The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice.

The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.

Tyrosine kinase 2 and interferon regulatory factor 5 polymorphisms are associated with discoid and subacute cutaneous lupus erythematosus.

Lupus erythematosus (LE) is a heterogeneous disease ranging from skin-restricted manifestations to a progressive multisystem disease. The specific skin lesions include chronic cutaneous, subacute cutaneous and acute cutaneous LE. Both genetic and environmental factors are involved in the development of LE. However, reports on the genetic background of cutaneous lupus erythematosus (CLE) forms, namely discoid (DLE) and subacute cutaneous lupus erythematosus (SCLE), are sparse. We investigated whether the known systemic LE (SLE) susceptibility genes also predispose to CLE. Altogether, 219 Finnish patients with DLE or SCLE and 356 healthy controls were recruited. Single nucleotide polymorphisms tagging reported risk genes were genotyped. Tyrosine kinase 2 (TYK2) rs2304256 was associated with increased risk of DLE (P = 0.012, OR = 1.47, 95% CI = 1.01-1.98). Expression of TYK2 was demonstrated by immunohistochemistry in macrophage-like cells and neutrophils and interferon regulatory factor 5 (IRF5) in macrophage- and fibroblast-like cells of DLE, SCLE and SLE skin. IRF5 rs10954213 showed association with DLE (P = 0.017, OR = 1.40, 95% CI = 1.06-1.86) and SCLE (P = 0.022, OR = 1.87, 95% CI = 1.09-3.21). A haplotype of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) showed association with DLE (P = 0.0065, OR = 2.51, 95% CI = 1.25-5.04). Our results show that the TYK2, IRF5 and CTLA4 genes previously associated with SLE also confer risk for DLE and SCLE, suggesting that different LE subphenotypes may share pathogenetic pathways.

Expression profiles of matrix metalloproteinases and their inhibitors in colonic inflammation related to pediatric inflammatory bowel disease.

OBJECTIVE: The differential diagnosis of chronic colitis in inflammatory bowel disease (IBD) is challenging and a distinction between Crohn's disease (CD) and ulcerative colitis (UC) is not always possible. Matrix metalloproteinases (MMPs) cleave components of the extracellular matrix and their dysregulation leads to damage to the mucosa. They are involved in inflammation in IBD, as well as in eventual tissue repair. We aimed to examine putative differences in the profiles of MMPs and their tissue inhibitors [tissue inhibitors of metalloproteinase (TIMPs)] in pediatric IBD to find better tools for differential diagnosis of various IBD subgroups at the tissue level in the colon. MATERIAL AND METHODS: Expression of MMPs -1, -7, -8, -9, -10, -12 and -26 and TIMPs -1 and -3 was studied by immunohistochemistry in colonic tissue samples of 32 pediatric patients with IBD and 11 non-IBD cases. RESULTS: In the colon, expression of MMP-7 in epithelium was greater in CD samples compared to UC samples (1.09 versus 0.33; P = 0.010). Furthermore, epithelial MMP-10 expression was elevated in CD and UC samples compared to non-IBD samples (1.55 versus 1.00; P = 0.041 and 1.58 versus 1.00; P = 0.025, respectively). TIMP-3 expression in the stroma was higher in both the CD and UC groups when compared to non-IBD samples (2.18 versus 1.36; P = 0.026 and 2.50 versus 1.36; P = 0.002, respectively), but differences between UC and CD could not be observed. CONCLUSIONS: Increased expression of epithelial MMP-10 and stromal TIMP-3 could serve as histological indicators of IBD etiology. Epithelial MMP-7 expression, on the other hand, could help to differentiate between CD-related colitis and UC.

Ultraviolet B radiation regulates cysteine-rich protein 1 in human keratinocytes.

