Consensus statement on the diagnosis and treatment of sclerosing diseases of the skin, Part 1: Localized scleroderma, systemic sclerosis and overlap syndromes.

The term 'sclerosing diseases of the skin' comprises specific dermatological entities, which have fibrotic changes of the skin in common. These diseases mostly manifest in different clinical subtypes according to cutaneous and extracutaneous involvement and can sometimes be difficult to distinguish from each other. The present consensus provides an update to the 2017 European Dermatology Forum Guidelines, focusing on characteristic clinical and histopathological features, diagnostic scores and the serum autoantibodies most useful for differential diagnosis. In addition, updated strategies for the first- and advanced-line therapy of sclerosing skin diseases are addressed in detail. Part 1 of this consensus provides clinicians with an overview of the diagnosis and treatment of localized scleroderma (morphea), and systemic sclerosis including overlap syndromes.

Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. OBJECTIVES: To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. METHODS: We examined the role of the basement membrane proteins laminin subunits α3, ß3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. RESULTS: Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, ß3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. CONCLUSIONS: These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma.

BACKGROUND: The incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. OBJECTIVES: To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. METHODS: The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real-time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. RESULTS: Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa-associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal-related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. CONCLUSIONS: These results provide evidence for the role of tumour-cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally. Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.

Tumor cell-specific Serpin A1 expression in vulvar squamous cell carcinoma.

PURPOSE: The two main etiological factors for vulvar squamous cell carcinoma (vSCC) are the vulvar dermatosis lichen sclerosus (LS) and high-risk human papillomavirus (hrHPV). Serpin A1 (α1-antitrypsin) is a serine protease inhibitor, which plays a role in the tumorigenesis of various cancer types. The aim of the study was to evaluate the expressions of Serpin A1 in LS, premalignant vulvar lesions, and vSCC using immunohistochemistry (IHC) and serum analysis, and to compare Serpin A1 stainings to the tumor markers p53 and p16. METHODS: In total, 120 samples from 74 patients were studied with IHC for Serpin A1, p53 and p16: 18 normal vulvar skin, 53 LS, 9 premalignant vulvar lesions (dVIN/HSIL) and 40 vSCC samples. Serum concentrations of Serpin A1 were analyzed from 30 LS, 44 vSCC and 10 control patients. Expressions were compared to clinical data. RESULTS: Tumor cell-specific Serpin A1 overexpression was detected in 88% of vSCC samples, independent of the etiology. The intensity of Serpin A1 expression was significantly higher in vSCC than in healthy vulvar skin, LS, or premalignant vulvar lesions. Serpin A1 showed an association with p53 positivity. No difference in overall survival was found between Serpin A1-, p53-, or p16-positive vSCC patients. Serum concentrations of Serpin A1 were equal in the LS, vSCC, and control groups. CONCLUSION: Tumor cell-specific Serpin A1 overexpression is a potential biomarker in vSCC.

European dermatology forum S1-guideline on the diagnosis and treatment of sclerosing diseases of the skin, Part 2: Scleromyxedema, scleredema and nephrogenic systemic fibrosis.

The term 'sclerosing diseases of the skin' comprises specific dermatological entities which have fibrotic changes of the skin in common. These diseases mostly manifest in different clinical subtypes according to cutaneous and extracutaneous involvement and can sometimes be difficult to distinguish from each other. The present guideline focuses on characteristic clinical and histopathological features, diagnostic scores and the serum autoantibodies most useful for differential diagnosis. In addition, current strategies in the first- and advanced-line therapy of sclerosing skin diseases are addressed in detail. Part 2 of this guideline provides clinicians with an overview of the diagnosis and treatment of scleromyxedema, scleredema (of Buschke) and nephrogenic systemic sclerosis (nephrogenic fibrosing dermopathy).

European Dermatology Forum S1-guideline on the diagnosis and treatment of sclerosing diseases of the skin, Part 1: localized scleroderma, systemic sclerosis and overlap syndromes.

