Genetic associations of psoriasis in a Pakistani population.

BACKGROUND: Genetic predisposition to psoriasis, an inflammatory skin disease affecting 0·2-4% of the world population, is well established. Thus far, 41 psoriasis susceptibility loci reach genome-wide significance (P ≤ 5 × 10(-8) ). Identification of genetic susceptibility loci in diverse populations will help understand the underlying biology of psoriasis susceptibility. OBJECTIVES: The primary objective of this study was to examine psoriasis susceptibility associations previously reported in Chinese and caucasian populations in a Pakistani cohort. METHODS: Blood samples and phenotype data were collected from psoriasis cases and controls in Islamabad, Pakistan. DNA was isolated and genotypes of selected susceptibility markers were determined. The data were analysed using χ(2) tests or logistic regression for psoriasis association. RESULTS: HLA-Cw6 showed the strongest association [odds ratio (OR) 2·43, P = 2·3 × 10(-12) ]. HLA-Cw1 showed marginally significant association (OR 1·66, P = 0·049), suggesting that the HLA-Cw1-B46 risk haplotype may be present in the Pakistani population. Three other loci (IL4/IL13, NOS2, TRAF3IP2) showed nominally significant association (P < 0·05). CONCLUSIONS: HLA-Cw6 is strongly associated with psoriasis susceptibility in the Pakistani population, as has been found in every other population studied. In addition, HLA-Cw1 showed marginal association, reflecting the relative geographical proximity and thus likely genetic relatedness to other populations in which the HLA-Cw1-B46 haplotype is known to be associated. A larger cohort and a denser marker set will be required for further analysis of psoriasis associations in the South Asian population.

Comparison of MHC class I risk haplotypes in Thai and Caucasian psoriatics shows locus heterogeneity at PSORS1.

Earlier studies have shown that psoriasis in Japan and Thailand is associated with two different major histocompatibility complex (MHC) haplotypes - those bearing HLA-Cw6 and those bearing HLA-Cw1 and HLA-B46. In an independent case-control sample from Thailand, we confirmed the association of psoriasis with both haplotypes. No association was seen in Thai HLA-Cw1 haplotypes lacking HLA-B46, nor was HLA-Cw1 associated with psoriasis in a large Caucasian sample. To assess whether these risk haplotypes share a common origin, we sequenced genomic DNA from a Thai HLA-Cw1-B46 homozygote across the ∼300 kb MHC risk interval, and compared it with sequence of a HLA-Cw6-B57 risk haplotype. Three small regions of homology were found, but these regions share equivalent sequence similarity with one or more clearly non-risk haplotypes, and they contain no polymorphism alleles unique to all risk haplotypes. Differences in psoriasis phenotype were also observed, including lower risk of disease, greater nail involvement, and later age at onset in HLA-Cw1-B46 carriers compared with HLA-Cw6 carriers. These findings suggest locus heterogeneity at PSORS1 (psoriasis susceptibility 1), the major psoriasis susceptibility locus in the MHC, with HLA-Cw6 imparting risk in both Caucasians and Asians, and an allele other than HLA-Cw1 on the HLA-Cw1-B46 haplotype acting as an additional risk variant in East Asians.

Genome-wide association scan yields new insights into the immunopathogenesis of psoriasis.

Psoriasis is a common, immunologically mediated, inflammatory and hyperproliferative disease of the skin and joints, with a multifactorial genetic basis. We earlier mapped PSORS1, the major psoriasis susceptibility gene in the major histocompatibility complex (MHC), to within or very near HLA-Cw6. In an effort to identify non-MHC psoriasis genes, we carried out a collaborative genome-wide association study. After the initial follow-up genotyping of 21 single nucleotide polymorphisms from 18 loci, showing strong evidence of association in the initial scan, we confirmed evidence of association at seven loci. Three of these loci confirm earlier reports of association (HLA-C, IL12B, IL23R) and four identify novel signals located near plausible candidate genes (IL23A, IL4/IL13, TNFAIP3 and TNIP1). In other work, we have also shown that interferon-gamma (IFN-gamma) treatment induces interleukin (IL)-23 mRNA and protein in antigen-presenting cells (APC), leading to the proliferation of CD4+ and CD8+ memory T cells expressing IL-17. Although functional variants remain to be identified, we speculate that genetic variants at the IL4/IL13 locus contribute to the Th1 bias that is characteristic of psoriasis, that Th1-derived IFN-gamma supports expansion of IL-17+ T cells through APC-derived IL-23 and that negative regulation of inflammatory signaling through the NF-kappaB axis is impaired because of genetic variants of TNFAIP3 and TNIP1.