BACKGROUND: Cysteine-rich protein 1 (CRP1) is a growth-inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body. METHODS: The effects of physiologically relevant doses of UVB radiation on CRP1 protein levels were studied in cultured primary keratinocytes and transformed cell lines (HaCaT, A-431) by immunoblotting. UVB-induced keratinocyte apoptosis was assessed by flow cytometry and monitoring caspase activity. Expression of CRP1 in human skin in vivo was studied by immunohistochemistry in samples of normal skin, actinic keratosis (AK) representing UV-damaged skin and squamous cell carcinoma (SCC), a UV-induced skin cancer. RESULTS: CRP1 expression increased by UVB radiation in primary but not in immortalized keratinocytes. Upon high, apoptosis-inducing doses of UV radiation, CRP1 was cleaved in a caspase-dependent manner. In normal skin, CRP1 was expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands and hair root sheath, but very little CRP1 was present in keratinocytes. CRP1 expression was elevated in basal cells in AK but not in SCC. CONCLUSION: CRP1 expression is regulated by UVB in human keratinocytes, suggesting a role for CRP1 in the phototoxic responses of human skin.

IL23R in the Swedish, Finnish, Hungarian and Italian populations: association with IBD and psoriasis, and linkage to celiac disease.

BACKGROUND: Association of the interleukin-23 receptor (IL23R) with inflammatory bowel disease (IBD) has been confirmed in several populations. IL23R also associates with psoriasis, suggesting that the gene may be an important candidate for many chronic inflammatory diseases. METHODS: We studied association of single-nucleotide variants in IL23R with IBD in Swedish patients, in both Crohn's disease (CD) and ulcerative colitis (UC) subsets. The same genetic variants were also studied in Finnish patients with psoriasis or celiac disease, and in Hungarian and Italian patients with celiac disease. RESULTS: Association of IL23R with IBD was replicated in our Swedish patients, and linkage and association of the IL23R region with psoriasis was found in the Finnish population. The IL23R region was also linked to celiac disease in Finnish families, but no association of IL23R variants with celiac disease was found in the Finnish, Hungarian or Italian samples. CONCLUSION: Our study is the first to demonstrate association of IL23R with CD and UC in Swedish patients with IBD. It is also the first study to report linkage and association of the IL23R region with psoriasis in the Finnish population. Importantly, this is the first report of linkage of the IL23R region to celiac disease, a chronic inflammatory condition in which IL23R has not been previously implicated.

MMP-10 (Stromelysin-2) and MMP-21 in human and murine squamous cell cancer.

The squamous cell cancers (SCC) of renal transplant recipients are more aggressive and metastasize earlier than those of the non-immunocompromised population. Matrix metalloproteinases (MMPs) have a central role in tumor initiation, invasion and metastasis. Our aim was to compare the expression of MMPs-10, -12 and -21 in SCCs from immunosuppressed (IS) and control patients and the contribution of MMPs-10 and -21 to SCC development in the FVB/N-Tg(KRT5-Nfkbia)3Rto mouse line. Immunohistochemical analysis of 25 matched pairs of SCCs, nine of Bowen's disease and timed back skin biopsies of mice with selective inhibition of Rel/NF-kappaB signalling were performed. Semiquantitatively assessed stromal MMP-10 expression was higher (P = 0.009) in the control group when compared with IS patients. Tumor cell-derived MMP-10, -12 and -21 expression did not differ between the groups but stromal fibroblasts of the control SCCs tended to express MMP-21 more abundantly. MMP-10 expression was observed already in Bowen's disease while MMP-21 was absent. MMP-10 and -21 were present in inflammatory or stromal cells in ageing mice while dysplastic keratinocytes and invasive cancer were negative. Our results suggest that MMP-10 may be important in the initial stages of SCC progression and induced in the stroma relating to the general host-response reaction to skin cancer. MMP-21 does not associate with invasion of SCC but may be involved in keratinocyte differentiation.

Association of psoriasis to PGLYRP and SPRR genes at PSORS4 locus on 1q shows heterogeneity between Finnish, Swedish and Irish families.

