Integrins of normal human epidermis: differential expression, synthesis and molecular structure

The expression of integrin cell surface receptors in normal skin and their synthesis and molecular structure in keratinocyte cultures was investigated. The reactivity of four different polypeptides of the integrin family (alpha 2-, alpha 3-, alpha 6- and beta 1-chains) was demonstrated in the basal cell layer of normal epidermis. Studies of labelled keratinocyte cell lines showed that the polypeptides were expressed as alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins. Only the alpha 6 beta 4 integrin showed polarization towards the basement membrane attachment site of basal layer keratinocytes, and was preferentially expressed at microvillous projections. In contrast, alpha 2 beta 1 and alpha 3 beta 1 integrins were equally expressed throughout the basal cell plasma membrane.     

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha5 chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha5 chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (alpha5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha3beta1 and alpha6beta4 integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.     

Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein

Basal keratinocytes in the epidermis and hair follicle are biologically heterogeneous but must include a stable subpopulation of epidermal stem cells. In animal models these can be identified by their retention of radioactive label due to their slow cycle (label-retaining cells) but human studies largely depend on in vitro characterization of colony forming efficiency and clonogenicity. Differential integrin expression has been used to detect cells of increased proliferative potential but further stem cell markers are urgently required for in vivo and in vitro characterization. Using LHM2, a monoclonal antibody reacting with a high molecular weight melanoma-associated proteoglycan core protein, a subset of basal keratinocytes in both the interfollicular epidermis and the hair follicle has been identified. Coexpression of melanoma-associated chondroitin sulfate proteoglycan with keratins 15 and 19 as well as beta 1 and alpha 6 integrins has been examined in adult and fetal human skin from hair bearing, nonhair bearing, and palmoplantar regions. Although melanoma-associated chondroitin sulfate proteoglycan coexpression with a subset of beta 1 integrin bright basal keratinocytes within the epidermis suggests that melanoma-associated chondroitin sulfate proteoglycan colocalizes with epidermal stem cells, melanoma-associated chondroitin sulfate proteoglycan expression within the hair follicle was more complex and multiple subpopulations of basal outer root sheath keratinocytes are described. These data suggest that epithelial compartmentalization of the outer root sheath is more complex than interfollicular epidermis and further supports the hypothesis that more than one hair follicle stem cell compartment may exist.     

Loss of integrin alpha9beta1 results in defects in proliferation, causing poor re-epithelialization during cutaneous wound healing

The wound microenvironment comprises constituents, such as the extracellular matrix (ECM), that regulate with temporal and spatial precision, the migratory, proliferative, and contractility of wound cells. Prompt closure of the wound is an early and critical phase of healing and beta1 integrins are important in this process. We previously reported a marked increase in integrin alpha9beta1 expression in epidermal keratinocytes in cutaneous and corneal wounds. However, the functional role of keratinocyte alpha9beta1 during re-epithelialization is unknown and analysis has been precluded by the lethal phenotype of integrin alpha9beta1 knockout mice. We now report that in conditional integrin alpha9 knockout (K14-alpha9 null) mice, normal proliferation occurs in epidermal keratinocytes and corneal basal cells. Normal epidermal keratinocyte morphology is also retained. However, corneal basal cell morphology and epithelial thickness are altered, suggesting that loss of integrin alpha9beta1 results in abnormal corneal differentiation. In cutaneous wounds, the number of proliferating epidermal keratinocytes is significantly reduced in K14-alpha9 null mice compared with alpha9(fl/-) mice, but not in Cre (control) mice. The decreased keratinocyte proliferation observed in K14-alpha9 null mice negatively impacts healing, resulting in a thinner migrating epithelium, demonstrating that alpha9beta1 is crucial for efficient and proper re-epithelialization during cutaneous wound healing.     

