Non-lesional Psoriatic Epidermis Research Articles

The Psoriasis Therapeutic Potential of a Novel Short Laminin Peptide C16.

Psoriasis is a chronic inflammatory skin disease characterized by excessive growth of keratinocytes and hyperkeratosis in the epidermis. An abnormality of the non-lesional epidermis at an early stage of psoriasis is involved in triggering inflammatory cell infiltration into the dermis. Integrin α5β1 acts as a receptor for fibronectin and has been found to be overexpressed in non-lesional psoriatic epidermis. To investigate whether α5β1 integrin has a potential as a drug target for psoriasis treatment, the α5β1 integrin-binding peptide, C16, was used to obstruct the HaCat keratinocyte cellular responses induced by fibronectin (Fn) in culture and psoriasis-like skin inflammation induced in mice by imiquimod (IMQ). The C16 exhibited antagonistic activity against α5β1 integrin in HaCat cells, with evidence of suppression of the Fn-mediated proliferative, cytoskeletal, and inflammatory responses. Topical treatment with C16 greatly reduced the IMQ-induced epidermal hyperplasia, infiltration of neutrophils/macrophages, and expression of pro-inflammatory mediators in mouse skin. The C16SP (C16-derived short peptide; DITYVRLKF) also exhibited antagonistic activity, suppressing α5β1 integrin activity in culture, and reducing IMQ-induced skin inflammation. Taken together, this study provides the first evidence that α5β1 integrin may be a potential drug target for psoriasis. The synthetic C16 peptide may serve as an agent for psoriasis therapy.

NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis.

Psoriatic skin differs distinctly from normal skin by its thickened epidermis. Most gene expression comparisons utilize full-thickness biopsies, with substantial amount of dermis. We assayed the transcriptomes of normal, lesional, and non-lesional psoriatic epidermis, sampled as split-thickness skin grafts, with 5′-end RNA sequencing. We found that psoriatic epidermis contains more mRNA per total RNA than controls, and took this into account in the bioinformatic analysis. The approach highlighted innate immunity-related pathways in psoriasis, including NOD-like receptor (NLR) signaling and inflammasome activation. We demonstrated that the NLR signaling genes NOD2, PYCARD, CARD6, and IFI16 are upregulated in psoriatic epidermis, and strengthened these findings by protein expression. Interestingly, PYCARD, the key component of the inflammasome, showed an altered expression pattern in the lesional epidermis. The profiling of non-lesional skin highlighted PSORS4 and mitochondrially encoded transcripts, suggesting that their gene expression is altered already before the development of lesions. Our data suggest that all components needed for the active inflammasome are present in the keratinocytes of psoriatic skin. The characterization of inflammasome pathways provides further opportunities for therapy. Complementing previous transcriptome studies, our approach gives deeper insight into the gene regulation in psoriatic epidermis.

Comparison of stress-induced PRINS gene expression in normal human keratinocytes and HaCaT cells

Psoriasis is a chronic inflammatory skin disease that affects approximately 2-4% of the population. We recently described a novel non-coding RNA, psoriasis susceptibility related RNA gene induced by stress (PRINS), that was overexpressed in non-lesional psoriatic epidermis, and its expression was induced by various stress factors such as serum starvation, contact inhibition, ultraviolet (UV)-B irradiation, viral infection and translational inhibition in HaCaT cells. In the present work we set out to compare the stress and microbial agent-induced PRINS expression in normal human keratinocytes (NHKs) and HaCaT cells. Since nuclear factor-κB (NF-κB) is involved in the cellular stress response, we sought to explore whether there is a connection between the NF-κB and PRINS-mediated signal transduction pathways in NHKs and HaCaT cells. We found that the PRINS expression responded differentially to various stress signals and microbial agents in HaCaT cells and in NHKs: after translational inhibition and UV-B treatment, similar induction of PRINS expression occurred with different time courses while after microbial agent treatment, the PRINS expression was significantly induced in HaCaT cells, whereas we could not detect similar changes in NHKs. To explore whether the known NF-κB abnormalities in HaCaT cells could be related to this differential PRINS expression, we silenced the PRINS gene expression with small interfering RNA (siRNA) in both HaCaT cells and in NHKs and monitored NF-κB signal transduction after lipopolysaccharide (LPS) treatment. Silencing of PRINS had no effect on LPS-induced NF-κB activity either in HaCaT cells or in NHKs. Our results indicate that PRINS probably affects keratinocytes functions independently of NF-κB signalling.