A susceptibility locus for psoriasis, PSORS4, has been mapped to chromosome 1q21 in the region of the epidermal differentiation complex. The region has been refined to a 115 kb interval around the loricrin (LOR) gene. However, no evidence of association between polymorphisms in the LOR gene and psoriasis has been found. Therefore, we have analysed association to three candidate gene clusters of the region, the S100, small proline-rich protein (SPRR) and PGLYRP (peptidoglycan recognition protein) genes, which all contain functionally interesting psoriasis candidate genes. In previous studies, the SPRR and S100 genes have shown altered expression in psoriasis. Also polymorphisms in the PGLYRP genes have shown to be associated with psoriasis. We genotyped altogether 29 single nucleotide polymorphisms (SNPs) in 255 Finnish psoriasis families and analysed association with psoriasis using transmission disequilibrium test. A five-SNP haplotype of PGLYRP SNPs associated significantly with psoriasis. There was also suggestive evidence of association to SPRR gene locus in Finnish families. To confirm the putative associations, selected SNPs were genotyped also in a family collection of Swedish and Irish patients. The families supported association to the two gene regions, but there was also evidence of allelic heterogeneity.

Changes in matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinases (TIMP) expression profile in Crohn's disease after immunosuppressive treatment correlate with histological score and calprotectin values.

BACKGROUND: Matrix metalloproteinases (MMPs) constitute a family of enzymes capable of degrading various extracellular matrices (ECM) and basement membrane components playing a role in ECM turnover. They activate and degrade signaling molecules, such as cytokines and chemokines. MMPs are involved in inflammation and have been implicated in tissue degradation and repair occurring in inflammatory bowel disease. The aim of this study was to investigate the MMP profile of intestinal Crohn's disease (CD) patients before and after immunosuppressive treatment (anti-TNF-alpha agents or corticosteroids and conventional immunosuppressants azathioprine or methotrexate) to learn more about the therapeutic pathways for immunosuppressive agents. METHODS: Expression of MMP-1, MMP-7, MMP-9, MMP-10, and MMP-26 and tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-3 was studied by immunohistochemistry in pretreatment and post-treatment tissue samples. Semiquantitative immunohistochemical scores were tested for correlations with fecal and serum inflammation markers as well as endoscopic and clinical disease activity scores. RESULTS: Neutrophil MMP-9 (p = 0.039) and MMP-26 (p = 0.030) and stromal TIMP-1 (p = 0.041) and TIMP-3 (p = 0.029) decreased along with treatment. However, expression of TIMP-3 by enterocytes tended to increase. Total histological score demonstrated positive correlation with neutrophil MMP-9 (p = 0.000), MMP-26 (p = 0.014), and macrophage TIMP-1 (p = 0.001). Calprotectin followed a similar pattern with stromal MMP-26 (p = 0.011), TIMP-1 (p = 0.000), and TIMP-3 (p = 0.001). Crohn's disease endoscopic index of severity (CDEIS) value correlated positively with macrophage TIMP-1 (p = 0.007) and stromal TIMP-3 (p = 0.005). Epithelial TIMP-3 presented with negative correlations with CDEIS (p = 0.006) and C-reactive protein values (p = 0.004). CONCLUSIONS: Our results suggest that immunosuppressive drugs modulate disease activity in CD by downregulation of MMP-9 and MMP-26 positive neutrophils and stromal TIMP-1 and TIMP-3.

Matrix metalloproteinase-26 is present more frequently in squamous cell carcinomas of immunosuppressed compared with immunocompetent patients.

BACKGROUND: Skin cancers are the most frequent malignancies in organ transplant recipients (OTRs). Squamous cell carcinomas (SCCs) occur 65-250 times more frequently in OTRs and tend to be aggressive in behavior. Because matrix metalloproteinases (MMPs) have a central role in tumorigenesis and invasion, we investigated the epithelial and stromal MMP and tissue inhibitor of MMP (TIMP) expression profile in SCCs of immunosuppressed (IS) compared with immunocompetent (IC) patients to determine if differences could explain the more aggressive behavior of SCCs in OTRs. METHODS: Matched pairs from 20 SCCs of IS and IC patients were studied using immunohistochemistry for MMP-1, MMP-7, MMP-8, MMP-9, MMP-13 and MMP-26 and TIMP-1 and TIMP-3. RESULTS: Among all MMPs studied, only staining for MMP-26 was significantly more intense in cancer cells of the post-transplant group compared with the IC group (p = 0.01), whereas MMP-9 expression was more abundant in stromal macrophages surrounding SCCs of IC patients (p = 0.02). MMP-26 expression in cancer cells (p = 0.04) and that of MMP-9 in neutrophils (p = 0.005) were more abundant in SCCs of patients using cyclosporine. CONCLUSIONS: We conclude that MMP-26 and MMP-9 may contribute to the more aggressive behavior of SCCs in OTRs.