Flotillas of lipid rafts in transit amplifying cell-like keratinocytes

Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids and are involved in the regulation of a variety of cellular processes, such as proliferation, apoptosis and cell motility. We have previously described that large lipid raft aggregates are readily detectable on cultured keratinocyte cell line HaCaT by staining with the fluorescein-tagged cholera toxin (CTx-FITC). In this paper we adopted this method for the detection of lipid rafts in human epidermis and keratinocytes in culture. Double labelling of showed the non-overlapping clusters of basal cells in human epidermis: CD29dimCTx-FITCbright cells in the deep rate ridges and CD29brightCTx-FITCdim cells at the tips of dermal papillae. A similar patchy, non-overlapping staining pattern was observed in cultured keratinocytes in vitro. CTx-FITCbright cells are mitotically active whereas a large proportion of CTx-FITCdim cells are quiescent. We conclude that the epidermal stem-like cells, previously shown to occupy the tips of dermal papillae and to exhibit high density of membrane beta1 integrin have a low content of lipid rafts. In contrast, the putative transit amplifying cells in deep rate ridges show enrichment in lipid rafts. Thus, lipid rafts may be a factor controlling the mitotic activity of epidermal keratinocytes and possibly the differentiation of stem cells into the transit amplifying cells.     

Phenotypical and functional differences in germinative subpopulations derived from normal and psoriatic epidermis

A model that explains how maintenance of normal homeostasis in human epidermis is achieved describes a heterogeneous cell population of stem cells (SC) and transit amplifying cells (TAC). There must be a tightly regulated balance between SC self-renewal and the generation of TAC that undergo a limited number of divisions before giving rise to postmitotic, terminally differentiated cells. To investigate whether this balance is disturbed in psoriatic epidermis, we have characterized flow sorted enriched SC and TAC using immunocytochemistry, flow cytometry, and real-time quantitative PCR. Our data demonstrate phenotypical and functional differences in SC (beta(1)-integrin bright) and TAC (beta(1)-integrin dim) enriched fractions between normal and psoriatic keratinocytes. Some of these were expected, such as mRNA levels of keratins 6 and 10 and of the Ki-67 antigen. Most remarkable were differences in phenotype of the psoriatic TAC compared with TAC from normal skin. These subpopulations also displayed striking differences following culture. Downregulation of markers associated with the regenerative phenotype (psoriasin, elafin, psoriasis-associated fatty acid binding protein) in cultured psoriatic dim cells in the absence of hyperproliferation suggests that proliferation and regenerative maturation are coupled. From these results, in combination with our earlier findings, we propose a model for epidermal growth control in which TAC play a crucial role.     

Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo

BACKGROUND: The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis. OBJECTIVES: We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. METHODS: (i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively. RESULTS: (i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation. CONCLUSIONS: We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.     

Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.     

In vitro modulation of keratinocyte wound healing integrins by zinc, copper and manganese

Although the trace elements zinc, copper and manganese are used in vivo for their healing properties, their mechanism of action is still only partially known. Some integrins expressed by basal layer keratinocytes play an essential part in healing, notably alpha2beta1, alpha3beta1, alpha6beta4 and alphaVbeta5, whose expression and distribution in epidermis are modified during the re-epithelialization phase. This study demonstrates how the expression of these integrins are modulated in vitro by trace elements. Integrin expression was studied in proliferating keratinocytes in monolayer cultures and in reconstituted skin that included a differentiation state. After 48 h incubation with zinc gluconate (0.9, 1.8 and 3.6 microg/mL), copper gluconate (1, 2 and 4 microg/mL), manganese gluconate (0.5, 1 and 2 microg/mL) and control medium, integrin expression was evaluated by FACScan and immunohistochemistry. Induction of alpha2, alpha3, alphaV and alpha6 was produced by zinc gluconate 1.8 microg/mL in monolayers, of alpha2, alpha6 and beta1 by copper gluconate 2 and 4 microg/mL and of all the integrins studied except alpha3 by manganese gluconate 1 microg/mL. Thus, alpha6 expression was induced by all three trace elements. The inductive effect of zinc was particularly notable on integrins affecting cellular mobility in the proliferation phase of wound healing (alpha3, alpha6, alphaV) and that of copper on integrins expressed by suprabasally differentiated keratinocytes during the final healing phase (alpha2, beta1 and alpha6), while manganese had a mixed effect.     