The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA, PRINS

Psoriasis Susceptibility-Related RNA Gene Induced by Stress (PRINS) is a non-coding RNA overexpressed in lesional and non-lesional psoriatic epidermis and induced by stress. Its function in healthy and psoriatic skin is still not known. Here, we report that PRINS regulates G1P3, a gene with anti-apoptotic effects in keratinocytes. siRNA-mediated inhibition of PRINS gene resulted in altered cell morphology and gene expression alterations, as demonstrated in a microarray experiment. One of the genes regulated by PRINS ncRNA was G1P3, an interferon-inducible gene with anti-apoptotic effects in cancer cells. Interestingly, we found that G1P3 was 400-fold upregulated in hyperproliferative lesional and ninefold upregulated in non-lesional psoriatic epidermis compared to healthy epidermis. In vitro, G1P3 protein levels were highest in proliferating keratinocytes and siRNA-mediated downregulation of G1P3 resulted in increased cell apoptosis. These data indicate that G1P3 inhibits spontaneous keratinocyte apoptosis and hence its high expression in psoriatic skin may contribute to the development of psoriatic lesions. We hypothesize that the deregulation of the PRINS ncRNA may contribute to psoriasis and results in decreased sensitivity to spontaneous keratinocyte apoptosis via the regulation of G1P3.

Expression of antiapoptotic protein c-FLIP is upregulated in psoriasis epidermis

The most characteristic change in psoriasis is markedly increased, persistent keratinocyte proliferation. The pathogenic mechanism underlying the hyperproliferation of keratinocytes in psoriasis is still not completely clarified. Cellular FLIP (cFLIP) is a close homologue of caspase 8 without the caspase activity that inhibits Fas signaling. The cFLIP protein is often expressed in human tumors and is believed to suppress antitumor immune responses involving the Fas system. PCNA is an auxiliary protein of DNA polymerase-5, which appears early in G1 and becomes more abundant in the S phase, thereafter declining during G2/M phases of the cell cycle. Thus, the PCNA staining profiles were used as markers of keratinocyte proliferation. Our objective was to obtain insight into the role of c-FLIP in kerarinocyte proliferation and to investigate further the pathogenesis of psoriasis. Using real time quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) and immunohistochemical staining, we studied the expression of c-FLIP mRNA and protein in skin biopsies from psoriatic patients and healthy subjects. Apoptotic cells were evaluated using the terminal deoxynucleotide transferase (TdT) mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. c-FLIP mRNA and protein expressions were significantly greater in lesional psoriatic epidermis compared with normal and non-lesional psoriatic epidermis (P < 0.01). c-FLIP was strongly expressed within all epidermal layers in lesional psoriatic skin, whereas weak c-FLIP staining was restricted to the basal and suprabasal layers in normal and non-lesional psoriatic epidermis. c-FLIP protein levels significantly correlated with PASI score, PCNA and apoptosis index (Spearman's rho = 0.83; rho = 0.61; rho = - 0.41; P < 0.05, respectively). We conclude that over-expression of c-FLIP in lesional psoriatic skin might contribute to abnormal keratinocyte proliferation due to a functional decrease in the apoptotic pathway.