Differential expression of stromal MMP-1, MMP-9 and TIMP-1 in basal cell carcinomas of immunosuppressed patients and controls.

Matrix metalloproteinases (MMPs) have an important role in the initiation, growth, and invasion of malignant tumors. Basal cell cancer (BCC) is the most common human malignancy. The risk of BCC is 10-16 times higher among organ transplant recipients compared with the nontransplanted population. The aim of this study was to compare the expression of several MMPs and their tissue inhibitors (TIMPs) in BCCs from kidney transplant recipients and controls. Expression of MMPs-1, -7, -8, -9, -10, -13, -26, and TIMPs-1 and -3 was evaluated by immunohistochemistry in 25 samples of BCC of kidney transplant recipients and 25 matched controls representing superficial and nodular subtypes. No significant differences were detected in MMP expression of BCC tumor cells between immunocompetent and immunodeficient patients. However, MMPs-1 and -9 and TIMP-1 were expressed more frequently in stromal macrophages in the BCCs of immunocompetent patients. When tumor subtypes were compared irrespective of the patient group, more MMP-1-positive fibroblasts and MMP-9-positive neutrophils were detected in the superficial subtype, while stromal MMP-10 expression was more abundant in nodular tumors. Our results suggest that abundant peritumoral expression of TIMP-1 in non-immunocompromised patients limits ECM degradation permissive for cancer cell migration.

The CCHCR1 (HCR) gene is relevant for skin steroidogenesis and downregulated in cultured psoriatic keratinocytes.

The HCR gene, officially called Coiled-Coil alpha-Helical Rod protein 1 (CCHCR1), located within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene for the risk effect. Recently, CCHCR1 was shown to promote steroidogenesis by interacting with the steroidogenic acute regulator protein (StAR). Here, we examined the role of CCHCR1 in psoriasis and cutaneous steroid metabolism. We found that CCHCR1 and StAR are expressed in basal keratinocytes in overlapping areas of the human skin, and CCHCR1 stimulated pregnenolone production in steroidogenesis assay. Overexpression of either the CCHCR1*WWCC risk allele or the non-risk allele enhanced steroid synthesis in vitro. Furthermore, the cytochrome P450scc enzyme was expressed in human keratinocytes and was induced by forskolin, a known activator of steroidogenesis, and forskolin also upregulated CCHCR1. CCHCR1 has an altered expression pattern in lesional psoriatic skin compared to normal healthy skin, suggesting its dysregulation in psoriasis. We found that the expression of CCHCR1 is downregulated twofold at the mRNA level in cultured non-lesional psoriatic keratinocytes when compared to non-psoriatic healthy cells. Our results also suggest a connection between CCHCR1 and vitamin D metabolism in keratinocytes. The expression of the vitamin D receptor (VDR) gene was lower in non-lesional psoriatic keratinocytes than in healthy cells. Furthermore, Vdr expression was downregulated in the keratinocytes of mice overexpressing the CCHCR1*WWCC risk allele when compared to keratinocytes from mice with the non-risk allele of CCHCR1. Finally, we demonstrate that other agents relevant for psoriasis and/or the regulation of steroidogenesis influence CCHCR1 expression in keratinocytes, including insulin, EGF, cholesterol, estrogen, and cyclosporin A. Taken the role of steroid hormones, including vitamin D and estrogen, in cell proliferation, epidermal barrier homeostasis, differentiation, and immune response, our results suggest a role for CCHCR1 in the pathogenesis of psoriasis via the regulation of skin steroid metabolism.

The human GIMAP5 gene has a common polyadenylation polymorphism increasing risk to systemic lupus erythematosus.