Defective beta1-integrins expression in arsenical keratosis and arsenic-treated cultured human keratinocytes

BACKGROUND: beta1-integrins, which localize to the basolateral surface of basal keratinocytes, are important in the differentiation control and proliferation of the epidermis. Many cutaneous diseases with perturbed differentiation, including arsenical keratosis, show altered patterns of integrin distribution and expression. Arsenic may induce arsenical keratosis through the differentiation and apoptosis aberration by integrins. The purpose of this study is to investigate the role of integrin and arsenic in the pathogenesis of arsenical keratosis. METHODS: Twenty-five specimens obtained from 25 patients with arsenical keratosis disease were studied. Immunohistochemistry staining to beta1, alpha2beta1, or alpha3beta1 integrins was performed in arsenical keratosis and clinically normal perilesional skin. Western blotting was used to assess the expression of integrin beta1 and focal adhesion kinase (FAK) in arsenic-treated cultured keratinocytes. RESULTS: A decreased expression of beta1, alpha2beta1, or alpha3beta1 integrins was demonstrated in arsenical keratosis and clinical normal perilesional skin in a large proportion of arsenical keratosis cases studied. The expressions of integrin beta1 and FAK were both decreased in arsenic-treated keratinocytes. CONCLUSIONS: Our results suggest that arsenic induces abnormal differentiation in arsenical keratosis via the effects of integrin expression in keratinocytes.     

Expression of beta 4 integrins in human skin: comparison of epidermal distribution with beta 1-integrin epitopes, and modulation by calcium and vitamin D3 in cultured keratinocytes.

The expression of beta 4 integrins in adult and fetal human skin as well as in cultured keratinocytes was studied by immunodetection with monoclonal antibodies, and compared with that of beta 1 integrins. The distribution of the beta 1 and beta 4 integrin epitopes was entirely different in the adult epidermis. As reported previously by us (J Clin Invest 84:1916, 1989), the beta 1 epitopes were present most prominently at lateral cell-cell contact points, whereas staining with the beta 4 antibody demonstrated a linear staining pattern polarizing to the basal surface juxtaposed to the dermal-epidermal basement membrane. In contrast, in fetal skin, the staining patterns both with beta 1 and beta 4 antibodies were similar and demonstrated the presence of epitopes surrounding the entire cell surface of both basal and suprabasal keratinocytes. Distinct polarization of beta 4 integrin epitopes was noted in cultured keratinocytes maintained in low-calcium (0.15 mM) medium, and the expression of these integrins was also noted in human papilloma virus-transformed keratinocytes. Switch of the cultures to high-calcium (1.2 mM) medium resulted in redistribution of the epitopes to a pattern suggesting their location at under-surface of the cells adjacent to the substratum. This Ca(++)-induced redistribution of beta 4 integrin epitopes could be counteracted by 10(-7) M vitamin D3. Finally, incubation with anti-beta 4 integrin antibody reduced the capacity of keratinocytes to attach to plastic substratum. The results provide further evidence for the role of beta 4 integrins in cell-matrix interactions.     

Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration

We have described previously that beta1 integrins, which mediate keratinocyte cell adhesion and migration, are in ligand-occupied conformation at the basal surface but not at the lateral and apical surfaces of keratinocytes. This led us to study the cellular localization and function of tetraspanin molecules, which have been postulated to modulate integrin activity. We found that CD9 and CD81 are highly expressed by keratinocytes clearly delineating filopodia at lateral and apical surfaces. CD63 and CD151 are largely expressed in the intracellular compartment, although some membrane expression is observed. We found accumulation of CD9, CD81, and CD151 together with alpha3 and beta1 integrins at intercellular junctions. In low calcium medium, this intercellular space is crossed by a zipper of filopodia enriched in alpha3beta1 and tetraspanin proteins. Interestingly, the expression of CD9, CD81, and beta1 and alpha3 integrins was detected in the footprints and rippings of motile keratinocytes, suggesting their role in both adhesion to extracellular matrix and keratinocyte motility. beta1 integrins were only partially activated in the rips, whereas cytoskeleton-linking proteins such as talin were completely absent. On the other hand, antitetraspanin antibodies did not stain focal adhesions, which contain talin. The involvement of tetraspanins in keratinocyte motility was assessed in a wound healing migration assay. Inhibition of cell migration was observed with antibodies to CD9, CD81, beta1, and alpha3, and, to a lesser extent, to CD151. Together these results indicate that tetraspanin-integrin complexes might be involved in transient adhesion and integrin recycling during keratinocyte migration, as well as in intercellular recognition.     