Altered Morphology and Distribution of Cellular Junction Proteins in Non-Lesional Psoriatic Epidermis: An Insight into Disease Severity

Psoriasis affects 2.7% of the world's population. Keratinocyte proliferation outside the basal layer suggests alterations in cell-cell interactions in affected epidermis. Anomalous expression of proteins forming intercellular junctions has been reported in lesional skin of psoriatic patients. In contrast, little is known about possible alterations in psoriatic non-lesional skin. Ten clinically diagnosed psoriasis vulgaris patients and ten controls were studied. All patients were diagnosed with active but controlled psoriatic plates (PASI 3 to 5) and had not received any systemic treatment. The mean age was 43 years for patients and 43.5 years for controls. Four-mm2 skin samples were taken from lesional and non-lesional zones in patients and from abdomen in controls. Five-mum sections were examined for integrity and structural organization by fluorescent labeling of actin filaments and nuclei. Specific antibodies were utilized to localize occludin, E-cadherin, beta-catenin, and proliferation-specific keratins in sections and epidermal sheets. Samples were also processed for immunoblotting with occludin antibody. Lesional and non-lesional psoriatic epidermis from all patients showed keratinocyte hyperproliferation, lack of rete ridges and dermal papillae in the dermal-epidermal junction in some areas. Proteins forming tight and adherens junctions in non-lesional skin keratinocytes from two patients who during the course of the study evolved to uncontrolled disease, showed similar alterations to those observed in lesional skin of all the patients. However, the occludin isoforms expressed were apparently the same in all samples. Analysis of non-lesional skin in psoriatic patients diagnosed with controlled disease may provide clues about incipient structural abnormalities in the pathogenesis of psoriasis, providing an early diagnostic indicator for evolution to a generalized form of the disease.

Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis.

Toll-like receptors (TLRs) are part of the innate immune system involved in the response to microbial pathogens. TLR2 recognizes various ligands expressed by Gram-positive bacteria, while TLR3, TLR4 and TLR5 are specific for double-stranded RNA, Gram-negative lipopolysaccharides and bacterial flagellin, respectively. To determine, firstly, whether epidermal keratinocytes of normal skin express TLRs and, secondly, whether modulation of TLR expression occurs in psoriasis, an inflammatory skin disease associated with certain microorganisms such as streptococci, staphylococci and yeasts. Eight samples of normal, and 15 samples of lesional and nonlesional psoriatic skin were stained with polyclonal antibodies specific for TLR1-5 using an avidin-biotin-peroxidase technique. Epidermal keratinocytes in normal skin constitutively expressed TLR1, TLR2 and TLR5, while TLR3 and TLR4 were, in most cases, barely detectable. Cytoplasmic TLR1 and TLR2 were expressed throughout the epidermis, with higher staining of the latter on basal keratinocytes, while TLR5 expression was concentrated in the basal layer. In contrast, in lesional epidermis from patients with psoriasis, TLR2 was more highly expressed on the keratinocytes of the upper epidermis than on the basal layer, while TLR5 was downregulated in basal keratinocytes compared with corresponding nonlesional psoriatic epidermis. In addition, nuclear TLR1 staining was observed in the upper layers of both nonlesional and lesional psoriatic epidermis, but not in that of normal skin. These findings suggest that TLRs expressed by epidermal keratinocytes constitute part of the innate immune system of the skin. The relevance of altered keratinocyte TLR expression in psoriasis remains to be determined.

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.

Upregulation of Connexin 26 is a Feature of Keratinocyte Differentiation in Hyperproliferative Epidermis, Vaginal Epithelium, and Buccal Epithelium

In epidermis, it has been suggested, intercellular communication through gap junctions is important in coordinating cell behavior. The connexins, may facilitate selective assembly or permeability of gap junctions, influencing the distribution of metabolites between cells. Using immunohistochemistry, we have compared the distribution of connexins 26 and 43 with that of proliferating cells (Ki67 labeling) in normal epidermis, hyperplastic epidermis (tape-stripped epidermis, psoriatic lesions, and viral warts), and vaginal and buccal epithelia. Connexin 43 was abundant in spinous layers of all epidermal specimens and in vaginal and buccal epithelia. Connexin 26 was absent from the interfollicular and interductal epidermis of normal hair-bearing skin, and nonlesional psoriatic epidermis but present at very low levels in plantar epidermis. Connexin 26 was prominent in lesional psoriatic epidermis and viral warts and in vaginal and buccal epithelia. In three independent experiments connexin 26 appeared in a patchy intercellular distribution in the basal epidermis within 24 h of tape stripping, proceeding to more extensive distribution in basal and suprabasal layers by 48 h. The increase in connexin 26 preceded that in cell proliferation. In vaginal epithelium, buccal epithelium, and viral warts connexin 26 was restricted mainly to suprabasal, nonproliferating cells. In psoriatic lesional epidermis connexin 26 was also located mainly in suprabasal, nonproliferating cells. Connexin 26 was present in a patchy distribution in the basal layer of psoriatic lesional epidermis, but double labeling for connexin 26 and Ki67 showed that many connexin 26 positive basal cells were nonproliferative, suggesting that connexin 26 may be related to differentiation rather than to proliferation. These observations would be consistent with a role for connexin 26 containing gap junctions during both early and later stages of keratinocyte differentiation in hyperplastic epidermis and in vaginal and buccal epithelia.