BACKGROUND: Several members of the GIMAP gene family have been suggested as being involved in different aspects of the immune system in different species. Recently, a mutation in the GIMAP5 gene was shown to cause lymphopenia in a rat model of autoimmune insulin-dependent diabetes. Thus it was hypothesised that genetic variation in GIMAP5 may be involved in susceptibility to other autoimmune disorders where lymphopenia is a key feature, such as systemic lupus erythematosus (SLE). MATERIAL AND METHODS: To investigate this, seven single nucleotide polymorphisms in GIMAP5 were analysed in five independent sets of family-based SLE collections, containing more than 2000 samples. RESULT: A significant increase in SLE risk associated with the most common GIMAP5 haplotype was found (OR 1.26, 95% CI 1.02 to 1.54, p = 0.0033). In families with probands diagnosed with trombocytopenia, the risk was increased (OR 2.11, 95% CI 1.09 to 4.09, p = 0.0153). The risk haplotype bears a polymorphic polyadenylation signal which alters the 3' part of GIMAP5 mRNA by producing an inefficient polyadenylation signal. This results in higher proportion of non-terminated mRNA for homozygous individuals (p<0.005), a mechanism shown to be causal in thalassaemias. To further assess the functional effect of the polymorphic polyadenylation signal in the risk haplotype, monocytes were treated with several cytokines affecting apoptosis. All the apoptotic cytokines induced GIMAP5 expression in two monocyte cell lines (1.5-6 times, p<0.0001 for all tests). CONCLUSION: Taken together, the data suggest the role of GIMAP5 in the pathogenesis of SLE.

Th1 response and cytotoxicity genes are down-regulated in cutaneous T-cell lymphoma.

PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.

Matrilysin-2 (matrix metalloproteinase-26) is upregulated in keratinocytes during wound repair and early skin carcinogenesis.

Matrilysin-2 (matrix metalloproteinase (MMP)-26) is a small protein of the MMP family expressed in some epithelial carcinomas and normal tissues. We studied its role in benign skin disorders characterized by epithelial proliferation, in wound repair, skin cancer, and regulation in keratinocyte (KC) cultures. MMP-26 is expressed by laminin-5-positive KC in the migrating area during wound repair, in benign skin disorders characterized by inflammation and microdisruptions of basement membrane, but in intact skin only in hair follicles. It was detected in occasional atypical KC in pre-malignant lesions but not in basal cell cancer islands. Although MMP-26 was expressed in grades I and II squamous cell cancers (SCC), it was not present in dedifferentiated grade III tumors. MMP-26 was neither co-expressed with its close homologue matrilysin-1 nor with the proliferation marker Ki-67. But in tissue samples it either co-localized or was detected in adjacent cells of same regions with the tumor suppressor p16. In KC and HaCaT cell cultures, 12-phorbol-13-myristate-acetate, epidermal growth factor, tumor necrosis factor-alpha, transforming growth factor-beta1, interleukin-1 (IL-1)beta, IL-6, insulin-like growth factor, gamma-IFN, retinoic acid, dexamethasone, four matrices or ras-transformation were unable to upregulate MMP-26 expression. The expression pattern of MMP-26 suggests that it may be upregulated in basal KC even without tumorigenesis because of altered cell-matrix interactions and inflammation and, unlike most MMP, becomes downregulated during histological dedifferentiation of SCC. Thus, lack of MMP-26 in SCC could be a marker of aggressive growth.

MMP-21 is upregulated at early stages of melanoma progression but disappears with more aggressive phenotype.

The expression of matrix metalloproteinases (MMPs) is frequently altered during malignant transformation. We examined the profile of three recently cloned MMPs, MMP-21, MMP-26, and MMP-28, in melanomas in vivo and in culture. Immunohistochemistry for MMPs-21, -26, -28, and -13 in melanoma specimens (27 nonmetastatic, 26 with nodal micrometastases, and 10 in situ melanomas) from 63 patients was performed. MMP-21 was expressed in melanoma cells in 29/53 cases, being more frequent in melanoma samples without micrometastases. Six out of ten in situ melanomas were positive, while five nevus samples were negative. MMP-26 and -28 were not generally expressed in melanoma cells. MMP-13 was detected in melanoma cells in 36/53 samples. MMP-21 was not found in sentinel nodes with metastases, while MMP-13 was seen in all of them. MMP-21 messenger RNA was variably expressed in all five melanoma cell lines investigated using reverse transcriptase-polymerase chain reaction. Our results suggest that expression of MMP-21 may serve as a marker of malignant transformation of melanocytes and does not associate with the presence of micrometastases.

