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J Lohi

Publications and source records attributed to J Lohi.

At least 19 recordsLinked to original sources
PubMed2001

Laminin-5 in the progression of carcinomas.

The basement membrane (BM) separates epithelial elements from the surrounding stroma. BM is dynamic in regulation of epithelial cells differentiation as well as their organization into 3-dimensional tissues. In these functions, among the molecules of the BM, laminins are especially dynamic. Laminins are distributed in a spatially and temporally regulated manner in various epithelial tissues. Various changes in the laminin distribution accompany the malignant transformation of epithelia. The role of the BM and laminins in the progression of carcinomas is not well understood. The BM has been suggested to act as a mechanical barrier against carcinoma cell invasion. BM laminins may play an active role in regulating the migration and proliferation of the carcinoma cells. Laminin isoform laminin-5 expression is typical for some invasive carcinomas and it may act as a ligand for invading carcinoma cells. Neoexpression of laminin-5 has also been associated to proliferative activity of the carcinoma cells. Integrins alpha(3)beta(1) and alpha(6)beta(4) are probable cell surface receptors acting with laminin-5 in the regulation of carcoma cell invasion and proliferation.

Basement Membrane↗
PubMed2001

Promoter characterization of the human and mouse epilysin (MMP-28) genes.

Epilysin (MMP-28) is a recently cloned member of the matrix metalloproteinase family (Lohi et al., J. Biol. Chem. 276 (2001) 10134). It is expressed at highest levels in the skin by basal and suprabasal keratinocytes, and in testis by developing germ cells. To characterize the epilysin promoter, we isolated a 3.0 kb fragment of human genomic DNA containing 5'-flanking sequence of the epilysin gene, and a corresponding 660 bp fragment from the mouse. The 5'-flanking sequences contain no typical TATA-boxes or CCAAT sequences close to the translation initiation sites. RNase protection assay revealed that two transcription start sites are utilized in the human epilysin gene, situated 210 and 230 bp upstream from the translation start site. The promoter contains a GT-box, situated 300 bp upstream from the translation start site, with homology to the consensus binding site for transcription factors of the Sp family. This site is perfectly conserved between the human and mouse promoters. For reporter gene assays a series of constructs with fragments of increasing length of the epilysin promoter were coupled to the firefly luciferase gene. Reporter gene assays indicated that deletion or mutation of the GT-box dramatically reduces the transcriptional activity both in keratinocytes and in spermatogonia. Gel mobility shift assays showed that several nuclear proteins bind specifically to this sequence. Supershift assays with antibodies specific for members of the Sp family identified Sp1 and Sp3 as components of these protein/DNA complexes and hence as possible regulators of the epilysin gene. Our results indicate that the epilysin promoter has distinctive structural and functional features, which may control the unique expression and regulation patterns of the epilysin gene.

Animals↗
PubMed2001

Epsin 3 is a novel extracellular matrix-induced transcript specific to wounded epithelia.

Using an in vitro model of keratinocyte activation by the extracellular matrix following injury, we have identified epsin 3, a novel protein closely related to, but distinct from previously described epsins. Epsin 3 contains a domain structure common to this gene family, yet demonstrates novel differences in its regulation and pattern of expression. Epsin 3 mRNA and protein were undetectable in keratinocytes isolated from unwounded skin, but induced in cells following contact with fibrillar type I collagen. The native triple helical structure of collagen was required to mediate this response as cells failed to express epsin 3 when plated on gelatin. Consistent with the reported function of other epsins, epsin 3 was evident in keratinocytes as punctate vesicles throughout the cytoplasm that partially co-localized with clathrin. In addition, epsin 3 exhibited nuclear accumulation when nuclear export was inhibited. In contrast to other known epsins, epsin 3 was restricted to keratinocytes migrating across collagen and down-regulated following cell differentiation, suggesting that expression was spatially and temporally regulated. Indeed, epsin 3 was localized specifically to migrating keratinocytes in cutaneous wounds, but not found in intact skin. Intriguingly, Northern hybridization and reverse transcriptase-polymerase chain reaction experiments indicated that epsin 3 expression was restricted to epithelial wounds or pathologies exhibiting altered cell-extracellular matrix interactions. Thus, we have identified a novel type I collagen-induced epsin that demonstrates structural and behavioral similarity to this gene family, yet exhibits restricted and regulated expression, suggesting that epsin 3 may serve an important function in activated epithelial cells during tissue morphogenesis.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Differential patterns of stromelysin-2 (MMP-10) and MT1-MMP (MMP-14) expression in epithelial skin cancers.

