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Supplementary Figure S1; Supplementary Tables S1-S2.

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.

In the acute phase of atopic dermatitis (AD), T-helper type 2 (Th2) cytokines characterize the inflammatory response in the skin. IL-33 is a new tissue-derived cytokine, which is mainly expressed by cells of barrier tissues, and is known to activate Th2 lymphocytes, mast cells, and eosinophils. IL-33 signals through a receptor complex consisting of IL-33-specific receptor ST2 and a co-receptor IL-1RAcP. As IL-33 is known to promote Th2-type immunity, we examined expression profiles of IL-33 and its receptor components in human AD skin, in the murine model of AD, and in various cell models. We found increased expression of IL-33 and ST2 in AD skin after allergen or staphylococcal enterotoxin B (SEB) exposure, as well as in the skin of 22-week-old filaggrin-deficient mice. In addition, skin fibroblasts, HaCaT keratinocytes, primary macrophages, and HUVEC endothelial cells efficiently produced IL-33 in response to the combined stimulation of tumor necrosis factor-α and IFN-γ, which was further enhanced by a mimetic of double-stranded RNA. Finally, the increased expression of IL-33 and ST2 caused by irritant, allergen, or SEB challenge was suppressed by topical tacrolimus treatment. These results suggest an important role for IL-33-ST2 interaction in AD and highlight the fact that bacterial and viral infections may increase the production of IL-33.

Cerebral amyloid angiopathy (CAA) is a frequent finding in the brains of patients with Alzheimer's disease (AD). CAA may be complicated with CAA-associated intracerebral haemorrhage (CAAH). Previous studies have revealed matrix metalloproteinase (MMP) expression in a mouse model of CAA and in human intracerebral haemorrhage. Here we studied the involvement of MMPs in human CAA and CAAH.To investigate the putative expression of MMPs in human CAA and CAAH (Step 1), immunohistochemistry (IHC) against MMPs-1, -2, -7, -9, -19 and -26 was applied on tissue microarray (TMA) constructed of cerebral samples from 29 individuals with AD, 15 with CAAH and 2 controls. The findings in TMA were confirmed (Step 2) in tissue samples from 64 individuals, 45 presenting with CAA (including 36 with CAAH) and 19 without CAA (including 11 with hypertensive cerebral haemorrhage).In Step 1, immunoreactivity against MMPs-19 and -26 was detected in cerebral blood vessels in CAA. The results were confirmed in Step 2, where CAA (p0.001) and intracerebral haemorrhage (p=0.045) were associated with vascular immunoreactivity against MMP-19. Multivariate analysis showed that the association between vascular MMP-19 and intracerebral haemorrhage was dependent from CAA. MMP-26 associated with CAA (p=0.021) but not with intracerebral haemorrhage.This is the first human study showing local MMP-19 immunoreactivity in the Aβ-amyloid-laden blood vessels in CAA, suggesting that MMPs may be involved in CAA.

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.

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.

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.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.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).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.

Objective.Several candidate genes have been implicated in susceptibility for systemic lupus erythematosus (SLE), a complex autoimmune disease. The proposed genes include members of the type I interferon (IFN) pathway and genes involved in immunological defense functions. Our aim was to systematically replicate 6 such genes, TYK2, IRF5, CTLA4, PDCD1, FCGR2A, and NOD2.Methods.Single-nucleotide polymorphisms in TYK2, IRF5, CTLA4, PDCD1, FCGR2A, and NOD2 were genotyped in 277 SLE patients and 356 healthy controls from Finland, giving a power of 42%–70% for different genes at published allele frequencies.Results.Significant association was seen for rs2304256 (p = 0.0001) and rs12720270 (p = 0.0031) in TYK2 and rs10954213 (p = 0.0043) in IRF5 in our samples, but not for the other genes. We found evidence for genetic interaction (p = 0.014) between rs2304256 in TYK2 and rs10954213 in IRF5, both members of the type I IFN pathway, strengthening the role of the type I IFN pathway in the pathogenesis of SLE.Conclusion.The IFN pathway genes IRF5 and TYK2 may act epistatically in increasing risk for SLE, but our lack of replication does not exclude effects of the other genes studied.

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.

Our objective was to characterize clinical features, laboratory findings, concomitant autoimmune diseases, and smoking habits of lupus erythematosus subgroups in genetically homogeneous patients from two Dermatology Departments of Finnish University hospitals. One hundred and seventy eight discoid lupus erythematosus, 55 subacute cutaneous lupus erythematosus, and 77 systemic lupus erythematosus patients were enrolled using patients’ charts from institutional database (1995–2006) and during routine control visits. Clustering analysis was performed to reveal natural groupings. Smoking at the onset of disease was significantly more common in all subgroups (57% for discoid lupus erythematosus, 35% for subacute cutaneous lupus erythematosus, and 34% for systemic lupus erythematosus) compared with the age/gender-matched prevalence in the Finnish population, suggesting smoking to be a trigger factor for cutaneous lupus. Leukopenia (38%) and lymphopenia (52%) were observed more often in patients with systemic lupus erythematosus than reported previously. Photosensitivity characterized all groups, especially patients with subacute cutaneous lupus erythematosus (87%). Of the autoimmune diseases, Sjögren’s syndrome was the most common (22% of patients with systemic lupus erythematosus), followed by autoimmune thyroid disease (13% of patients with subacute cutaneous lupus erythematosus). The clustering analysis showed environmental factors (smoking) to be more involved in disease development in discoid lupus erythematosus, whereas immunological factors were more significant in initiating systemic lupus erythematosus. The high prevalence of autoimmune thyroid disease, together with photosensitivity, and the clustering profiles suggest that lupus erythematosus subtypes, especially discoid lupus erythematosus, are heterogeneic in their pathomechanisms.

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.