Interferon alpha-inducible protein 27 (IFI27) is upregulated in psoriatic skin and certain epithelial cancers.

IFI27 is an interferon alpha-inducible protein found to be upregulated in lesional and non-lesional psoriatic skin in a gene array study. To further characterize its function, we studied by in situ hybridization whether IFI27 is expressed in psoriasis, other inflammatory skin diseases, and wound repair in vivo. We also examined its regulation by different growth factors and anti-psoriatic agents using quantitative RT-PCR (TaqMan). IFI27 mRNA expression was highly upregulated in lesional psoriatic epidermis and also detected in non-lesional keratinocytes. It was also expressed in lichen planus, chronic eczema, cutaneous squamous cell cancers, and during normal wound repair when IFI27 was found in the proliferating subpopulation of keratinocytes. A 3-17-fold upregulation of IFI27 mRNA expression was observed when keratinocytes were stimulated with IFN-gamma, TNF-alpha, or TGF-beta1 while retinoids and vitamin D downregulated its expression. Our results suggest that IFI27 is a novel marker of epithelial proliferation and cancer.

Matrix metalloproteinase inhibitor BB-3103 unlike the serine proteinase inhibitor aprotinin abrogates epidermal healing of human skin wounds ex vivo.

Several matrix metalloproteinases and serine proteinases are upregulated in migrating keratinocytes during cutaneous wound repair. Single cell culture studies indicate the necessity for matrix metalloproteinases but not for serine proteinases in keratinocyte locomotion. To account for epithelial-mesenchymal interactions, an ex vivo human skin wound model was used to investigate the contribution of matrix metalloproteinases and serine proteinases to wound healing by treatment with broad-spectrum inhibitors of matrix metalloproteinases (BB-3103) or serine proteinases (aprotinin). Human skin explants with circular 3 mm superficial defects were incubated in culture medium without (controls) or with the proteinase inhibitors for 7 d. BB-3103 abrogated epithelialization (p < 0.001), whereas aprotinin-treated wounds and controls were covered with new epithelium. Lack of epithelialization was unlikely due to cytotoxicity because the matrix metalloproteinase inhibitor did neither influence viability of cultured epidermal keratinocytes nor apoptosis in wounds. Involvement of specific matrix metalloproteinases in epithelialization was analyzed by gelatin zymography, western blotting, immunohistochemistry, and in situ hybridization. Wound healing was accompanied by active matrix metalloproteinase-1 and increased active matrix metalloproteinase-2 but irrespectively of active matrix metalloproteinase-9. BB-3103 blocked activation of matrix metalloproteinase-2 and matrix metalloproteinase-9 but not of matrix metalloproteinase-1. Active matrix metalloproteinase-2 localized solely to the dermis, whereas matrix metalloproteinase-9 was consistently found in new epithelium. Membrane-type 1 matrix metalloproteinase was undetectable in wound keratinocytes. BB-3103 and aprotinin reduced tumor necrosis factor-alpha in media but did not appreciably alter amounts of other soluble regulators of matrix metalloproteinases and epithelialization. Our findings demonstrate that keratinocyte migration is associated with active matrix metalloproteinase-2 but occurs independently of serine proteinases and active matrix metalloproteinase-9 in fibrin-deficient skin wound healing.

Epilysin (MMP-28) expression is associated with cell proliferation during epithelial repair.