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumours. To investigate the role of stromelysin-2 (MMP-10) in growth and invasion of skin tumours, we studied cutaneous carcinomas with high metastatic capacity (squamous cell carcinomas, SCCs), only locally destructive tumours (basal cell carcinomas, BCCs) and pre-malignant lesions (Bowen's disease and actinic keratosis) using in situ hybridization. Expression of MMP-10 was compared with that of stromelysin-1 (MMP-3) and of MT1-MMP, the expression of which has been shown to correlate with tumour invasiveness. MMP-10 was expressed in 13/21 SSCs and 11/19 BCCs only in epithelial laminin-5 positive cancer cells, while premalignant lesions were entirely negative. MT1-MMP mRNA was detected in 19/21 SCCs both in epithelial cancer cells and stromal fibroblasts and in 14/18 BCCs only in fibroblasts. The level of MMP-10 was upregulated in a cutaneous SCC cell line (UT-SCC-7) by transforming growth factor-alpha and keratinocyte growth factor, and by interferon-gamma in combination with transforming growth factor-beta1 and tumour necrosis factor-alpha both in UT-SCC-7 and HaCaT cells. Our results show that MMP-10 expression does not correlate with the invasive behaviour of tumours as assessed by their histology and MT1-MMP expression, but may be induced by the wound healing and inflammatory matrix remodelling events associated with skin tumours.

Basal Cell Carcinoma↗
PubMed2001

Effects of Lactobacillus rhamnosus GG and Lactobacillus reuteri R2LC on acetic acid-induced colitis in rats.

BACKGROUND: Certain lactobacilli reduce the severity of experimental colitis. The aim of this study was to compare the effects of a human strain Lactobacillus rhamnosus GG and a rat strain Lactobacillus reuteri R2LC on acetic acid-induced colitis in rats. METHODS: Lactobacillus rhamnosus GG, Lactobacillus reuteri R2LC or sulphasalazine were given orally to the rats. Colitis was assessed 72 h after induction with acetic acid. RESULTS: Lactobacillus reuteri R2LC significantly antagonized body weight loss caused by inflammation compared with Lactobacillus rhamnosus GG and sulphasalazine, and oedema formation in the colon compared with sulphasalazine. Lactobacillus reuteri R2LC reduced the median value of macroscopic ulceration and the protein content of inducible nitric oxide synthase by 50% and the median of the protein content of inducible cyclooxygenase by 30% compared with that of the colitis control group, and Lactobacillus rhamnosus GG reduced the median of inducible nitric oxide protein content by 40% and increased the median of inducible cyclooxygenase protein content by 30% compared with the median value of the colitis control group, but these differences were not statistically significant. CONCLUSIONS: The rat strain Lactobacillus reuteri R2LC, but not the human strain Lactobacillus rhamnosus GG, is of benefit in reducing the severity of acetic acid-induced colitis in rats. These results suggest that it is not the total amount of Lactobacillus but the particular species or strain of Lactobacillus that is important in attenuating experimental colitis.

Acetic Acid↗
PubMed2001

[Headache attacks].

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Calcium Channel Blockers↗
PubMed2000

Epilysin, a novel human matrix metalloproteinase (MMP-28) expressed in testis and keratinocytes and in response to injury.

We have cloned a new human matrix metalloproteinase (MMP-28, epilysin) from human keratinocyte and testis cDNA libraries. Like most MMPs, epilysin contains a signal sequence, a prodomain with a PRCGVTD sequence, a zinc-binding catalytic domain with an HEIGHTLGLTH sequence, and a hemopexin-like domain. In addition, epilysin has a furin activation sequence (RRKKR) but has no transmembrane sequence. The exon-intron organization and splicing pattern of epilysin differ from that of other MMP genes. It has only 8 exons, and 5 exons are spliced at sites not used by other MMPs. Another novel feature of epilysin is that exon 4 is alternatively spliced to a transcript that does not encode the N-terminal half of the catalytic domain. Northern hybridization of tissue RNA indicated that epilysin is expressed at high levels in testis and at lower levels in lungs, heart, colon, intestine, and brain. RNase protection assay with various cell lines indicated that epilysin was selectively expressed in keratinocytes. Recombinant epilysin degraded casein in a zymography assay, and its proteolytic activity was inhibited by EDTA and by batimastat, a selective MMP inhibitor. Immunohistochemical staining showed expression of epilysin protein in the basal and suprabasal epidermis of intact skin. In injured skin, prominent staining for epilysin was seen in basal keratinocytes both at and some distance from the wound edge, a pattern that is quite distinct from that of other MMPs expressed during tissue repair. These findings suggest that this new MMP functions in several tissues both in tissue homeostasis and in repair.