Background: Pyoderma gangrenosum (PG) is a non-infectious, autoimmune, chronic ulcer of the skin, often co-existing with inflammatory bowel disease (IBD). Matrix metalloproteinases (MMPs) have been implicated as mediators of tissue destruction in chronic cutaneous and intestinal wounds. Methods: Twenty-four skin biopsies with clinically and histologically confirmed PG and acute wounds were immunostained for MMP-1, -7, -8, -9, -10 and -26; tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -3 and tumor necrosis factor-α (TNF-α). Results: MMP-1 was generally expressed by keratinocytes distal from the wound edge, whereas MMP-10 was detected abundantly in the epithelium. MMP-26 was positive in 42% at the migratory front. Abundant stromal expression was evident for MMP-1, -9 and -10, TIMP-1 and -3 and TNF-α. In acute wounds, stromal MMP-1, -9 and -10 and TNF-α were sparse. Conclusions: Unlike in normally healing cutaneous wounds, MMP-1 and -26 were detected bordering the wound in only a minority of PGs and their lack may thus retard epithelial repair. Particularly, MMP-9 and -10 and TNF-α would be suitable therapeutic targets as they may contribute to the degradation of provisional matrices needed for migration in healing wounds. The presence of MMP-1, -9, -10 and -26 in both PG and IBD ulcers may suggest a similar pathogenesis for cutaneous and mucosal inflammation.

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.

Receptor tyrosine kinases expressed in endothelial cells are potential targets for therapy with specific tyrosine kinase inhibitors. Endothelial cell KIT expression has not been systematically evaluated in human cancer. In the present study, endothelial cell KIT expression was assessed in 345 tumours consisting of 34 different histological types using a tissue microarray technique. Marked KIT expression occurred in the tumour endothelial cells only in primary glioblastomas in the microarray. Moderate to strong KIT and phosphorylated KIT expression was detected in the tumour endothelial cells in six (16%) and seven (19%) of the 37 primary glioblastomas examined, respectively. In whole tissue sections, KIT and phosphorylated KIT were expressed in tumour endothelial cells in 13 (59%) and 11 (50%) of the 22 glioblastomas examined, respectively. RNA in situ hybridization showed KIT mRNA expression in most glioblastomas both in tumour vessel endothelial cells and in perinecrotic palisading glioblastoma cells, whereas little KIT mRNA was found in the endothelial cells of colon or pancreatic carcinomas. Phosphorylated KIT, its ligand stem cell factor, and the downstream signalling molecules phosphorylated Akt and mTOR were often expressed in glioblastoma cells located in the perinecrotic tumour areas that often also contained abundant HIF-1α. It is concluded that marked KIT and phosphorylated KIT expression is frequently present in the endothelial cells of glioblastomas, which are known to harbour florid microvascular proliferation with characteristic morphological features. Glioblastomas also express phosphorylated KIT and its activated downstream signalling molecules in the tumour cells. Lower levels of KIT and phosphorylated KIT are present in endothelial cells of other tumour types and in normal tissues. Endothelial cell and tumour cell expression of activated KIT might explain in part the responsiveness of glioblastomas to the combination of imatinib (an inhibitor of KIT) and hydroxyurea. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The PSORS1 locus is the consistently replicated genetic risk factor for psoriasis. Clinical associations with the main marker allele of PSORS1, HLA-Cw6, have been addressed in a number of studies, but clinical associations have not been used as a way to distinguish the effects of the neighbouring candidate genes in PSORS1. Our results show that HLA-Cw6 and CCHCR1 risk allele associations with clinical features of psoriasis are predictably highly similar in a Finnish nationwide cohort of 379 psoriasis patients. The clinical profiling of a small group of patients (n=34) who were HLA-Cw6- but CCHCR1*WWCC positive suggested that no great differences existed between them and HCR-Cw6- patients. HCR+ genotype (as well as Cw6+ genotype) correlated for the first time positively with female sex and, in contrast with previous studies, negatively with disease severity. Presence of psoriatic arthritis was more pronounced in HCR- psoriasis (as well as in Cw6- psoriasis). Clinical profiling may be a useful approach to distinguishing genetic effects of candidate genes even within a locus in sufficiently large cohorts.

Matrix metalloproteinase (MMP)-21 and MMP-26 (matrilysin-2) are two recently cloned epithelial metalloproteases. Here we examined their expression in various benign skin disorders, in which macrophages and fibroblasts have been implicated as well as in cultures of these cells. Expression of MMP-21 was detected by immunohistochemistry in a subset of macrophages of granulomatous skin lesions and in fibroblasts in dermatofibromas. MMP-21 mRNA was found in THP-1, U937, HEL 299 and Hs68 cells. Furthermore, MMP-21 protein was detected by immunohistochemistry in cultures of the same cell lines. In culture MMP-21 was upregulated by phorbol myristate acetate in THP-1 cells and by retinoic acid (RA) in U937 cells, and downregulated by transforming growth factor beta 1 (TGF-beta1) in HEL 299 as assessed by Taqman quantitative polymerase chain reaction (PCR). Expression of MMP-26 was detected by immunohistochemistry in granulomatous skin diseases and actinic elastosis. MMP-26 at both mRNA and protein levels was only found in HEL 299 cells. In culture it was downregulated by TGF-beta1, RA and IL-1beta as assessed by Taqman quantitative PCR. Our results suggest these two novel MMPs are not only associated with cancer but may be important in connective tissue remodelling and pathobiology of various benign skin disorders.

The neuropeptide S (NPS)-NPS receptor 1 (NPSR1) pathway has recently been implicated in the pathogenesis of asthma. The purpose of this study was to identify downstream gene targets regulated by NPSR1 upon NPS stimulation. A total of 104 genes were found significantly up-regulated and 42 down-regulated by microarray analysis 6 h after NPS administration. By Gene Ontology enrichment analysis, the categories 'cell proliferation', 'morphogenesis' and 'immune response' were among the most altered. A TMM microarray database comparison suggested a common co-regulated pathway, which includes JUN/FOS oncogene homologs, early growth response genes, nuclear receptor subfamily 4 members and dual specificity phosphatases. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), exhibited a significant NPS dose-response relationship as confirmed by quantitative reverse-transcriptase-PCR and for MMP10 by immunoassay. Immunohistochemical analyses revealed that MMP10 and TIMP metallopeptidase inhibitor 3 (TIMP3) were both strongly expressed in bronchial epithelium, and macrophages and eosinophils expressed MMP10 in asthmatic sputum samples. Because remodeling of airway epithelium is a feature of chronic asthma, the up-regulation of MMP10 and TIMP3 by NPS-NPSR1 signaling may be of relevance in the pathogenesis of asthma.