Zinc, copper and manganese enhanced keratinocyte migration through a functional modulation of keratinocyte integrins

The migration of keratinocytes plays an important role in the re-epithelialization of cutaneous wounds. Zinc, copper and manganese are used in vivo for their healing properties and their mechanism of action is still only partially known. Thus, they have been shown both to promote keratinocyte proliferation and to modulate integrins expression. The aim of this study was to determine if trace elements induce an increase of the migration of keratinocytes and if this effect is related to the modulation of integrins. Two independent migration assays were used to study keratinocyte migration: the scratch assay using normal human keratinocytes and the modified Boyden chamber using HaCaT cells. Inhibition studies using function-blocking antibodies directed to alpha3, alpha6, alpha(v) and beta1 subunits were performed to investigate the modulator effect of trace elements on integrin function. In this way, zinc and copper gluconates increased alpha3, alpha(v) and beta1 function whereas manganese gluconate seems mainly able to modulate the function of alpha3 and beta1. The stimulating effect of these trace elements on keratinocyte migration does not appear related to alpha6 subunit. Thus, zinc, copper and manganese enhanced keratinocyte migration and one of the mechanisms was going through a modulation of integrin functions.     

Lack of intrinsic polarity in the ligandbinding ability of keratinocyte β1 integrins

Abstract: Within the basal layer of the epidermis the β1 integrins have a pericellular distribution. Two monoclonal antibodies, 15/7 and 12G10, that detect a conformation of the β1 integrin subunit that is induced following cation or ligand occupancy selectively recognized β1 integrins at the basement membrane zone in vivo and in focal adhesions of cultured keratinocytes; they did not recognize integrins on the apical and upper lateral membranes of basal keratinocytes nor integrins on the suprabasal keratinocytes of hyperproliferative epidermis. Inhibition of intercellular adhesion did not induce the 15/7 epitope on the lateral and apical membrane domains. The surface distribution of the epitopes was consistent with the antibodies acting as reporters of ligand-binding; in addition, the 15/7 epitope was exposed on unglycosylated, immature β1 integrins. Although the apical membrane of basal keratinocytes is not normally in contact with extracellular matrix proteins, we found that it was capable of binding fibronectin-coated beads and that the 15/7 epitope was exposed on plasma membrane in contact with the beads. When a chimeric molecule consisting of the extracellular domain of CD8 and the cytoplasmic domain of the β1 integrin subunit, used to mimic a constitutively active β1 heterodimer, was introduced into keratinocytes it localized to the basal, lateral and apical membrane domains. We conclude that although the conformation of the keratinocyte β1 integrins differs between the basal and the lateral/apical membrane domains there is no intrinsic polarity in the ligand binding potential of the receptors.     

Assessment of epidermal subpopulations and proliferation in healthy skin, symptomless and lesional skin of spreading psoriasis

BACKGROUND: The margin zone in spreading psoriatic lesions has frequently been used as a model to study the changes in epidermal proliferation, keratinization and inflammation during the transition from symptomless to lesional skin. However, the dynamics of the changes in the epidermal subpopulations-basal cells, transit amplifying cells and differentiated cells-have not been studied in the transition between symptomless and lesional skin. OBJECTIVES: To quantify in a dynamic model of the margin zone in psoriasis the characteristics of these subpopulations with respect to epidermal proliferation and differentiation. METHODS: From seven patients with active psoriasis, biopsies were taken from the distant uninvolved skin, outer margin, inner margin and centre of a spreading psoriatic plaque. Frozen sections were labelled immunofluorescently using direct immunofluorescence for Ki-67 and beta1 integrin and the Zenon labelling technique for keratin 6, 10 and 15. Digital photographs of the stained sections were quantitatively analysed. RESULTS: In the distant uninvolved skin the expression of beta1 integrin was decreased and keratin 15 expression was lost. In this area suprabasal cells expressed beta1 integrin and in the outer margin suprabasal cells expressed Ki-67. From the outer to the inner margin of the psoriasis plaque, which coincided with the appearance of the clinical lesion, there was a significant change in the various markers. The patchy expression of keratin 6 in the inner margin became homogeneous in the centre of the psoriasis plaque and here was also coexpression of keratin 6 and keratin 10 in a single cell. CONCLUSIONS: The present study provides additional evidence that the distant uninvolved skin has a prepsoriatic phenotype, which is the first step in a psoriatic cascade. The cascade between symptomless and lesional skin comprises first an abnormality in inflammation with involvement of beta1 integrin-dim cells (transit amplifying cells) subsequently eliciting an enlarged germinative compartment with increased recruitment of cycling epidermal cells and focal expression of proliferation-associated keratins, ultimately culminating in a more-or-less homogeneous epidermis with massive recruitment of cycling epidermal cells and proliferation-associated keratinization.     