Epilysin (MMP-28) is the newest member of the matrix metalloproteinase enzyme family. Several members of this enzyme family have been associated with various aspects of wound repair and cancer invasion. The aim of this study was to characterize in different types of wounds, skin cancers, and keratinocyte cultures factors that contribute to epilysin expression in vivo, as well as how and where it is induced in relation to other matrix metalloproteinases. Our results indicate that epilysin is produced by the mitotic Ki-67-positive keratinocytes distal from the wound edge in both acute and chronic wounds and that it does not generally colocalize with collagenase-1, stromelysin-2, or 92 kDa gelatinase in migrating keratinocytes. An injury of epidermis was needed for epilysin induction as it was upregulated in ulcerated pyogenic granulomas and in suction blisters but was not detected in intact acanthotic or normal skin. Unlike many other matrix metalloproteinases, epilysin was not detected in the invading cancer cell nests of sclerosing basal or squamous cell cancers of various grades. When primary keratinocytes were stimulated with tumor necrosis factor alpha, upregulation of epilysin mRNA was evident within 24-48 h as measured by quantitative reverse transcription polymerase chain reaction. In primary keratinocyte, HaCaT, and A431 carcinoma cell cultures none of the 10 other growth factors or extracellular matrices studied were able to upregulate epilysin expression. Our results suggest that epilysin expression is tightly spatially and temporally regulated during wound repair. Although the in vivo substrates of epilysin are not known at present, its expression pattern suggests that it may be needed to restructure the basement membrane or to degrade adhesive proteins between keratinocytes to supply new cells for the migrating front.

Matrix metalloproteinase-19 expression in dermal wounds and by fibroblasts in culture.

Here, we have examined the expression of matrix metalloproteinase-19 (MMP-19) in human cutaneous wounds and by human skin fibroblasts in culture. Expression of MMP-19 was detected by immunohistochemistry in fibroblasts, capillary endothelial cells, and macrophages in the dermal layer of large granulating wounds, as well as in chronic venous and decubitus ulcers. MMP-19 mRNA expression and pro-MMP-19 production by dermal fibroblasts in culture was potently enhanced by tumor necrosis factor-alpha (TNF-alpha). Induction of MMP-19 expression by TNF-alpha was prevented partially by blocking the activation of extracellular signal-regulated kinase (ERK)-1/2 by PD98059 and p38 activity by SB203580. Activation of ERK1/2 by adenovirus-mediated delivery of constitutively active MAPK/ERK kinase 1 resulted in the induction of MMP-19 expression. Activation of p38 alone by adenovirally delivered constitutively active MAPK kinase 3b (MKK3b) and MKK6b also enhanced MMP-19 production, and the most potent induction of MMP-19 expression was noted when ERK1/2 was activated in combination with p38. Activation of c-Jun N-terminal kinase (NK). Abundant pro-MMP-19 production by fibroblasts was associated with proteolytic processing of secreted pro-MMP-19. These results suggest a role of MMP-19 in cutaneous wound repair and identify three distinct signaling pathways, which coordinately mediate induction of MMP-19 expression in fibroblasts: mitogen-activated ERK1/2 pathway and stress-activated JNK and p38 pathways, of which control proteolytic activity of dermal fibroblasts.

HCR, a candidate gene for psoriasis, is expressed differently in psoriasis and other hyperproliferative skin disorders and is downregulated by interferon-gamma in keratinocytes.

We have previously shown that HCR is a good candidate gene for psoriasis based on its location in the PSORS1 locus, predicted secondary structure change of the associated allele, and expression pattern. To understand better the function of HCR, we studied how HCR expression is altered in hyperproliferative skin diseases other than psoriasis and in cancers. We examined also its regulation by different cytokines, growth factors, and antipsoriatic agents using quantitative RT-PCR (TaqMan) analysis and its location by immunostaining of keratinocyte cultures. Compared to psoriasis, HCR protein had a different distribution in chronic dermatitis, pityriasis rubra pilaris, mycosis fungoides, and chronic skin ulcers. In three of six grade III squamous cell carcinomas of the skin, four of four adenocarcinomas of the lung, and two of two ductal breast adenocarcinomas, positive cytoplasmic staining in cancer cells was detected. As in psoriasis, Ki67 did not colocalize with HCR. In cell cultures, HCR staining was detected perinuclearly in the cytoplasm and in the nuclei, suggesting that the protein may have a role in both compartments. A 2-fold downregulation of HCR mRNA expression was observed on stimulation with interferon-gamma. Based on the observations that HCR is detected in cancers of epithelial origin in Ki67-negative areas and that interferon-gamma downregulates its expression, we suggest it to have an antiproliferative function.