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.

Drug screening procedures should preferably utilize experimental settings mimicking the in vivo situation. The aim of this study was to evaluate a skin explant model as a tool to identify topical agents with anti-proliferative properties in human epidermis. Re-epithelialization was initiated from a skin punch biopsy explanted onto de-epidermized dermis and cultured at the air-liquid interface in the presence of epidermal growth factor receptor inhibitor PKI166, tacrolimus or established topical anti-psoriatic drugs: betamethasone, calcipotriol, dithranol and tazarotene. Neo-epidermal extension was traced by fluorescence microscopy prior to histomorphometric analysis. PKI166 at 1 microM decreased the mean radial outgrowth rate (-19%), frequency of BrdU-positive (-37%) and laminin 5-positive (-45%) cells, indicating reduced proliferation and migration of neo-epidermal keratinocytes. However, the papillomatosis index and epithelial thickness were not significantly affected. Calcipotriol at 1 microM had a similar effect on the outgrowth rate (-15%) and fraction of laminin 5-stained keratinocytes (-40%). Furthermore, calcipotriol significantly reduced mean neo-epidermal thickness. Equimolar concentrations of the other test compounds had no apparent effect on histology or outgrowth parameters. This study exemplifies the versatility of combined dynamic and morphological analysis and emphasizes the potential of epidermal growth factor receptor-directed inhibition in hyperproliferative disorders of the epidermis.

Keratoacanthomas are rapidly growing hyperproliferative skin tumors that may clinically or histologically be difficult to distinguish from well-differentiated squamous cell cancers (SCCs). UV light, trauma, and immune suppression represent their etiological factors. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated the expression profile of several cancer-related MMPs to find markers that would differentiate keratoacanthomas from SCCs and shed light to the pathobiology of keratoacanthoma. Samples from 31 keratoacanthomas and 15 grade I SCCs were studied using immunohistochemistry for MMP-2, -7, -8, -9, -10, -13, and -19 and p16 and laminin-5gamma2 chain. In situ hybridization for MMP-7, -10, and -13 was performed in a subset of tumors. Keratinocytes with atypia, presence of neovascularization, and composition of the inflammatory infiltrate were graded from hematoxylin-eosin stainings. MMP-7 was present in the epithelium of 4/31 keratoacanthomas and 9/15 SCCs, MMP-8 in 3/30 keratoacanthomas and 0/15 SCCs, but MMP-13 in 16/31 keratoacanthomas and 10/15 SCCs, and MMP-10 in 28/31 keratoacanthomas and all cancers. MMP-9 was detected in the epithelium in 5/31 keratoacanthomas and 8/15 SCCs, whereas MMP-2 was only present in fibroblasts in both tumors. MMP-19 was upregulated in proliferating epithelium of keratoacanthomas as was p16. Cytoplasmic laminin-5gamma2 was particularly abundant in keratinocytes at the pushing border of MMP-13-positive keratoacanthomas. We conclude that although some MMPs (MMP-10 and -13) are abundantly expressed in keratoacanthomas, the presence of MMP-7 and -9 in their epithelial pushing border is rare and should raise suspicion of SCC. Further, the loss of MMP-19 and p16 could aid in making the differential diagnosis between well-differentiated SCC and keratoacanthoma. Frequent expression of the transformation-specific MMP-13 in keratoacanthomas suggests that they are not benign tumors but incomplete SCCs.

Summary Background The risk of squamous cell carcinoma (SCC) is significantly increased in chronic leg ulcers. Very little is known about the molecular pathogenesis of these tumours, which are often undiagnosed for a long time. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated whether the pattern of epithelial MMP expression can predict development of SCC from pseudoepitheliomatous hyperplasia of chronic wounds. Methods Samples from nine patients with SCCs that had arisen in chronic wounds and 31 with venous leg ulcers were studied using immunohistochemistry for MMP-7, MMP-8, MMP-9, MMP-13, MMP-19 and the tumour suppressor p16. In situ hybridization was performed for MMP-1, MMP-3, MMP-7, MMP-12 and MMP-13. Results MMP-7 was expressed by malignantly transformed epithelium, while it was absent from chronic wounds. MMP-9 was detected in the epithelium in both SCCs and chronic wounds. Epithelial MMP-13 expression was strong in SCC, but was absent in chronic wounds. MMP-12 was expressed in the epithelium in two SCCs, while macrophages were positive in chronic wounds. MMP-19 was induced in proliferating epithelium of wounds, but was absent from invasive areas of SCC. p16 was expressed by keratinocytes in half of the chronic wounds and at superficial margins of SCCs, while invasive areas were negative. Conclusions Our results suggest that epithelial expression of MMP-7, MMP-12 and MMP-13, but not that of MMP-1, MMP-3, MMP-8, MMP-9 and MMP-10, in chronic wounds provides a diagnostic clue for distinguishing SCCs from nonmalignant wounds. The loss of MMP-19 and p16 from the epithelium could aid in making the differential diagnosis between well-differentiated SCCs and nonmalignant chronic wounds.

Objectives: Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants characterized histologically by extensive tissue injury and inflammation. Matrix metalloproteinases (MMP) are involved in tissue remodeling and cell migration, both being important aspects of inflammatory disease. The aim of this study was to investigate whether MMPs play a role in the pathogenesis of NEC. Methods: Expression of MMP-1, -7, -9, -10, -12, -19 and -26 was studied using in situ hybridization/immunohistochemistry in samples intestinal tissue removed from 15 patients with NEC; in 7 of them control samples were obtained at closure of stomas. Six intestinal samples from patients with intestinal atresia and four samples of necrosis were also included in the material examined. Laminin-5 was immunostained to find migrating enterocytes and cytokeratin to delineate mucosal epithelium. Results: MMP-7 protein was upregulated in the epithelium of 12/18 NEC samples. MMP-26 was induced in stromal cells of 12/17 NEC specimens. Stromal expression was found for MMP-1 and -12 mRNAs in 7/18 samples. MMP-1 was also detected in the epithelium of regenerating areas. Both NEC and stoma samples expressed MMP-9 in inflammatory cells. Epithelial MMP-19 was downregulated in NEC. Conclusions: Our results suggest that several MMPs may be major factors in tissue destruction and remodeling in NEC. Targeted inhibition of matrilysins, using synthetic MMP inhibitors or blockers of their signal transduction pathways, may represent a novel therapeutic option for the treatment of intestinal inflammation associated with NEC.