Overexpression of transforming growth factor alpha in psoriatic epidermis.

Transforming growth factor alpha (TGF-alpha) is produced by and required for the growth of epithelial cells and is angiogenic in vivo. Since epidermal hyperplasia and angiogenesis are hallmarks of psoriasis, TGF-alpha gene expression was analyzed in epidermal biopsies of normal and psoriatic skin. TGF-alpha messenger RNA and protein are much more abundant in lesional psoriatic epidermis than in normal-appearing skin of psoriatic patients or in normal epidermis. In contrast, messenger RNA levels of transforming growth factor beta 1 (TGF-beta 1), which inhibits epithelial cell growth, are not significantly different in normal, uninvolved, and lesional psoriatic epidermis. Thus, psoriatic epidermal hyperplasia may involve increased expression of a keratinocyte mitogen (TGF-alpha) rather than deficient expression of a growth inhibitor (TGF-beta 1).

Obesity in psoriasis: leptin and resistin as mediators of cutaneous inflammation.

BACKGROUND: Obesity is a significant risk factor for psoriasis and body mass index (BMI) correlates with disease severity. Objectives To investigate the relationship between obesity and psoriasis, focusing on the role of adipokines such as leptin and resistin. PATIENTS/METHODS: Patients with psoriasis (n = 30) were recruited and their BMI, waist circumference and disease severity [Psoriasis Area and Severity Index (PASI)] were recorded. Fasting serum samples were obtained on enrolment and after a course of ultraviolet (UV) B treatment. Age-, sex- and BMI-matched healthy controls were also recruited. RESULTS: On enrolment, serum leptin and soluble leptin receptor levels were not raised compared with the controls. However, resistin, interleukin (IL)-1beta, IL-6, and chemokines CCL2, CXCL8 and CXCL9 were all significantly elevated in the patient group and serum resistin correlated with disease severity (r = 0.372, P = 0.043). Improvement after UVB treatment was accompanied by decreased serum CXCL8. In vitro, both leptin and resistin could induce CXCL8 and tumour necrosis factor-alpha production by blood monocytes, and leptin could additionally induce IL-1beta and IL-1 receptor antagonist production. Leptin also dose dependently increased secretion of the growth factor amphiregulin by ex vivo-cultured lesional psoriasis skin. CONCLUSIONS: These data support the view that leptin and resistin may be involved in the pathogenesis of psoriasis in overweight individuals, possibly by augmenting the cytokine expression by the inflammatory infiltrate.

The psoriasis-protective TYK2 I684S variant impairs IL-12 stimulated pSTAT4 response in skin-homing CD4+ and CD8+ memory T-cells.

Tyrosine kinase 2 (TYK2) belongs to the Janus kinase (JAK) family of tyrosine kinases, which transmit signals from activated cytokine receptors. GWAS have consistently implicated TYK2 in psoriasis susceptibility. We performed an in-depth association analysis of TYK2 using GWAS and resequencing data. Strong genetic association of three nonsynonymous variants in the exonic regions of the TYK2 gene (rs34536443, rs12720356, and rs2304256) were found. rs12720356 encoding I684S is predicted to be deleterious based on its location in the pseudokinase domain. We analyzed PBMCs from 29 individuals representing the haplotypes containing each of the significantly associated signals. STAT4 phosphorylation was evaluated by phospho-flow cytometry after CD3/CD28 activation of cells followed by IL-12 stimulation. Individuals carrying the protective I684S variant manifested significantly reduced p-STAT4 levels in CD4 + CD25 + CD45RO+ (mean Stimulation Index (S.I.) 48.08, n = 10) and CD8 + CD25 + CD45RO + cells (S.I. 55.71, n = 10), compared to controls homozygous for the ancestral haplotype (S.I. 68.19, n = 10 (p = 0.002) and 76.76 n = 10 (p = 0.0008) respectively). Reduced p-STAT4 levels were also observed in skin-homing, cutaneous lymphocyte associated antigen (CLA)-positive CD4 and CD8 cells from I684S carriers. No significant changes in p-STAT4 for the psoriasis-associated variant rs34536443 was found. These data establish the functional significance of the TYK2 I684S variant in psoriasis susceptibility.

