medium and sequenced tryptic peptides. These sequences were used to identify the motility factor as heregulin-alpha, which is released by skin keratinocytes. Heregulin-alpha induces spreading, motility, and chemotaxis of SK-BR-3 cells, as does motility factor. Motility factor activities of heregulin-alpha are inhibited by monoclonal antibody AB2, directed against the extracellular domain of HER2/NEU, which blocks the binding of heregulin-alpha. We used in situ hybridization to show that normal epidermal cells produce heregulin-alpha messenger RNA and that heregulin receptors, HER3 and/or HER4, as well as their coreceptor HER2/NEU, are expressed by Paget cells. Heregulin-alpha is a motility factor that is produced and released by normal epidermal keratinocytes and thus plays a key role in the pathogenesis of Paget's disease. Paget cells express heregulin receptors HER2/NEU, as well as HER3 and/or HER4, both of which function as a co-receptor of HER2/NEU. Binding of heregulin-alpha to the receptor complex on Paget cells results in the chemotaxis of these breast cancer cells, which eventually migrate into the overlying nipple epidermis.

Evidence for intracellular cleavage of plasminogen activator inhibitor type 2 (PAI-2) in normal epidermal keratinocytes.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain primarily active, nonglycosylated PAI-2. In keratinocytes, the vast majority of PAI-2 is retained within the cell, supporting the hypothesis that PAI-2 may serve specific intracellular function(s) through interaction with an unknown cytoplasmic proteinase. During interaction with the target proteinase, cleavage of PAI-2 within its reactive site loop leads to the formation of a more stable, "relaxed" conformation (PAI-2r). Using a monoclonal antibody specific for PAI-2r, we demonstrate here that PAI-2r is present in keratinocytes of the granular and basal layers of normal human epidermis. In addition, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiating cells. These data provide evidence for the presence of an endogenous, keratinocyte-derived proteinase that constitutively cleaves intracellular PAI-2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this proteinase may reflect specific intracellular action of PAI-2 in normal cells. Finally, we demonstrate that a commercially available anti-PAI-2 monoclonal antibody (#3750, American Diagnostica, Greenwich, CT), under native experimental conditions, preferentially recognizes the uncleaved, active form of PAI-2 and does not efficiently detect PAI-2r.

CD2-mediated CD59 stimulation in keratinocytes results in secretion of IL-1alpha, IL-6, and GM-CSF: implications for the interaction of keratinocytes with intraepidermal T lymphocytes.

Normal epidermal keratinocytes are here shown to express membrane-associated complement inhibitory protein CD59 in vitro that protects keratinocytes from damage by complement because preincubation with blocking antibodies to CD59 renders the cells susceptible to complement mediated lysis. CD59 expression in keratinocytes is constitutive and not modulated by inflammatory cytokines, phorbol myristate acetate (PMA), and a number of other agents tested. Antibody mediated cross-linking of CD59, however, revealed an additional function of CD59: keratinocytes in vitro are activated to secrete the cytokines IL-1alpha, IL-6, and GM-CSF. CD59 mediated induction of these cytokines is regulated at the transcriptional level. Binding of keratinocytes to HL60 cells that express CD59 ligand CD2 induced the same pattern of secreted cytokines whereas binding to CD2-negative HL60 cells did not. Induction of cytokine secretion was completely blocked by preincubation of keratinocytes with both anti-CD58 and anti-CD59 antibodies together. The results demonstrate that CD2-mediated CD59 stimulation in human keratinocytes leads to synthesis of a particular set of cytokines implying a potential activation pathway in the interaction of keratinocytes with intraepithelial CD2+ T cells.

Elaboration of Matrix Metalloproteinase Inhibitors by Human Skin in Organ Culture and by Skin Cells in Monolayer Culture: Relationship to Invasion

Functional and immunochemical approaches were used to assess matrix metalloproteinase (MMP) inhibitors, e.g., tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2), in organ cultures of normal human skin maintained under growth factor free conditions or in medium supplemented with a combination of growth factors including epidermal growth factor, insulin, and pituitary extract. It has previously been shown that under growth factor free conditions, normal skin structure and function are maintained for several days, while in the presence of these exogenous growth factors, the epithelial cells invade the stroma [Invasion and Metastasis 1993;13:225–233]. TIMP-1 was detected in equivalent amounts in organ culture fluids under both conditions. TIMP-2 was not detected under either condition. Normal epidermal keratinocytes, normal dermal fibroblasts, and three different epithelial tumor cell lines were also examined for MMP inhibitor expression. Keratinocytes and fibroblasts produced high levels of both TIMP-1 and TIMP-2, but in neither cell type was there a significant difference between growth factor free and growth factor containing conditions. In contrast, the three epithelial tumor cell lines produced low to undetectable levels of both TIMP-1 and TIMP-2. These data suggest that acquisition of local invasive capacity is not dependent on a reduction in MMP inhibitor expression. A reduction in MMP inhibitors may accompany the transition from invasive to metastatic tumors.