Differential Expression of Peroxisome Proliferator-Activated Receptor Subtypes During the Differentiation of Human Keratinocytes

The expression of mRNA encoding peroxisome proliferator-activated receptor (PPAR) subtypes in human keratinocytes was determined by semiquantitative reverse transcriptase-polymerase chain reaction. When normal human keratinocytes were induced to differentiate by shifting the culture medium to high Ca2+ concentration, the expression of PPAR-alpha and -gamma mRNA was increased, whereas that of PPAR-delta remained unchanged. At the protein level, the expression of PPAR in cultured human keratinocytes was demonstrated by a DNA mobility shift assay and the functionality of the receptor subtypes was assessed by transactivation experiments. In epidermis reconstructed in vitro, the level of PPAR-alpha and -gamma mRNA was also associated with keratinocyte differentiation. In lesional compared with nonlesional psoriatic epidermis, the expression of PPAR-alpha and -gamma mRNA was reduced, indicating that these two subtypes are tightly linked to the epidermal differentiation process.

Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis.

Down-regulation of transforming growth factor-beta receptors I and II is seen in lesional but not non-lesional psoriatic epidermis.

Transforming growth factor-beta s (TGF-beta s) are a family of growth factors with inhibitory effects on epithelial cell proliferation. Their effects are mediated by two interacting receptors, of which type I (T beta R-I) mediates signal transduction after interaction with type II (T beta R-II) carrying the TGF-beta ligand. We have studied the expression of T beta R-I and T beta R-II in psoriatic and normal human skin by using polyclonal rabbit antisera and immunohistochemistry. Immunohistochemical analysis revealed an intense immunoreactivity for both receptors in the basal and often also suprabasal layer of normal and non-lesional psoriatic epidermis. In contrast, all psoriatic lesions studied lacked detectable immunoreactivity of either receptor in the epidermis. The results suggest that lack of TGF-beta-mediated growth inhibition by down-regulation of TGF-beta receptor expression may play an important part in the pathogenesis of psoriasis.

The IL-1 system in psoriatic skin: IL-1 antagonist sphere of influence in lesional psoriatic epidermis.

The epidermal expression of IL-1 in psoriasis is clearly altered, but data are still incomplete and poorly understood. To thoroughly study the IL-1 system in psoriasis, we semiquantitatively analyzed the expression of all currently characterized IL-1 isoforms and their receptors in parallel in both lesional (PP) and nonlesional psoriatic (PN) epidermis. Immunostaining of skin sections showed that IL-1alpha, located in the basal keratinocytes of normal control (NN) and PN epidermis, was significantly decreased to negligible levels in PP epidermis. IL-1 receptor antagonist (IL-1ra) and IL-1R type II (IL-1RII) were both significantly overexpressed in mutually exclusive compartments of PP epidermis, the suprabasal and basal compartment, respectively. A significant inverse correlation was found between the expressions of IL-1alpha and these two IL-1 antagonists, which may be inherent to the accelerated terminal differentiation of the psoriatic keratinocyte. In situ hybridization of IL-1(R) mRNAs confirmed the staining results. Levels of IL-1ra mRNA, however, were not increased in PP epidermis, suggesting that the overexpression of IL-1ra protein may be explained at the level of translation. The more sensitive PCR demonstrated a clearly increased expression of IL-1beta mRNA in PP epidermal cells (EC), which may be related to the inflammatory response in psoriasis. IL-1RI mRNA was clearly present in both PP and NN EC. The mRNA levels of the secreted IL-1ra isoform, but not intracellular IL-1raI and II, and IL-1RII were elevated in PP EC and paralleled those of IL-1beta. In summary, this study provides a defined phenotype of the complete epidermal IL-1 system in psoriasis; it shows that the expressions of IL-1(R) isoforms are coordinately altered, resulting in a predominance of IL-1 antagonists, which may represent a negative feedback response to IL-1 agonists, leading to a decreased IL-1 responsiveness.