The term 'sclerosing diseases of the skin' comprises specific dermatological entities, which have fibrotic changes of the skin in common. These diseases mostly manifest in different clinical subtypes according to cutaneous and extracutaneous involvement and can sometimes be difficult to distinguish from each other. The present guideline focuses on characteristic clinical and histopathological features, diagnostic scores and the serum autoantibodies most useful for differential diagnosis. In addition, current strategies in the first- and advanced-line therapy of sclerosing skin diseases are addressed in detail. Part 1 of this guideline provides clinicians with an overview of the diagnosis and treatment of localized scleroderma (morphea), and systemic sclerosis including overlap syndromes of systemic sclerosis with diseases of the rheumatological spectrum.

Matrix metalloproteinase-7 activates heparin-binding epidermal growth factor-like growth factor in cutaneous squamous cell carcinoma.

BACKGROUND: Tumour-specific expression of matrix metalloproteinase (MMP)-7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB). OBJECTIVES: To examine the potential role of MMP-7 in shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in RDEB-associated and sporadic SCCs. METHODS: Tissue microarrays of RDEB-associated SCC (n = 20), non-EB SCC (n = 60) and Bowen disease (n = 28) were immunostained for MMP-7, CD44 variant 3 (CD44v3) and HB-EGF. Shedding of HB-EGF was studied in vitro using two cutaneous SCC cell lines. RESULTS: Immunohistochemical analysis showed that HB-EGF was absent in tumour cells when MMP-7 and CD44v3 colocalized, and that the absence of HB-EGF was more pronounced in RDEB-associated SCCs than in non-EB SCCs. The loss of HB-EGF in MMP-7-CD44v3 double-positive areas was interpreted to indicate shedding and activation of HB-EGF; this was also detected in Bowen disease indicating its importance in the early phase of SCC development. Specific knockdown of MMP-7 expression in human cutaneous SCC cells by small interfering RNA inhibited shedding of HB-EGF and resulted in diminished activation of the EGF receptor (EGFR) and ERK1/2, and in reduced proliferation of SCC cells. CONCLUSIONS: These findings provide evidence for the role of MMP-7 in promoting the growth of cutaneous SCCs by shedding HB-EGF, and identify EGFR signalling as a potential therapeutic target in RDEB-associated SCC and unresectable sporadic cutaneous SCC.

Proteinases in cutaneous wound healing.

Cutaneous wound healing is a complex and highly coordinated process where a number of different cell types participate to renew the damaged tissue under the strict regulation of soluble and insoluble factors. One of the most versatile processes involved in wound repair is proteolysis. During cell migration, proteins of extracellular matrix are cleaved, often creating biologically active cleavage products, and proteolysis of cellular contacts leads to increased cell motility and division. Moreover, proteases activate various growth factors and other proteases in wound and regulate growth factor signaling by shedding growth factor receptors on cell surface. Normally, proteolysis is strictly controlled, and changes in protease activity are associated with alterations in wound closure and scar formation. Here, we present the current view on the role of metalloproteinases and the plasmin-plasminogen system in normal and aberrant cutaneous wound repair and discuss their role as potential therapeutic targets for chronic ulcers or fibrotic scars.

Extended release of adenovirus from silica implants in vitro and in vivo.

Despite promising preclinical results, the clinical benefits of cancer gene therapy have been modest heretofore. The main obstacle continues to be the level and persistence of gene delivery to sufficiently large areas of the tumor. One approach for overcoming this might entail extended local virus release. We studied the utility of silica gel monoliths for delivery of adenovirus to advanced orthotopic gastric and pancreatic cancer tumors. Initially, the biochemical properties of the silica-virus matrix were studied and nearly linear release as a function of time was detected. Virus stayed infective for weeks at +37 degrees C and months at +4 degrees C, which may facilitate storage and distribution. In vivo, extended release of functional replication deficient and also replication-competent, capsid-modified oncolytic viruses was seen. Treatment of mice with pancreatic cancer doubled their survival (P<0.001). Also, silica gel-based delivery slowed the development of antiadenovirus antibodies.

Integrin alpha2beta1 mediates isoform-specific activation of p38 and upregulation of collagen gene transcription by a mechanism involving the alpha2 cytoplasmic tail.