RANTES expression in psoriatic skin, and regulation of RANTES and IL-8 production in cultured epidermal keratinocytes by active vitamin D3 (tacalcitol).

The chemokine RANTES is a chemoattractant for eosinophils, T lymphocytes of memory phenotype and monocytes, suggesting that it plays an important part in chronic inflammatory and allergic diseases. In various types of cells, RANTES production is markedly induced by tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma in combination. Psoriasis vulgaris is a chronic cutaneous inflammatory disease. Cytokines and chemokines produced by T cells and epidermal keratinocytes, such as interleukin (IL) 8, are involved in the pathogenesis of psoriasis. T-cell clones obtained from psoriatic skin have been shown to produce the Th1 cytokine IFN-gamma. In addition, abnormal expression of proinflammatory cytokines including TNF-alpha has been observed in psoriatic lesions. These reports led us to hypothesis that psoriatic skin could provide epidermal keratinocytes with TNF-alpha and IFN-gamma, so that keratinocytes could produce RANTES. In this study, we addressed the question as to whether RANTES was involved in psoriasis vulgaris. Immunohistochemistry of skin biopsies showed RANTES was present in the intercellular spaces between epidermal keratinocytes, in the fully developed lesions from the middle to the edge of psoriatic plaques, but not in the perilesional uninvolved and healthy control skin. Further, we confirmed the production of RANTES, together with IL-8, by cultured normal human epidermal keratinocytes, using an enzyme-linked immunosorbent assay. Stimulation with TNF-alpha and IFN-gamma in combination synergistically increased the RANTES production in this system. These results clearly demonstrate the expression of RANTES in psoriatic lesions and suggest the involvement of this chemokine in the outcome of cutaneous inflammatory diseases. Tacalcitol (1 alpha,24(R)-dihydroxyvitamin D3), an active vitamin D3 analogue, inhibited RANTES and IL-8 production in cultured normal epidermal keratinocytes. This result indicates that active vitamin D3 is effective in the regulation of chemokine production by epidermal keratinocytes, which may partly account for its action as an antipsoriatic drug.

CD44 variant expression in cutaneous T-cell lymphoma.

Expression of the lymphocyte homing receptor CD44 and its splice variants have been linked to tumour dissemination and poor prognosis in non-Hodgkin's lymphoma. Specifically, the in vitro expression of variant exon V6 confers metastatic potential in rat pancreatic carcinoma cell lines. In this study, we investigated the expression of CD44 splice variants in cutaneous T-cell lymphomas, including patients with mycosis fungoides (MF), Sezary syndrome (SS), large-cell anaplastic lymphoma (LCAL) and HTLV1-associated cutaneous lymphoma. In addition, 4 involved lymph nodes from 2 patients with MF and 1 patient with SS were examined. Inflammatory dermatoses, lichen planus and psoriasis, and normal skin were also studied. Immunohistochemistry was performed using a panel of monoclonal antibodies, including those with specificity for CD44H (standard isoform) and variant exons V3, V6 and V8-9. Normal epidermal keratinocytes were consistently CD44H and CD44 V3, V6 and V8-9 positive. In all the different clinicopathological subtypes and stages of cutaneous T-cell lymphomas, including involved lymph nodes, tumour cells consistently expressed CD44H, but were CD44 V3 and V6 negative. CD44 V8-9 was expressed on a majority of tumour cells in 2/5 LCAL and on occasional tumour cells in 2/5 LCAL. Occasional V8-9 positive tumour cells were also identified in 6/13 MF, 1/4 SS and 3/4 HTLV1. In 2/3 lymph node samples from 2 patients with tumour-stage MF, CD44 V8-9 expression was found on a small percentage of atypical mononuclear cells. Scattered V8-9 positive dermal mononuclear cells were present in sections of lichen planus and psoriasis. We have found no evidence to suggest that the metastasis-associated CD44 variant exon (V6) is expressed in cutaneous T-cell lymphoma, or that CD44H expression is associated with an adverse prognostic group. It is not clear whether the strong expression of CD44 V8-9 in 2 patients with CD30 positive LCAL reflects activation status or metastatic potential.