Increased Epidermal Growth Factor Receptor in fsn/fsn Mice

Epidermal growth factor receptors (EGF-Rs) are elevated in active human psoriatic lesions, but decrease in resolving lesions. Similar biologic responses in EGF-R levels have been demonstrated within human psoriatic skin grafted onto mice. We tested the hypothesis that flaky-skin mice (fsn/fsn), one proposed genetic animal model of psoriasis, would display EGF-R levels comparable to human psoriatic epidermis and show similar biologic responses. EGF-R levels were characterized in unperturbed sites in fsn/fsn skin and +/? skin by enzyme-linked immunosorbent assay, 125I-EGF binding, and immunostaining. Altered EGF-R levels were noted after mild trauma (tape stripping) or under resolving conditions (30 doses of 50 mJ/CM2 ultraviolet B, 2.5 mg/kg oral cyclosporin A, or daily 30 microg/ml topical EGF). Increased EGF-R immunostaining was observed in involved flaky epidermal sites before treatment. To determine whether a hyperproliferative (Koebner) reaction could be induced, we tape stripped fsn/fsn tail and non-flaky dorsal sites. EGF-R levels in dorsal epidermis increased by days 3-4 after injury by enzyme-linked immunoabsorbent assay methods. When fsn/fsn mice received one of three different treatments for 6 weeks, the skin returned to a normal phenotype both grossly and microscopically. Immunoreactive EGF-R in treated, but not untreated, sites decreased to either normal or nondetectable levels. These data indicate that fsn/fsn mice exhibit an EGF-R profile identical to that of lesional and nonlesional human psoriatic epidermis. Modulations of the flaky phenotype in response to injury and three different treatments suggest that fsn/fsn is a useful in vivo model for examining new treatment modalities for psoriasiform skin diseases.

Epidermal fatty-acid-binding protein in psoriasis, basal and squamous cell carcinomas: an immunohistological study.

In human keratinocytes, we have recently characterized a low-molecular-weight cytosolic protein of 15 kD that specifically binds fatty acids (FAs) with high affinity, the epidermal FA-binding protein (E-FABP). The distribution of E-FABP in skin diseases is not known. To localize by immunohistochemistry the expression of E-FABP in psoriasis, basal and squamous cell carcinomas in order to obtain indirect information, at the cellular level, on the transport of the FAs. E-FABP was localized in the upper stratum spinosum and stratum granulosum in normal and non-lesional psoriatic skin. In contrast, lesional psoriatic epidermis strongly expressed E-FABP in all suprabasal layers, like nonkeratinized oral mucosa. The basal layer did not express E-FABP reactivity in any of these samples. Accordingly, basal cell carcinomas were E-FABP negative whereas only well-differentiated cells of squamous cell carcinomas expressed E-FABP. It is unlikely that E-FABP plays a significant role in FA uptake by basal cells. Our data rather indicate that E-FABP expression is related to the commitment of keratinocyte differentiation and that the putative role of E-FABP should not be restricted to the formation of the skin lipid barrier. Since the pattern of E-FABP expression mimics cellular FA transport, our results suggest that lesional psoriatic skin and oral mucosa have a higher metabolism/transport for FAs than normal and non-lesional psoriatic epidermis.