Two collagen receptors, integrins alpha1beta1 and alpha2beta1, can regulate distinct functions in cells. Ligation of alpha1beta1, unlike alpha2beta1, has been shown to result in recruitment of Shc and activation of the Ras/ERK pathway. To identify the downstream signaling molecules activated by alpha2beta1 integrin, we have overexpressed wild-type alpha2, or chimeric alpha2 subunit with alpha1 integrin cytoplasmic domain in human osteosarcoma cells (Saos-2) lacking endogenous alpha2beta1. The chimeric alpha2/alpha1 chain formed a functional heterodimer with beta1. In contrast to alpha2/alpha1 chimera, forced expression of alpha2 integrin resulted in upregulation of alpha1 (I) collagen gene transcription in response to three-dimensional collagen, indicating that the cytoplasmic domain of alpha2 integrin was required for signaling. Furthermore, signals mediated by alpha2beta1 integrin specifically activated the p38alpha isoform, and selective p38 inhibitors blocked upregulation of collagen gene transcription. Dominant negative mutants of Cdc42, MKK3, and MKK4 prevented alpha2beta1 integrin-mediated activation of p38alpha. RhoA had also some inhibitory effect, whereas dominant negative Rac was not effective. Our findings show the isoform-specific activation of p38 by alpha2beta1 integrin ligation and identify Cdc42, MKK3, and MKK4 as possible downstream effectors. These observations reveal a novel signaling mechanism of alpha2beta1 integrin that is distinct from ones previously described for other integrins.

p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells.

Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38alpha and p38delta specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.

Activation of Smad signaling enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells.

Squamous cell carcinoma (SCC) cells of the head and neck specifically express collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the expression of which correlates with their invasion capacity. Transforming growth factor-beta (TGF-beta) enhances MMP-13 and collagenase-1 (MMP-1) expression and invasion of SCC cells via p38 mitogen-activated protein kinase. Here, we have examined the role of Smad signaling in regulating MMP-13 expression and in invasion of head and neck SCC cells. Treatment with TGF-beta resulted in activation of Smad2 and Smad3 in SCC cells, but had no effect on their proliferation or viability. Basal activation of Smad3 and p38 was noted in SCC cells without exogenous TGF-beta stimulation, and adenoviral delivery of Smad7 and dominant-negative Smad3 inhibited p38 activation in these cells. Adenoviral overexpression of Smad3 augmented the upregulatory effect of TGF-beta on MMP-13 expression by SCC cells. Disruption of Smad signaling by adenoviral expression of kinase-defective TGF-beta type I receptor (activin-receptor-like kinase-5), Smad7, and dominant-negative Smad3 potently suppressed the basal and TGF-beta-induced expression of MMP-13 and MMP-1 in SCC cells, and inhibited their basal and TGF-beta-induced invasion through Matrigel and type I collagen. Adenoviral overexpression of Smad7 in cutaneous and oral SCC cells significantly inhibited their implantation in skin of SCID mice and growth of xenografts in vivo, as compared to LacZ adenovirus-transduced control cells. Together, these results show that Smad signaling plays an important role in promoting the invasive phenotype of human head and neck SCC cells by upregulating their collagenase expression.

Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels.

Regulation of human type I procollagen gene expression was studied in cultured fibroblasts both at the transcriptional and posttranscriptional level. Transcriptional regulation was examined in cultures transfected with a human pro alpha 2(I) collagen promoter/reporter gene (chloramphenicol acetyltransferase) construct, while posttranscriptional regulation was assessed by parallel determinations of type I procollagen mRNA steady-state levels. Transforming growth factor-beta 1 (TGF-beta 1) elicited a marked, approximately 5-23-fold, enhancement of pro alpha 2(I) collagen promoter activity, which was accompanied by an elevation of type I procollagen mRNA levels. This enhancement of gene expression was suppressed by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), as determined at mRNA steady-state level, but two distinct mechanisms were involved. TNF-alpha suppressed the pro alpha 2(I) collagen promoter activity, whereas IFN-gamma had only a minimal effect at transcriptional level. The effects of TNF-alpha and IFN-gamma were synergistic, suggesting that combination of these two factors may potentially provide pharmacologic means to counteract tissue deposition of collagen in diseases involving TGF-beta.

p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression.