Chromatin structure and developmental expression of the human alpha-globin cluster.

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

Translocation of an erythroid-specific hypersensitive site in deletion-type hereditary persistence of fetal hemoglobin.

Hereditary persistence of fetal hemoglobin (HPFH) can involve large deletions which eliminate the 3' end of the beta-like globin gene cluster and more than 70 kilobases (kb) of flanking DNA. Blot hybridization revealed a DNase I-hypersensitive site extending from 1.1 to 1.4 kb downstream of the HPFH-1 3' deletion endpoint. The site was found in normal fetal and adult nucleated erythroid cells and in two erythroleukemia cell lines but not in nonerythroid cells and tissues. Simian virus 40 core enhancer-like sequences were found nonrandomly distributed within the boundaries of the site, which is contained in a fragment of known enhancer activity (E. A. Feingold and B. G. Forget, Blood, in press). A second hypersensitive site was found 0.5 kb upstream of the HPFH-1 3' deletion endpoint but was not erythroid specific. A third site, most prominent in fetal liver-derived erythroid cells, was found 1 kb upstream of the HPFH-2 deletion endpoint. As predicted by the locations of the deletion endpoints, the first two sites were translocated to within 12 kb of the A gamma gene in erythroid colonies derived from an HPFH-2 heterozygote and in hybrid mouse-human erythroid cells carrying the HPFH-2 deletion chromosome. Further analysis of this region showed that it was DNase I sensitive in erythroid and myeloid cells, indicating that it resides in an open chromatin domain. These observations suggest that alterations of chromatin structure flanking the fetal globin genes may contribute to abnormal gene regulation in deletion-type HPFH.

Analysis of RUNX1 binding site and RAPTOR polymorphisms in psoriasis: no evidence for association despite adequate power and evidence for linkage.

BACKGROUND: A previous study identified two peaks of allelic association between psoriasis and single nucleotide polymorphisms (SNPs) mapping to distal chromosome 17q, including a disease associated SNP that leads to loss of a RUNX1 transcription factor binding site, and additional SNPs in the third intron of the RAPTOR gene. Another study found an association with SNPs in the RAPTOR gene, but not with the RUNX1 binding site polymorphism. METHODS: In an effort to confirm these observations, we genotyped 579 pedigrees containing 1285 affected individuals for three SNPs immediately flanking and including the RUNX1 binding site, and for three SNPs in the RAPTOR gene. RESULTS: Here we report further evidence for linkage to distal chromosome 17q, with a linkage peak mapping 1.7 cM distal to the RUNX1 binding site (logarithm of the odds 2.26 to 2.73, depending upon statistic used). However, we found no evidence for association to individual SNPs or haplotypes in either of the previously identified peaks of association. Power analysis demonstrated 80% power to detect significant association at genotype relative risks of 1.2 (additive and multiplicative models) to 1.5 (dominant and recessive models) for the RUNX1 binding site, and 1.3 to 1.4 for the RAPTOR locus under all models except dominant. CONCLUSIONS: Our data provide no support for the previously identified RUNX1 binding site or for the RAPTOR locus as genetic determinants of psoriasis, despite evidence for linkage of psoriasis to distal chromosome 17q.

FOXM1 allows human keratinocytes to bypass the oncogene-induced differentiation checkpoint in response to gain of MYC or loss of p53.