Immunohistochemical Localization of SKALP/Elafin in Psoriatic Epidermis

Recently we have reported the purification and biochemical characterization of a new, inducible elastase inhibitor [skin-derived antileukoproteinase (SKALP)], which could be extracted in high amounts from psoriatic skin but not from normal human skin. Here we demonstrate the immunohistochemical localization of SKALP in psoriatic epidermis. SKALP was found exclusively in the upper layers of the suprabasal compartment and stratum corneum of lesional psoriatic epidermis. Basal keratinocytes were always negative. No immunoreactive SKALP was found in normal epidermis and non-lesional psoriatic epidermis, in accordance with findings in functional assays. Western blots of skin extracts from psoriatic and normal skin confirmed the immunohistochemical findings and revealed two major bands with apparent molecular weights of 10.5 and 11.5 kDa. We would hypothesize that SKALP could act as a modulator of epidermal inflammation by interfering with polymorphonuclear leukocyte trafficking, and that it could protect structural proteins against elastase-mediated damage.

Increased Phosphatidylinositol Kinase Activity in Psoriatic Epidermis

Phosphatidylinositol (PI) kinase is activated by growth factors, such as epidermal growth factor (EGF), and is thought to be involved in cellular proliferation. Psoriasis is a hyperproliferative epidermal disease in which EGF receptor expression is altered and phospholipase C activity is increased. Considering the potential importance of growth factor stimulated phosphoinositide metabolism in the genesis of abnormal growth, we measured PI kinase activity in epidermal keratome biopsies from normal skin and the lesional and nonlesional skin of psoriatic patients. The PI kinase activity in 10 psoriatic involved plaques was increased 6.7-fold (Vmax = 67.1 +/- 23.9 pmol formed/min/mg protein +/- SE) when compared with 11 normal epidermal biopsies (Vmax = 10.0 +/- 1.3 pmol/min/mg protein, p less than 0.025). Similar results were noted when enzyme activity was standardized using DNA content. The apparent Km of PI kinase for ATP in involved psoriatic biopsies (0.45 +/- 0.14 mM) was also significantly (p less than 0.025) increased compared with normals (0.11 +/- 0.02 mM). The PI kinase activity in 11 biopsies of nonlesional psoriatic epidermis was not statistically different from normal epidermis. Both psoriatic and normal PI kinases required Mg++ and were inhibited by Ca++. The polyamine, spermine, a known activator of PI kinase in other tissues, stimulated normal but not psoriatic epidermal PI kinase. Both normal and psoriatic PI kinase activities had an apparent mol wt of 85,000. Increased synthesis of phosphoinositides by PI kinase in psoriatic tissue may provide more substrate for phospholipase C; a key enzyme in growth factor-mediated signal transduction.

FITC-insulin binding to normal and psoriatic epidermis

FITC-insulin was prepared and applied to frozen human skin sections. In normal and nonlesional psoriatic epidermis, the binding was cytoplasmic in suprabasal cells. In psoriatic lesions, fairly stained or even unstained cells were littered in suprabasal epidermis indicating the expansion of selected basal cell qualities into the stratum spinosum.

Modulation of phospholipase A2 activity in extracts of lesion-free psoriatic epidermis by alkaline phosphatase and a protein phosphatase inhibitor.

Phospholipase A2 activity is raised in non-lesional psoriatic epidermis compared with normal epidermis. It has been shown that the activity of this enzyme is controlled by an inhibitory protein the inhibitory effect of which is increased by dephosphorylation. Treatment of epidermal extracts with alkaline phosphatase reduced the phospholipase A2 activity, both in normal and in lesion-free psoriatic epidermis. Inclusion of pyrophosphate, a protein phosphatase inhibitor, in the homogenizing medium caused the activity of phospholipase A2 in epidermal extracts from normal and lesion-free epidermis to be raised to the same high level. These results are consistent with the hypothesis that the raised phospholipase A2 activity in psoriatic epidermis is due to hyperphosphorylation of an endogenous inhibitor as a result of defective control of a phosphorylation/dephosphorylation mechanism. The relevance of these findings to other work is discussed.