Background: Extramammary Paget's disease (EMPD) is a rare malignant neoplasm of apocrine gland bearing skin characterized by intraepidermal proliferation of adenocarcinoma cells. Tumor growth depends on the ability of tumor cells to migrate by proteolysis and on angiogenesis. The matrix metalloproteinase (MMP) enzymes have been implicated in both of these processes in other types of skin cancer. Methods: The expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, and MMP-19 was analyzed by immuno- histochemistry and/or in situ hybridization in 27 EMPD and five mammary PD (MMPD) specimens. The distribution of laminin-5 (LN-5) and tenascin-C, two extracellular matrix proteins associated with tumor invasion, was studied by immunohistochemistry. Results: MMP-7 (matrilysin-1) and MMP-19 were the most frequently expressed MMPs in Paget's cells. Overexpression of MMP-2, MMP-9, or MMP-13, which is seen in many cancers, was not evident in EMPD. LN-5 and tenascin-C positivity did not correlate with the level of invasion. MMP-7, MMP-13, and MMP-19 were detected abundantly in MMPD, while MMP-9 was absent. Conclusions: MMP expression did not generally associate with the level of invasion of EMPD. In three samples positive for MMP-7 and four samples positive for MMP-19, an underlying carcinoma was detected, suggesting the importance of these two MMPs as predictors of secondary EMPD or the putative origin of Paget's cells from the dermal adenocarcinoma cells of apocrine duct origin.

Genetic susceptibility for psoriasis is regulated to the greatest extent by the PSORS1 locus. Three psoriasis-associated susceptibility alleles have been identified within it, namely, HLACw6, HCR*WWCC and CDSN*5, but strong linkage disequilibrium between them has made it difficult to distinguish their individual genetic effects, and animal models to study their effects are not known. To study the function of HCR, we engineered transgenic mice with either a non-risk allele of HCR or the HCR*WWCC risk allele under the control of the cytokeratin-14 promoter. These choices were motivated by the apparently dominant effect of PSORS1 on psoriasis susceptibility and the physiological expression of HCR in basal keratinocytes. Transgenic mice appeared phenotypically normal and histologically their skin was indistinguishable from wild-type mice. Expression studies using Affymetrix arrays suggested that the HCR risk allele has specific functional consequences relevant to the pathogenesis of psoriasis. Comparison of gene expression changes between non-risk and risk allele mice revealed similarities to previous observations in human psoriatic skin, including upregulation of cytokeratins 6, 16 and 17 in risk allele mice. We also observed changes in the expression of genes associated with terminal differentiation and formation of the cornified cell envelope. Our results support the concept that HCR may constitute an essential gene in the PSORS1 locus. These observations are also compatible with a model that a susceptibility gene for psoriasis induces changes that are contributory but not sufficient by itself to produce the clinical phenotype.

Integrin alphavbeta6 is an epithelial cell-specific receptor that is not normally expressed by resting epithelium but its expression is induced during wound healing. The function of alphavbeta6-integrin in wound repair is not clear. In the present study, we showed that beta6-integrin expression was strongly up-regulated in the epidermis in human chronic wounds but not in different forms of skin fibrosis. To test whether increased beta6-integrin expression plays a role in abnormal wound healing we developed four homozygous transgenic mouse lines that constitutively expressed human beta6-integrin in the epithelium. The mice developed normally and did not show any histological abnormalities in the skin. The rate of experimental skin wound closure was unaltered and the wounds healed without significant scar formation. However, during breeding program 16.1 to 27.0% of transgenic mice developed spontaneous, progressing fibrotic chronic ulcers. None of the wild-type animals developed these lesions. The chronic lesions had areas with severe fibrosis and numerous activated macrophages and fibroblasts expressing transforming growth factor (TGF)-beta. The level of TGF-beta1 was significantly increased in the lesions as compared with normal skin. The findings suggest that increased alphavbeta6-integrin in keratinocytes plays an active part in abnormal wound healing possibly through a mechanism involving increased activation of TGF-beta.

Squamous cell carcinoma (SCC) of the oral cavity is a highly invasive tumour of stratified squamous epithelium that spreads through degradation of the basement membrane (BM) and extracellular matrix (ECM). There are currently no reliable tissue or serum markers to predict whether the tumour has metastasized at the time of diagnosis. Verrucous carcinoma (VC) of the oral cavity is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. Many matrix metalloproteinases (MMPs), such as MMP-1, -2, -7, -9, -13, and -14, as well as integrin receptors have been implicated in cancer invasion. Integrin alphavbeta6 is induced in SCC and appears to be involved in up-regulation of MMP-9 expression by oral keratinocytes and promotion of their migration. The aim of this study was to investigate whether the pattern of MMP expression or that of alphavbeta6 integrin contributes to the differences in the biological behaviour of oral SCC and VC. The results show that the less aggressive nature of oral VC may be connected to its MMP expression profile. Typically, VCs were devoid of epithelial MMP-3, -7, -9, -12 and -13 expression, compared with SCCs. MMP-19 was expressed by epithelial keratinocytes in hyperproliferative areas of verrucous hyperplasia, VC, and SCC, but was absent in the invasive cancer cell nests of SCC. MMP-26 was expressed by hyperproliferative keratinocytes in VC as well as by invasive cancer cells in SCCs. MMP-10 was expressed widely in the epithelium of all SCC specimens. alphavbeta6 integrin expression was also detected in some cases of epithelial hyperplasia but was significantly more abundant in cancers at the invasive front. The absence of MMP-7, -9 and -12 from epithelial cells may serve as a good prognostic marker of non-invasive oral carcinoma. Blocking the activity of invasion-specific MMPs or alphavbeta6 integrin might offer novel therapeutic modalities in early-stage oral carcinoma.