Tumour suppressor p53 or proto-oncogene MYC is frequently altered in squamous carcinomas, but this is insufficient to drive carcinogenesis. We have shown that overactivation of MYC or loss of p53 via DNA damage triggers an anti-oncogenic differentiation-mitosis checkpoint in human epidermal keratinocytes, resulting in impaired cell division and squamous differentiation. Forkhead box M1 (FOXM1) is a transcription factor recently proposed to govern the expression of a set of mitotic genes. Deregulation of FOXM1 occurs in a wide variety of epithelial malignancies. We have ectopically expressed FOXM1 in keratinocytes of the skin after overexpression of MYC or inactivation of endogenous p53. Ectopic FOXM1 rescues the proliferative capacity of MYC- or p53-mutant cells in spite of higher genetic damage and a larger cell size typical of differentiation. As a consequence, differentiation induced by loss of p53 or MYC is converted into increased proliferation and keratinocytes displaying genomic instability are maintained within the proliferative compartment. The results demonstrate that keratinocyte oncogene-induced differentiation is caused by mitosis control and provide new insight into the mechanisms driving malignant progression in squamous cancer.

CaN19 expression in benign and malignant hyperplasias of the skin and oral mucosa: evidence for a role in regenerative differentiation.

CaN19, a member of the S100 family of calcium-binding proteins, is known to be "underexpressed" in cultured breast carcinoma-derived cell lines relative to their normal counterparts. By Northern blotting, we confirm these results and find that CaN19 is also markedly "underexpressed" in several carcinoma-derived cell lines of the skin, oral mucosa, and urogenital tract. However, exceptions to the inverse correlation between CaN19 expression and malignancy have been identified, bringing into question the hypothesis that CaN19 functions as a tumor suppressor gene. Unexpectedly, CaN19 mRNA was strongly expressed in bulk specimens of basal and squamous cell carcinomas of the skin and oral cavity. However, in situ hybridization revealed only limited CaN19 expression in tumor cells themselves; the bulk of expression is localized to hyperplastic perilesional epidermis. Tumor cell expression of CaN19 was similar in primary and locally metastatic tumors, indicating that this gene is not necessarily down-regulated during tumor progression. Coordinate overexpression of CaN19 and the "hyperproliferalive" keratin K6a was observed only in tissues undergoing squamous differentiation. Taken together with other recent results from our laboratory, these findings suggest the hypothesis that CaN19 participates in an epidermal growth factor receptor-dependent pathway of regenerative squamous differentiation.

The EGF receptor ligand amphiregulin controls cell division via FoxM1.

Epidermal growth factor receptor (EGFR) is central to epithelial cell physiology, and deregulated EGFR signaling has an important role in a variety of human carcinomas. Here we show that silencing of the EGF-related factor amphiregulin (AREG) markedly inhibits the expansion of human keratinocytes through mitotic failure and accumulation of cells with ⩾ 4n DNA content. RNA-sequencing-based transcriptome analysis revealed that tetracycline-mediated AREG silencing significantly altered the expression of 2331 genes, 623 of which were not normalized by treatment with EGF. Interestingly, genes irreversibly upregulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. Moreover, a significant proportion of the irreversibly downregulated genes featured upstream binding sites recognized by forkhead box protein M1 (FoxM1), a key transcription factor in the control of mitosis that is widely dysregulated in cancer. The downregulation of FoxM1 and its target genes preceded mitotic arrest. Constitutive expression of FoxM1 in AREG knockdown cells normalized cell proliferation, reduced the number of cells with ⩾ 4n DNA content and rescued expression of FoxM1 target genes. These results demonstrate that AREG controls G2/M progression and cytokinesis in keratinocytes via activation of a FoxM1-dependent transcriptional program, suggesting new avenues for treatment of epithelial cancer.

EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL.

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.

Differential utilization and localization of ErbB receptor tyrosine kinases in skin compared to normal and malignant keratinocytes.

Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals.

EGF stimulates transcription of CaN19 (S100A2) in HaCaT keratinocytes.