Degradation of collagenous extracellular matrix by collagenase 1 (also known as matrix metalloproteinase 1 [MMP-1]) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, chronic ulcers, and tumor invasion and metastasis. Here, we have investigated the role of distinct mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-1 gene expression. The activation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (designated ERK1,2) pathway by oncogenic Ras, constitutively active Raf-1, or phorbol ester resulted in potent stimulation of MMP-1 promoter activity and mRNA expression. In contrast, activation of stress-activated c-Jun N-terminal kinase and p38 pathways by expression of constitutively active mutants of Rac, transforming growth factor beta-activated kinase 1 (TAK1), MAPK kinase 3 (MKK3), or MKK6 or by treatment with arsenite or anisomycin did not alone markedly enhance MMP-1 promoter activity. Constitutively active MKK6 augmented Raf-1-mediated activation of the MMP-1 promoter, whereas active mutants of TAK1 and MKK3b potently inhibited the stimulatory effect of Raf-1. Activation of p38 MAPK by arsenite also potently abrogated stimulation of MMP-1 gene expression by constitutively active Ras and Raf-1 and by phorbol ester. Specific activation of p38alpha by adenovirus-delivered constitutively active MKK3b resulted in potent inhibition of the activity of ERK1,2 and its upstream activator MEK1,2. Furthermore, arsenite prevented phorbol ester-induced phosphorylation of ERK1,2 kinase-MEK1,2, and this effect was dependent on p38-mediated activation of protein phosphatase 1 (PP1) and PP2A. These results provide evidence that activation of signaling cascade MKK3-MKK3b-->p38alpha blocks the ERK1,2 pathway at the level of MEK1,2 via PP1-PP2A and inhibits the activation of MMP-1 gene expression.

Adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells.

We have used adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinase (TIMP)-1, -2, and -3 to examine their effect on the invasion capacity of metastatic melanoma cell lines SK-Mel-5 and A2058. Infection of melanoma cells with recombinant replication-deficient adenoviruses coding for TIMP-1, TIMP-2, and TIMP-3 resulted in marked secretion of TIMP-1 and TIMP-2 to culture medium and accumulation of TIMP-3 to matrix. Overexpression of TIMP-3 inhibited invasion of SK-Mel-5 and A2058 cells through reconstituted basement membrane (Matrigel) even more potently than TIMP-1 and TIMP-2. In addition, overproduction of TIMP-3 reduced attachment of melanoma cells to type I and IV collagen and fibronectin and resulted in apoptosis in both SK-Mel-5 and A2058 cells. These results propose a novel role for TIMP-3 in regulation of invasion and survival of malignant cells and suggest potential use for TIMP-3 in adenovirus-mediated gene therapy of malignant melanoma.

Activation of fibroblast collagenase-1 expression by tumor cells of squamous cell carcinomas is mediated by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase-2.

Collagenase-1 [matrix metalloproteinase (MMP)-1] is expressed by stromal fibroblasts of various invasive malignant tumors. Here, we have examined the molecular mechanisms of tumor-induced expression of MMP-1 by stromal fibroblasts. Treatment of fibroblasts with conditioned media of tumor cells derived from squamous cell carcinomas (SCCs) of the oral cavity and larynx resulted in activation of fibroblast MMP-1 expression at the transcriptional level. The induction of MMP-1 expression correlates with activation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase and phosphorylation of c-Jun and activating transcription factor-2 (ATF-2) and is dependent on the activity of p38 mitogen-activated protein kinase. Furthermore, using fibroblasts derived from JNK2-/- mice, we show that JNK2 is required for induction of fibroblast collagenase-3 expression in response to conditioned SCC tumor cell medium. Together, these results provide evidence that stress-activated p38 and JNK pathways play a crucial role in paracrine regulation of collagenolytic capacity of stromal fibroblasts in SCCs and suggest JNK2 as a novel target for inhibition of MMP-1 expression and tumor invasion.

Suppression of TGFß and Angiogenesis by Type VII Collagen in Cutaneous SCC.

BACKGROUND: Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFß signaling and angiogenesis in vitro and in vivo. METHODS: Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFß pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided. RESULTS: Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2ß1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFß signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFß markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFß markers, n = 6; VEGF, n = 21). Inhibition of TGFß receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTßRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS: Type VII collagen suppresses TGFß signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.