Our understanding of MMP expression during corneal repair has previously relied upon animal models, isolated human biopsy specimens and cell culture studies. The aim of this study was to determine the temporal and spatial expression of matrix metalloproteinases following wounding of cultured human corneal tissue. Human corneas were cultured and cut into six pieces. The epithelium was removed with a corneal brush. The tissue was then re-cultured and tissue pieces were fixed up to 7 days post-wounding. Matrix metalloproteinases were detected by in situ hybridisation and immunohistochemistry. Intracellular laminin-5, a marker of migratory epithelial cells, was located immunohistologically. In the time scale studied tissue series from nine corneas achieved coverage of the stroma with epithelial cells and partial repair of damaged basement membrane, demonstrated by the Periodic acid-Schiff reaction and haematoxylin and eosin counter-staining. By day 3, migrating epithelial cells and stromal cells beneath the wounded area expressed collagenase-1 (MMP-1). Stromelysin-1 (MMP-3) was expressed only by fibroblast-like stromal cells. Stromelysin-2 (MMP-10) was detected in migrating epithelial cells and remained when the stroma was surrounded by a monolayer of epithelial cells. By day 7, development of multi-layered epithelium around the tissue coincided with cessation of MMP expression in both epithelial and stromal cells, except for MMP-9, which remained in epithelial basal cells. Tissue inhibitor of matrix metalloproteinase-1 was mainly associated with stromal cells and was reduced upon formation of a multi-layered epithelium. This study demonstrates matrix metalloproteinase expression in epithelial and fibroblast-like cells following wounding of human corneal tissue in culture where the cells remain in contact with their natural matrices.

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.

Many normal biological processes, such as reproduction, fetal development and wound healing, are critically dependent on controlled degradation of extracellular matrix (ECM) macromolecules. However, excessive degradation of matrix components occurs in pathologic tissue destruction, e.g. in atherosclerosis, rheumatoid arthritis, and cancer. Matrix metalloproteinases (MMPs) are degradative enzymes that play an important role in all aspects of tumor progression by enhancing tumor-induced angiogenesis and destroying local tissue architecture and basement membranes to allow tumor invasion and metastasis. Efficient breakdown of the ECM surrounding invasive cancer islands involves interplay between tumor cells, stromal cells, and inflammatory cells, all of which express a distinct set of MMPs. Besides the classical role of MMPs in degradation of ECM, MMPs may also indirectly influence the tumor microenvironment through the release of growth factors, cryptic sites or angiogenic factors, or through the generation of matrix fragments that inhibit tumor cell proliferation, migration and angiogenesis. This makes the contribution of MMPs to tumorigenesis much more complex than initially thought. Currently, a number of clinical studies have focused on testing MMP inhibitors as potential antineoplastic agents. In this review we discuss the present role of MMPs in the development and progression of cancer, focusing on non-melanoma skin cancers basal (BCC) and squamous (SCC) cell carcinoma, and the possible influence of MMPs in their differences.

MMP-19 (also designated RASI) is a recently discovered member of a large family of zinc-dependent proteolytic enzymes, most of which have been implicated in cancer growth and metastasis. It differs from the others by its chromosomal location and structure and is expressed by endothelial and vascular smooth muscle cells in vivo. Our aim was to study the putative role of MMP-19 in skin cancer. We also examined its regulation in keratinocyte cultures using quantitative TaqMan RT-PCR. Our results show that MMP-19 can also be detected in stimulated keratinocytes by Northern and Western analyses. In wounds, it was found in keratinocytes outside the migrating area, while in BCC and SCC, it was present in the hyperproliferative (p63-positive), E-cadherin-negative epidermis at the tumor surface but downregulated in invasive cancer islands. Expression was also evident in endothelial cells of neoangiogenic regions and in occasional stromal fibroblasts. Of the 12 tested cytokines/growth factors, only TNF-alpha and PMA were able to stimulate the expression of MMP-19 mRNA in primary keratinocytes. No MMP-19 mRNA was detected by Northern analysis in cultured HaCaT or A5 cells or in an SCC cell line established from head-and-neck cancer. Our data suggest that, unlike most MMPs, MMP-19 expression in the epidermis is downregulated during transformation and histologic dedifferentiation.

Human metalloelastase (MMP-12) has been implicated in elastin degradation and macrophage migration in many pathological conditions. It also generates angiostatin, thus having a potential to prevent tumour angiogenesis. It has previously been shown that transformed epithelial cells express MMP-12 in skin cancer. The aim of this study was further to elucidate the role of metalloelastase in squamous cell cancer (SCC) progression. By in situ hybridization, expression of MMP-12 mRNA was detected in 28/33 vulvar SCC samples in CD-68-positive macrophages, while 10 samples had positive cancer cells. By immunohistochemistry, MMP-12 protein was seen in the same area as the mRNA. MMP-12 mRNA expression in tumour cells correlated with more aggressive histology (p = 0.0099). In contrast, macrophage-derived MMP-12 mRNA was more abundant in well-differentiated grade I than grade III tumours (p = 0.01). However, the level of MMP-12 mRNA, regardless of its origin, did not correlate with metastasis or patient survival. No significant correlation was found between macrophage-derived MMP-12 mRNA and a low amount of blood vessels, as quantitated after von Willebrand staining. In agreement with vulvar SCCs in vivo, MMP-12 was expressed in cultured SCC cells by northern and western blot analysis. In HaCaTs and epithelial MCF-10f cells, MMP-12 mRNA was induced by transforming growth factor-beta1 (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) as measured by quantitative RT-PCR (TaqMan). Two MMPs capable of generating angiostatin in vivo, matrilysin (MMP-7) and gelatinase B (MMP-9), were also examined in these tumours. MMP-7 mRNA was mainly expressed by epithelial tumour cells, particularly in less differentiated tumours. MMP-9 was usually expressed by neutrophils and macrophages; epithelial protein was predominantly found in grade II/III tumours. These results suggest a dual role for MMP-12 in tumour progression.

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.