CaN19 (S100A2), a member of the S100 family of calcium-binding proteins, was originally isolated in a screen for tumor suppressor genes. Recent work from our laboratory suggests that CaN19 is likely to be an effector of the regenerative hyperplasia pathway of epidermal differentiation. As other work from our laboratory in a human skin organ culture model suggests that this response is mediated by activation of the epidermal growth factor (EGF) receptor and/or related receptors of the ErbB family, we asked whether CaN19 expression could be increased by organ culture and by EGF treatment of human keratinocytes. CaN19 was strongly induced after 24 h of organ culture, and its induction could be blocked by PD153035, a specific inhibitor of EGF receptor tyrosine kinase activity. EGF treatment of immortalized human keratinocytes (HaCaT cells) increased CaN19 mRNA levels by 4.5-fold within 8 h, and a corresponding increase in CaN19 protein was observed by western blotting. EGF treatment had no effect on the expression of five other members of the S100A gene cluster. As assessed by nuclear run-off assay, CaN19 transcription increased rapidly in response to EGF, reaching a maximum induction of 16-fold after 2 h. In contrast, EGF treatment had no detectable effects on the decay of CaN19 transcripts, which were long lived (t1/2 > 6 h) in the presence or absence of EGF. PD153035 also blocked CaN19 transcription and the accumulation of CaN19 mRNA and protein in HaCaT cells. These results demonstrate that EGF receptor activation selectively stimulates CaN19 gene expression at the transcriptional level in human keratinocytes, and support the hypothesis that CaN19 is an important mediator of regenerative epidermal hyperplasia.

Epidemiology and the genetics of psoriasis.

Several lines of compelling epidemiologic evidence indicate that susceptibility to psorasis is inherited, albeit not in a simple monogenic fashion. Psoriasis is one of a number of diseases with a presumed autoimmune pathogenesis that display significant human leukocyte antigen (HLA) associations. However, only a small fraction of those who carry the implicated HLA susceptibility alleles develop disease. Taken together with the epidemiologic data indicative of high heritability, this observations suggests that one or more loci in addition to HLA are necessary for the development of psoriasis. As the identity of these other genes is unknown, genetic linkage analysis offers an attractive strategy for their identification. To this end, we have initiated a large linkage study of multiplex psoriasis kindreds, and PCR-based genotyping of CA repeat polymorphisms has been performed for several markers in the HLA region (6p21.3). As expected given the hypothesis of oligogenic inheritance, these analyses have thus far failed to reveal tight linkage of psoriasis to the 6p21 region. Nevertheless, the substantial homogeneity of the psoriatric phenotype and the clear evidence for increased HLA association and heritability in juvenile onset disease (40 years) indicate that, like insulin-dependent diabetes mellitus, psoriasis is an HLA-associated, genetically complex disease whose etiology is potentially amenable to elucidation through linkage analysis.

Biochemical characterization of S100A2 in human keratinocytes: subcellular localization, dimerization, and oxidative cross-linking.

S100A2 is a calmodulin-like protein of unknown function, whose transcription is positively regulated in response to ErbB and p53 signaling. Expression of S100A2 is markedly increased in the context of ErbB-driven reactive epidermal hyperplasia, and decreased in the context of hypofunctional p53 mutations in carcinoma cell lines and tumors. This bimodal pattern of regulation suggests an important function for S100A2 in keratinocyte differentiation and carcinogenesis. Taking the biochemical approach to the determination of S100A2 function, we have characterized its physical state and subcellular localization in normal human keratinocytes. S100A2 in hypotonic lysates remained soluble after centrifugation at 100 000 x g, indicating that it is not associated with cell membranes. Permeabilization experiments confirmed the lack of membrane association and revealed a digitonin-insoluble nuclear fraction of S100A2, which was confirmed by immunofluorescence microscopy. Pulldown assays of epitope-tagged S100A2 and yeast two-hybrid screening revealed that S100A2 displays a strong propensity to homodimerize. Naturally expressed S100A2 dimers in normal human keratinocytes readily underwent intermolecular disulfide cross-linking unless a strong denaturant was present during cell lysis. Treatment of intact normal human keratinocytes with hydrogen peroxide strongly promoted S100A2 cross-linking. These results demonstrate that native S100A2 is a homodimer that does not depend on disulfide cross-linking for stability, but undergoes intermolecular cross-linking at cysteine residues in response to oxidative stress. Based on these findings, we propose that S100A2 may protect normal keratinocytes against carcinogens by participating in the cellular proof-reading response to oxidative stress.

GRO-alpha mRNA is selectively overexpressed in psoriatic epidermis and is reduced by cyclosporin A in vivo, but not in cultured keratinocytes.