Isolating a pure population of epidermal stem cells for use in tissue engineering

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.     

In vitro differences between keratinocyte stem cells and transit-amplifying cells of the human hair follicle

Epithelial stem cells within the human hair follicle are critical for hair development, hair cycling, wound healing, and tumorigenesis. We and others have previously shown that the hair follicle bulge area contains keratinocyte stem cells, whereas the hair matrix represents the proliferating and differentiating transit-amplifying (TA) cell compartment. In order to better characterize the phenotypic differences between human keratinocyte stem cells and their daughter TA cells, we compared the in vitro properties of cell adhesion, cell migration, clonogenicity, and in vitro life span. Epithelial outgrowths from the hair matrix appeared within 2 d of explant, whereas stem cell outgrowths appeared between 7 and 10 d after explant. Both populations form colonies; however, stem cells from telogen follicles formed more total colonies, and more colonies greater than 3 mm. Upon subculture, stem cells formed colonies until passage 6 and terminally differentiated at passage 7, whereas TA cells only formed colonies until passage 2. Stem cells express more beta1 integrin and adhere more rapidly to collagen IV. Most strikingly, TA cells showed a 7-fold greater mobility on migration assays than stem cells (0.704 vs 0.102 microm per min). These results help define the human hair follicle stem cell and TA cell phenotypes and correlate with the in vivo properties of these compartments.     

E‐FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro

Epidermal fatty acid‐binding protein (E‐FABP) is a lipid carrier, originally discovered in human epidermis. We show that E‐FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E‐FABP induces overexpression of K10 and involucrin. On the other hand, E‐FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF‐κB and JNK signalling pathways. E‐FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E‐FABP as compared to the same population in normal epidermis. E‐FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high‐calcium conditions, E‐FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E‐FABP plays an important role in keratinocyte differentiation. Moreover, E‐FABP modulates differentiation in psoriatic keratinocytes.     

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.     

Suprabasal expression of epidermal α2β1 and α3β1 integrins in skin treated with topical retinoic acid

In normal adult human skin, expression of epidermal integrins is confined to keratinocytes in the basal layer. However, suprabasal expression of α2, α3 and β1 integrin subunits is noted in hyperproliferative epidermis in wound repair and psoriasis. In this study, we examined the effect of topical all- trans -retinoic acid (RA), known to induce epidermal hyperplasia, on expression of integrins in human epidermis. Immunostaining of vehicle-treated skin revealed expression of α2, α3 and β1, as well as α6 and β4 integrin subunits entirely on basal keratinocytes. Topical application of RA (0.1%) for 2 weeks resulted in marked suprabasal expression of α2, α3 and β1 integrin subunits, whereas α6 and β4 staining remained on basal keratinocytes. Staining for putative ligands of α2β1 and α3β1 integrins, i.e. type IV collagen, laminin-5 and fibronectin, was not detected in the epidermal layer in RA- or vehicle-treated skin. Treatment of HaCaT keratinocytes in culture with RA (1 μmol/L) enhanced α2 and β1 mRNA abundance. Furthermore, RA slightly up-regulated the expression of α2, α3 and β1 integrin subunits on primary epidermal keratinocytes and HaCaT cells in culture with no effect on cell proliferation. These results provide evidence that RA-elicited epidermal hyperplasia is associated with aberrant suprabasal expression of α2β1 and α3β1 integrins, and that this also involves direct stimulation of keratinocyte integrin expression by RA.