Differential regulation of interstitial collagenase (MMP-1) gene expression by ETS transcription factors.

Expression of interstitial collagenase (MMP-1) has been detected in stromal fibroblasts of various malignant tumors. Here, we have studied the effect of three structurally different ETS transcription factors (ETS-1, ERGB/Fli-1, and PU.1) on MMP-1 promoter activity in NIH3T3 fibroblasts. ETS-1 increased the activity of 3.8 kb MMP-1 promoter construct up to tenfold, while ERGB/Fli-1 or PU.1 alone had no marked effect on basal promoter activity. ETS-1 also markedly potentiated enhancement of MMP-1 promoter by both c-Jun and JunB, whereas ERGB/Fli-1 augmented only the effect of c-Jun. Interestingly, PU.1 abolished induction of MMP-1 promoter by both c-Jun and JunB. Stimulation of MMP-1 promoter by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid was differentially augmented by ETS-1 and ERGB/Fli-1, and abrogated by PU.1. Co-transfection studies with MMP-1 promoter 5'-deletion constructs revealed that AP-1 site was necessary for PU.1-elicited suppression. As compared to control cell lines, PU.1-positive stable cells exhibited clearly weaker binding of c-Jun and JunD containing AP-1 complexes to MMP-1 promoter AP-1 element, as well as marked reduction in basal level and induction of c-jun mRNA by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid, suggesting a novel mechanism for PU.1-mediated inhibition of AP-1 dependent gene expression. These results show that three structurally distinct ETS transcription factors differently modulate AP-1 dependent upregulation of MMP-1 gene expression.

Inhibition of collagenase-3 (MMP-13) expression in transformed human keratinocytes by interferon-gamma is associated with activation of extracellular signal-regulated kinase-1,2 and STAT1.

Collagenase-3 (MMP-13) is characterized by an exceptionally wide substrate specificity and restricted expression. MMP-13 is specifically expressed by transformed human keratinocytes in squamous cell carcinomas in vivo and its expression correlates with their invasion capacity. Here, we show, that interferon-gamma (IFN-gamma) markedly inhibits expression of MMP-13 by human cutaneous SCC cells (UT-SCC-7) and by ras-transformed human epidermal keratinocytes (A-5 cells) at the transcriptional level. In addition, IFN-gamma inhibits collagenase-1 (MMP-1) expression in these cells. IFN-gamma abolished the enhancement of MMP-13 and MMP-1 expression by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), and inhibited invasion of A-5 cells through type I collagen. IFN-gamma also rapidly and transiently activates extracellular signal-regulated kinase 1,2 (ERK1,2) and blocking ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by specific MEK1,2 inhibitor PD98059 partially (by 50%) prevents Ser-727 phosphorylation of STAT1 and suppression of MMP-13 expression by IFN-gamma. Furthermore, Ser-727 phosphorylation of STAT1 by ERK1,2, or independently of ERK1,2 activation is associated with marked reduction in MMP-13 expression. These observations identify a novel role for IFN-gamma as a potent inhibitor of collagenolytic activity and invasion of transformed squamous epithelial cells, and show that inhibition of MMP-13 expression by IFN-gamma involves activation of ERK1,2 and STAT1.

Interleukin-1 increases collagen production and mRNA levels in cultured skin fibroblasts.

In the present study we show that highly purified human interleukin-1 increases collagen production nearly 2-fold and mRNA levels of type I and III collagen over 2.5-fold in cultured normal human dermal fibroblasts. To minimize the effects of transient prostaglanding E2 production in fibroblasts treated with interleukin-1, the cell cultures were preincubated for 24 h before these measurements were made. The effects of interleukin-1 were also tested on scleroderma fibroblasts exhibiting increased collagen production. Although collagen synthesis was stimulated by interleukin-1 to some degree, the cells grown from both affected and unaffected skin areas were found to be relatively unresponsive to the effects of interleukin-1, suggesting a role for this monokine in the earlier stages of the disease process. The results also suggest that interleukin-1 has a role in stimulation of collagen synthesis under certain normal and pathological conditions in addition to stimulating fibroblast proliferation.