Interleukin (IL)-8 and gro peptides are members of the intercrine-alpha family of chemotaxins known to be present in biologically active form in psoriasis lesions. However, the relative contribution of the three different gro genes to the expression of this material is unknown, as is the stimulus for gro overexpression in psoriatic lesions. To address these questions, Northern blot and semiquantitative polymerase chain reaction analysis were performed on RNA extracted from keratome biopsies of normal skin, untreated plaques of psoriasis, or plaques treated for 1 week with low-dose cyclosporin A (CsA). Northern blot analysis revealed a significant correlation between gro and IL-8 mRNA levels in psoriasis lesions from 26 different individuals (r = 0.61, p = 0.0009), and overexpression of gro was markedly reduced by CsA prior to detectable clinical improvement (79.3%, p = 0.01, n = 22). To determine which form(s) of gro were overexpressed in psoriatic lesions, total keratome RNA (1 microgram) was analyzed by semiquantitative reverse transcription-polymerase chain reaction (SQRT-PCR). In five patients known to markedly overexpress gro and IL-8 mRNAs by Northern blotting, gro-alpha was approximately six times more abundant than gro-beta, and 25 times more abundant than gro-gamma. In cultured human keratinocytes, all three forms of gro mRNA were increased by IL-1 alpha or by interferon (IFN)-gamma plus tumor necrosis factor (TNF)-alpha. However, in contrast to the situation in vivo, CsA had no inhibitory effect on cytokine-stimulated gro expression in cultured keratinocytes. Taken together, these results demonstrate that the gro-alpha gene is selectively overexpressed in psoriatic lesions and strongly suggest that overexpression of gro is a keratinocyte response to activated T cells in psoriasis.

Sphingosine inhibits phorbol ester-induced inflammation, ornithine decarboxylase activity, and activation of protein kinase C in mouse skin.

Tumor promoting phorbol esters, such as 12-0-tetradecanoylphorbol-13-acetate (TPA), when applied topically to mouse skin cause inflammation and hyperplasia. The major cellular phorbol ester receptor is a calcium and phospholipid dependent protein kinase, protein kinase C (PK-C). PK-C is directly activated by TPA and most of the responses of cells to TPA appear to be mediated by PK-C. This suggests that PK-C may play a key role as a mediator of inflammation and growth in TPA treated mouse skin. Sphingosine has been reported to be a potent inhibitor of PK-C in vitro and in intact leukocytes. We therefore have investigated the effects of sphingosine upon TPA-induced inflammation, hyperplasia, induction of ornithine decarboxylase (ODC) activity and ODC mRNA, and activation of PK-C in mouse skin. The results demonstrate that sphingosine is a potent inhibitor of all of the TPA-induced responses examined. These data are compatible with the hypothesis that PK-C is a major mediator of the phorbol ester response in mouse skin. Furthermore, PK-C inhibitors may have therapeutic potential in inflammatory skin diseases such as psoriasis.

Stimulus-selective induction of CRABP-II mRNA: a marker for retinoic acid action in human skin.

Acute topical treatment of human skin with retinoic acid (RA) results in a pleiotropic response, some aspects of which are mimicked by non-specific irritants. To identify reliable cutaneous markers of retinoid action, it is important to determine which aspects of this response are specifically due to the presence of RA. We have previously demonstrated a rapid and pronounced increase in steady-state cellular RA-binding protein II (CRABP-II)mRNA levels after topical RA treatment. Here we characterize the dose dependence and kinetics of this response, and compare the effects of a well-known irritant, sodium dodecylsulfate (SDS), to those of RA and its vehicle. The induction of CRABP-II mRNA in response to 0.1% RA cream was maximal by 16 h (elevenfold relative to untreated skin), and persisted at near-maximal levels (eight-fold) for up to 4 d. RA was potent in eliciting this response, as approximately half-maximal stimulation was observed after 16 h of treatment with 0.001% RA. Treatment for 4 d with 0.1% RA cream versus 2% SDS in RA vehicle resulted in nearly identical levels of cutaneous erythema, spongiosis, and epidermal thickening. However, the CRABP-II mRNA response to 2% SDS was no greater than that observed in response to vehicle alone (2.9 times relative to occluded skin control at 4 d). SDS also had no effect upon either CRABP-II or RAR-beta mRNA levels in quiescent human dermal fibroblasts in vitro, whereas RA elicited both responses at 1000-times lower concentrations than SDS. Taken together, these data identify the CRABP-II mRNA response as a reliable, rapid, and selective marker for retinoid activity in human skin.