We have studied the effect of a newly identified tumor suppressor tissue inhibitor of metalloproteinases- 3 (TIMP-3) on the growth of human melanoma and squamous-cell carcinoma (SCC). Adenoviral delivery of the TIMP-3 gene to human melanoma (A2058) and SCC (UT-SCC-7) cells ex vivo inhibited tumorigenesis after subcutaneous (s.c.) injection of the infected cells into SCID/SCID mice. Three daily consecutive intratumoral injections of 1.4×10 9 plaque-forming units (pfu) of TIMP-3 adenovirus (RAdTIMP-3) inhibited the growth of preestablished melanoma and SCC xenografts in SCID/SCID mice, whereas growth of control virus-injected tumors was not affected. The antitumor effect of RAdTIMP-3 was obtained with in vivo adenoviral transduction efficiency of 8–10%, and it was more potent than that of adenovirally delivered p53. Adenovirusmediated expression of TIMP-3 potently reduced gelatinolytic activity, increased the number of apoptotic cells, and inhibited vascularization of melanomas. Escalation of the adenoviral dose to three rounds of three daily consecutive injections with 1.4×10 9 pfu of RAdTIMP-3 every 6 days entirely inhibited growth of injected melanomas for 32 days. Mixing RAdTIMP-3-infected A2058 cells with uninfected cells in 1:1 ratio in culture resulted in death of all cells in 96 hours. Adenovirally delivered TIMP-3 was also expressed by A2058 cells in soluble form into the culture medium, where it exerted a cytotoxic effect on uninfected A2058 cell cultures after relocating to the cell layer. These results identify TIMP-3 as a novel type of secreted tumor suppressor, which has antiinvasive, antiangiogenic, and proapoptotic effects in vivo , and which displays a potent bystander effect validating further exploration of its applicability in human cancer gene therapy.

PSORS1, near HLA-C, is the major genetic determinant of psoriasis. We present genetic and structural evidence suggesting a major role for the HCR gene at the PSORS1 locus. Genotyping of 419 families from six populations revealed that coding single-nucleotide polymorphisms of HCR formed a conserved allele HCR*WWCC that associated highly significantly with psoriasis and with the HLA-Cw6 allele in all populations. Because of strong linkage disequilibrium between HLA-Cw6 and HCR*WWCC, the two genes could not be genetically distinguished by this sample size. However, the variant HCR allele was predicted to differ in secondary structure from the wild-type protein. HCR protein expression in lesional psoriatic skin differed considerably from that observed in normal skin. These results provide strong evidence for the HCR*WWCC allele as a major genetic determinant for psoriasis, probably by a mechanism impacting on keratinocyte proliferation.

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.

Fetal development and tumor progression both require a complex system of extracellular matrix (ECM) synthesis and breakdown, which is mediated by, for instance, the matrix metalloproteinases (MMPs). Human metalloelastase (MMP-12) is an MMP, the expression of which has so far been documented in macrophages associated with atherosclerosis, wound repair, and certain cancers. In this study we first examined the expression of MMP-12 during human fetal development. By in situ hybridization MMP-12 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column, in ribs, and in extremities undergoing ossification, beginning at the gestational age of 8 weeks. Also, periosteal cells expressed MMP-12 at 11 weeks. No expression of MMP-12 mRNA could be noted in other fetal tissues, including the skin, lungs, intestine, kidney, and liver. Expression of MMP-12 mRNA could not be detected in adult normal cartilage or osteosarcomas, but in chondrosarcomas both macrophages (8 of 19 samples) (identified by CD68 immunostaining) and chondrosarcoma cells (8 of 19) were positive. MMP-12 was also demonstrated in the tumors by western blotting and it was expressed in the same regions as MMP-13 mRNA. By immunostaining, MMP-12 mRNA colocalized with the protein in both fetal and chondrosarcoma specimens. Unlike basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha) induced MMP-12 mRNA production in chondrosarcoma-derived HTB-94 cells. Our results suggest that MMP-12 plays an important role in ECM remodeling during fetal bone development and is induced when chondrocytes undergo malignant transformation.

Matrix metalloproteinases (MMPs) degrade extracellular proteins during epithelialization of wounds. To evaluate the biological significance of MMPs in epidermal healing, the synthetic broad-spectrum MMP inhibitor GM 6001 (also called Galardin and Ilomastat) was applied topically to standardized human wounds. GM 6001 (10 microg/microl) or vehicle alone was applied every second day onto 4 de-roofed 6 mm suction blister wounds on the volar forearm of healthy male volunteers for 12 days. GM 6001 delayed healing by 2-4 days as assessed macroscopically and microscopically. In situ hybridization or immunohistochemistry showed that MMP-1 (interstitial collagenase) was present in and MMP-2 (gelatinase A) close to laterally migrating keratinocytes whereas MMP-9 (gelatinase B) was seen during maturation of new epidermis. MMP-1 was undetectable in blister roofs (normal epidermis) and found in low levels in normal skin. Total MMP-1 activities increased about 100-fold in wounds, independent of treatment, compared to normal skin as analyzed by specific ELISA-based activity assay. By gelatin zymography, MMP-2, but not MMP-9, was detected in blister roofs and wound healing was associated with increased active MMP-2 and latent MMP-9 levels. GM 6001 prevented activation of MMP-2 and increased latent MMP-9 levels. GM 6001 delayed re-appearance of laminin-5, the synthesis of which correlated with epidermal regeneration. Restoration of stratum corneum, measured indirectly by transepidermal water loss, was also impaired (P

Congenital ichthyoses are a group of heterogeneous disorders of cornification. Autosomal recessive congenital ichthyosis (ARCI) can be clinically subdivided into congenital ichthyosiform erythroderma and lamellar ichthyosis. Ultrastructurally, ARCI is classified into four groups: ichthyosis congenita (IC) types I-IV. The genetic background of the ARCI disorders is heterogeneous, but only one disease gene, transglutaminase 1, has been detected so far. We describe six patients with severe congenital ichthyosis from six different Scandinavian families. They could not be classified ultrastructurally into the four IC groups because of atypical findings of electron microscopy. These included abnormal lamellar bodies, alterations in keratohyalin, remnant organelles and lipid inclusions in the upper epidermal cells, which resembled the ultrastructural findings of harlequin ichthyosis (HI), although the HI phenotype was not present at birth. Some clinical features, such as thick scales, erythroderma, alopecia and ectropion were common to all patients. Ichthyosis was usually accentuated in the scalp and four patients had clumped fingers and toes. None of the patients carried the transglutaminase 1 mutation. We conclude that ultrastructural findings resembling those detected in previous HI cases (type 1 and 2) can also be found in patients who do not have classic clinical features of that rare ichthyosis. This may be due to lack of specificity of ultrastructural markers for HI or to its clinical heterogeneity.

