Downregulated miR-187 contributes to the keratinocytes hyperproliferation in psoriasis

Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. However, the pathogenesis of psoriasis is multifactorial and is not fully understood. MicroRNAs (miRNAs) represent a promising class of small, noncoding RNA molecules that have a large impact on cellular functions by regulating gene expression. Here we reported that microRNA-187 (miR-187), which is one of the most dynamic microRNAs identified in the deep screening miRNAs profile, is downregulated in inflammatory cytokines-stimulated keratinocytes and psoriatic skins. By luciferase activity assay and gain-of-function studies, we showed that miR-187 inhibits keratinocytes hyperproliferation by targeting CD276. Moreover, overexpression of miR-187 decreases acanthosis and reduces the disease severity in psoriasis mouse models. Taken together, the results of our study implies miR-187 as a critical factor in psoriasis pathogenesis, which could be a potent target for psoriasis treatment.     

Histochemical analysis of macrophage migration inhibitory factor in psoriasis vulgaris

Psoriasis is a persistent cutaneous disease characterized by skin inflammation and infiltration of immunocytes such as lymphocytes and monocytes/macrophages, concomitant with abnormal epidermal hyperproliferation. We previously showed that the serum level of macrophage migration inhibitory factor (MIF) and its production by peripheral blood mononuclear cells of patients with psoriasis were closely correlated with the severity of clinical symptoms; however, the precise role of MIF in psoriatic epidermis remains to be clarified. The current study was carried out to elucidate the possible involvement of MIF in psoriasis, using immunohistochemistry and in situ hybridization. In contrast to elevated serum MIF in psoriasis, MIF-positive staining in the lesional psoriatic epidermis was significantly decreased, as demonstrated by immunohistochemical analysis using an anti-MIF antibody. Consistent with this finding, we found, by in situ hybridization, that MIF mRNA concomitantly decreased in the psoriatic lesions. Although the reason for the different MIF levels in the psoriatic epidermis and in the circulation remains unknown, it is hypothesized that MIF, a potential growth factor, might be decreased in psoriatic lesions to counterregulate the abnormal epidermal proliferation caused by dysregulation of cytokines and growth factors.     

Gene expression in skin and lymphoblastoid cells: Refined statistical method reveals extensive overlap in cis-eQTL signals

Psoriasis, an immune-mediated, inflammatory disease of the skin and joints, provides an ideal system for expression quantitative trait locus (eQTL) analysis, because it has a strong genetic basis and disease-relevant tissue (skin) is readily accessible. To better understand the role of genetic variants regulating cutaneous gene expression, we identified 841 cis-acting eQTLs using RNA extracted from skin biopsies of 53 psoriatic individuals and 57 healthy controls. We found substantial overlap between cis-eQTLs of normal control, uninvolved psoriatic, and lesional psoriatic skin. Consistent with recent studies and with the idea that control of gene expression can mediate relationships between genetic variants and disease risk, we found that eQTL SNPs are more likely to be associated with psoriasis than are randomly selected SNPs. To explore the tissue specificity of these eQTLs and hence to quantify the benefits of studying eQTLs in different tissues, we developed a refined statistical method for estimating eQTL overlap and used it to compare skin eQTLs to a published panel of lymphoblastoid cell line (LCL) eQTLs. Our method accounts for the fact that most eQTL studies are likely to miss some true eQTLs as a result of power limitations and shows that ～70% of cis-eQTLs in LCLs are shared with skin, as compared with the naive estimate of < 50% sharing. Our results provide a useful method for estimating the overlap between various eQTL studies and provide a catalog of cis-eQTLs in skin that can facilitate efforts to understand the functional impact of identified susceptibility variants on psoriasis and other skin traits.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.     

MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R

Background

Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder.

Methodology/Principal Findings

Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca⁺⁺, miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a.

Conclusions/Significance

We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.     

A sequential double immunoenzymic staining procedure to obtain cell kinetic information in normal and hyperproliferative epidermis

Summary A sequential double immunoenzymic staining procedure was developed using the monoclonal antibody anti-BrdUrd and Ki67 in order to determine whether hyperproliferative skin disorders, such as psoriasis, are characterized by an increased growth fraction rather than a much shorter cell cycle time of all germinative cells. Ki67 binds to a proliferation-associated nuclear antigen in a variety of human cell types, and anti-BrdUrd can be used to identify DNA-synthesizing cells. Although in hyperproliferative epidermis the absolute numbers of BrdUrd-positive cells as well as Ki67-positive cells were grossly increased, the ratio of these values was not changed compared to the ratio found in the epidermis of the clinically uninvolved skin of psoriatic patients and in normal epidermis. This suggests an increased growth fraction in hyperproliferative epidermis.Our data show that immunohistochemical double-staining techniques can be a valuable tool in the study of cell cycle kinetics in epithelial tissues.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.     

Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB) phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Summary Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

IFN-gamma promoter gene polymorphism in psoriasis vulgaris.

This study was performed to investigate the association between interferon (IFN)-gamma single nucleotide polymorphism (SNP) and susceptibility for psoriasis vulgaris. DNA from 78 patients with psoriasis vulgaris (54 patients with type I psoriasis, 24 with type II psoriasis) and 74 healthy volunteers was investigated. IFN-gamma promoter gene SNP in position 874 was evaluated by polymerase chain reaction with sequence-specific primers (PCR-SSP) and the results were compared between a group of psoriatic patients, divided into early onset of psoriasis (type I) and late onset of psoriasis (type II) subgroups, and healthy control subjects. A significant difference in the genotype frequencies between psoriasis patients and healthy controls was found (p < 0.02) and no significant differences were observed analyzing subsets of psoriatic patients (gender, type of disease) also in carriage and allele frequencies. The results suggest that IFN-gamma polymorphism is associated with susceptibility to psoriasis vulgaris.