In skin biology, matrix metalloproteinases (MMPs) have been implicated in inflammatory matrix remodeling, neovascularization, wound healing and malignant transformation. Psoriasis is histologically characterized by keratinocyte hyperproliferation, infiltration of inflammatory cells, neoangiogenesis and production of cytokines, such as TNF-alpha, IL-1beta, TGF-alpha, and IFN-gamma, also capable of regulating MMP transcription. To investigate the role of stromelysins-1 and -2, matrilysin, metalloelastase, collagenases-1 and -3 and 92-kDa gelatinase as well as their inhibitors, TIMPs-1 and -3, in psoriasis, we performed in situ hybridization using 35S-labeled cRNA probes on 29 psoriatic lesions and 9 samples of normal looking skin from psoriatic patients. Metalloelastase mRNA was detected in 21/27 samples in macrophages that had migrated into the epidermis or in the inflammatory infiltrates of the superficial dermis. A quantity of 92-kDa gelatinase was found in macrophages and neutrophils (25/27). Stromelysin-1 mRNA was detected in basal keratinocytes in 4/21 lesions. Intracellular laminin-5 immunosignal in basal keratinocytes of the same samples, suggested that stromelysin-1 might participate in remodeling of the basement membrane zone. No signal for stromelysin-2 or collagenase-3 was found and only sweat glands were positive for matrilysin. TIMP-1 was more abundantly expressed than TIMP-3 in the inflammatory infiltrates and endothelial cells of dermal papillae (22/29). TIMP-3 was expressed perivascularly in 9/16 samples. Our results suggest that overexpression of the investigated MMPs by keratinocytes is not associated with psoriasis. However, macrophages express MMPs in psoriatic skin. Also TIMPs, particularly TIMP-1, were abundantly expressed, suggesting that mere MMP overexpression is unlikely to contribute to psoriatic tissue changes.

Stromelysin-2 is a matrix metalloproteinase that degrades in vitro several protein components relevant to wound repair such as collagens III and IV, gelatin, nidogen, laminin-1, proteoglycans, and elastin. Furthermore, it can activate other matrix metalloproteinases, such as collagenase-1 (matrix metalloproteinase-1) and collagenase-2 (matrix metalloproteinase-8), as well as 92 kDa gelatinase. The aim of this study was to determine in a large variety of wounds (normally healing dermal and mucosal wounds, suction blisters, ex vivo cultures, diabetic, decubitus, rheumatic, and venous ulcers) and keratinocyte cultures, which factors contribute to stromelysin-2 expression and how it is induced in relation to other matrix metalloproteinases. Our results show that stromelysin-2 mRNA and protein are upregulated later (at 3 d) than matrix metalloproteinase-1 in normally healing wounds and ex vivo explants, in which stromelysin-2 is invariably expressed by keratinocytes migrating over dermal matrix. The number of keratinocytes expressing stromelysin-2 was greatest in chronic inflamed diabetic and venous ulcers compared with rheumatoid and decubitus ulcers, six of which had no signal. In keratinocyte cultures, tumor necrosis factor-alpha, epidermal growth factor, and transforming growth factor-beta1 induced stromelysin-2 expression as measured by quantitative reverse transcriptase-polymerase chain reaction, whereas different matrices did not upregulate the mRNA. Immunostaining demonstrated stromal transforming growth factor-beta1 in contact with the stromelysin-2-positive keratinocytes. Our results suggest that stromelysin-2 expression is important for the normal repair process and is upregulated by cytokines rather than cell-matrix interactions. Stromelysin-2 is most likely to participate in the remodeling of the newly formed basement membrane, and is not overexpressed in retarded wound healing.

A second distinct family of anion transporters, in addition to the classical SLC4 (or AE) family, has recently been delineated. Members of the SLC26 family are structurally well conserved and can mediate the electroneutral exchange of Cl(-) for HCO(-)(3) across the plasma membrane of mammalian cells like members of the SLC4 family. Three human transporter proteins have been functionally characterized: SLC26A2 (DTDST), SLC26A3 (CLD or DRA), and SLC26A4 (PDS) can transport with different specificities the chloride, iodine, bicarbonate, oxalate, and hydroxyl anions, whereas SLC26A5 (prestin) was suggested to act as the motor protein of the cochlear outer hair cell. We report the expansion of the SLC26 family with five new members in chromosomes 3, 6, 8, 12, and 17 and mapping of SLC26A1 to 4p16.3. We have characterized one of them, SLC26A6, in more detail. It maps to chromosome 3p21.3, encodes a predicted 738-amino-acid transmembrane protein, and is most abundantly expressed in the kidney and pancreas. Pancreatic ductal cell lines Capan-1 and Capan-2 express SLC26A6, and immunohistochemistry localizes SLC26A6 protein to the apical surface of pancreatic ductal cells, suggesting it as a candidate for a luminal anion exchanger. The functional characterization of the novel members of this tissue-specific gene family may provide new insights into anion transport physiology in different parts of the body.

Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis and several matrix metalloproteinases have previously been implicated in this process. Using immunohistochemistry and in situ hybridization, we have studied the localization of two elastolytic matrix metalloproteinases, matrilysin (matrix metalloproteinase-7) and human macrophage metalloelastase (matrix metalloproteinase-12) in solar damage. Human macrophage metalloelastase protein was detected in the superficial dermis in areas of elastotic material. Matrix metalloproteinase-7 was seen in the mid-dermis in regions with less damaged elastic fibers and morphologically better preserved collagen as well as in a band-like pattern below basal keratinocytes in eight of 18 solar elastosis. In samples taken from healthy volunteers 3 d after repeated ultraviolet A or ultraviolet B photoprovocation, occasional immunopositive cells for human macrophage metalloelastase (stromal) or matrix metalloproteinase-7 (sweat gland epithelium) were detected. In samples taken 1 d after ultraviolet B exposure, however, basal keratinocytes were matrix metalloproteinase-7 immunopositive, explaining the linear immunostaining below basal keratinocytes noted particularly in ultraviolet B treated 3 d specimens. Upregulation of metalloelastase was also demonstrated in the skin of hairless mice after repeated ultraviolet exposure. In normal skin, no staining for human macrophage metalloelastase or matrix metalloproteinase-7 was observed in association with elastin. The amount of immunoreactivity for the substrates of matrix metalloproteinase-7, versican, and tenascin, was clearly increased in solar elastosis and photoprovocated skin; versican but not tenascin was detected in the same areas as matrix metalloproteinase-7. Our results suggest that both matrix metalloproteinase-7 and -12 may contribute to remodeling of elastotic areas in sun-damaged skin.

Autosomal recessive congenital ichthyosis (ARCI) is a group of inherited disorders of cornification in which progress has recently been made in the identification of pathogenic mechanisms causing the disease. Transglutaminase 1 (TGM1) has been found as a defective gene in a large fraction of patients with lamellar ichthyosis (LI), a severe inherited scaling disorder of the skin. We have previously performed molecular genetic studies of 38Finnish ARCI families and found six different mutations in 13 families of 38 (34%). In this study we compared the molecular genetic alterations with clinical and electron microscopic findings of these patients. Families were classified by electron microscopy in ichthyosis congenita (IC) types I, II, III, IV and a non-defined group. TGM 1 gene mutation was found in all of the IC type II and 1/3 of the IC type 1 families. Although electron microscopy is not always used to classify ARCI patients, it can distinguish groups which are parallel with molecular genetic findings. This finding might be useful in the classification of ARCI patients for further linkage studies. Clinically typical phenotype of the TGM1 mutation carrier includes large, thick, brownish scales, but ichthyosis of some of these patients tends to be milder.

Since proteolysis of the dermal collagenous matrix and basement membranes is required for local invasive growth and early metastasis formation of cutaneous melanomas, we have analysed the activities/expression levels of certain metalloproteinases in melanomas and cultured melanoma cells by in situ hybridization and Northern analysis. In addition to collagenases-1 and -3 that have been implicated in invasive growth behaviour of various malignant tumours, we analysed the levels of 72-kDa gelatinase and its activators MT1-MMP and TIMP-2 in cultured melanoma cells. The lesions examined included three cases of lentigo maligna and 28 cases of Clark grade I–V melanomas. The premalignant as well as the grade I tumours were consistently negative for collagenase-1 and -3 and TIMP-1 and -3. The collagenases were predominantly expressed in the cancer cells of Clark grade III and IV tumours. TIMP-1 and -3 were abundantly expressed in the cancer and/or stromal cells of grade III and IV melanomas, while TIMP-2 protein was detected also in melanomas representing lower invasive potential. Northern analysis of seven melanoma cell lines showed that the expression of collagenase-1 and TIMPs-1 and -3 was associated with 72-kDa gelatinase positivity. All melanoma cell lines were positive for MTI-MMP and TIMP-2 mRNAs. Our results suggest that overexpression of collagenases-1 and -3 and TIMPs -1 and -3 is induced during melanoma progression. Expression of TIMPs may reflect host response to tumour invasion in an effort to control MMP activity and preserve extracellular matrix integrity. © 1999 Cancer Research Campaign

Controlled degradation of the extracellular matrix (ECM) is crucial for the growth, invasive capacity, metastasis and angiogenesis of tumours. Matrix metalloproteinases (MMPs), a family of zinc-dependent neutral endopeptidases that are collectively capable of degrading essentially all ECM components, apparently play an important role in all of these aspects of tumour development. In addition, there is recent evidence that MMPs are also important for tumour cell survival. At present, therapeutic intervention on tumour growth and invasion based on the inhibition of MMP activity is under intensive investigation, and several MMP inhibitors are already being used on malignant tumours of various organs in clinical trials. In this review we discuss the role of MMPs and their inhibitors in tumour invasion as a basis for prognostic purposes and for targeted therapeutic intervention in cancer.

Excessive accumulation of fibrillar collagens is a hallmark of the cutaneous fibrosis in both systemic and localized scleroderma. Turnover of the collagenous extracellular matrix is dependent on the balance between collagenolytic matrix metalloproteinases and their specific inhibitors. We have examined the expression of the novel, matrix associated tissue inhibitor of metalloproteinases-3 (TIMP-3) in normal and scleroderma skin fibroblasts in culture and in vivo. The levels of TIMP-3 mRNA were elevated up to 2.5-fold in five of seven systemic sclerosis fibroblast strains, whereas TIMP-1 mRNA expression was elevated up to 1.8-fold in two and TIMP-2 mRNA expression up to 1.8-fold in two systemic sclerosis strains. Using in situ hybridization, TIMP-3 mRNA was detected in seven of 12 localized scleroderma skin samples, specifically in fibroblasts within fibrotic collagen fibers or in the vicinity of inflammatory cells. TIMP-1 mRNA was detected in three of eight scleroderma skin samples in fibroblasts adjacent to inflammatory cells. The expression of TIMP-3 mRNA by systemic sclerosis and normal skin fibroblasts was enhanced to a similar extent (by 8.6- and 8.1-fold, respectively) by transforming growth factor-β, and suppressed down to 34 and 54%, respectively, by tumor necrosis factor-α. Specific activation of TIMP-3 gene expression in scleroderma skin fibroblasts in culture and in vivo suggests a role for TIMP-3 in the pathogenesis of dermal fibrosis via inhibition of turnover of fibrotic dermal extracellular matrix, possibly due to upregulation of TIMP-3 expression by transforming growth factor-β. Keywords: fibrosis/matrix metalloproteinase. J Invest Dermatol 110:416–421 1998