Adult↗
PubMed2000

Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain.

Membrane-type-1 matrix metalloproteinase (MT1-MMP) has transmembrane and cytoplasmic domains, which target it to invasive fronts. We analyzed the role of the cytoplasmic tail by expressing wild type MT1-MMP and three mutants with progressively truncated C termini in human Bowes melanoma cells. We examined gelatinase A activation and the localization and processing of recombinant proteins in stable cell clones using gelatin zymography, immunoblotting, and immunofluorescence. Cell invasion was analyzed in vitro by Matrigel invasion assays. Gelatinase A was activated in all cell clones. However, the localization of MT1-MMP to the leading edge of migrating cells and cell invasion through Matrigel were strongly enhanced only in cells expressing either wild type or truncated MT1-MMP lacking 6 C-terminal amino acid residues (Delta577). Truncations of 10 or 16 amino acid residues in the cytoplasmic domain (Delta567 and Delta573, respectively) disturbed MT1-MMP localization. The expression of wild type and Delta577 MT1-MMPs induced also their cleavage to 43-kDa cell surface forms and the release of soluble, approximately 20-kDa N-terminal fragments containing the catalytic center. A synthetic MMP inhibitor but not a gelatinase inhibitor prevented the processing, suggesting that autocatalytic cleavage occurs. Purified soluble MT1-MMP was also autoproteolytically processed to 43- and 20-kDa forms in vitro. Our results indicate that the cytoplasmic domain has an important role in cell invasion by controlling both the targeting and degradation/turnover of MT1-MMP.

Cloning, Molecular↗
PubMed2000

Structural analysis and promoter characterization of the human membrane-type matrix metalloproteinase-1 (MT1-MMP) gene.

Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades extracellular matrix components directly and indirectly by activation of other matrix metalloproteinases (MMPs). In the present study, we have isolated and characterized the human MT1-MMP gene and its promoter. The gene consists of 10 exons and nine introns spanning more than 10 kilobases (kb). The locations of two exon-intron splicing sites are distinct from the preserved positions among other known MMP genes. Primer extension and RNAse and S1 nuclease protection analyses indicated that there are four major and several minor transcription start sites. The 5'-flanking sequence of the gene contains putative regulatory elements, including one Sp-1 site and four CCAAT-boxes, whereas there is no TATA-box. The Sp-1 binding site was functional, as shown by gel shift and supershift analyses. Transfection studies with promoter constructs containing 0.1 to 7.2 kb of 5'-flanking sequence coupled to a luciferase reporter gene indicated that the promoter contains additional positive and negative regulatory sequences. Deletion of the Sp-1 binding site by site-directed mutagenesis reduced luciferase activity by about 90%, demonstrating the crucial role of this element in maintaining MT1-MMP transcription. Our findings indicate that the human MT1-MMP promoter has distinctive structural and functional features compared with other MMP genes, which may lead to a unique expression pattern and regulation during physiological and pathological processes.

Base Sequence↗
PubMed2000

Expression and purification of soluble and inactive mutant forms of membrane type 1 matrix metalloproteinase.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteinase and a cell-surface receptor and activator of gelatinase A in normal and neoplastic cells. We have expressed and purified a soluble deletion mutant of MT1-MMP lacking the transmembrane and cytoplasmic domains and an inactive mutant of the soluble MT1-MMP, where the active-site glutamic acid(240) was substituted by alanine (E240A). A baculovirus transfer vector coding for amino acids 21-539 of MT1-MMP (DeltaTM) and a similar vector coding for the mutation (E240ADeltaTM) were constructed for expression in insect cells. Both DeltaTM and E240ADeltaTM were secreted to the culture medium of infected High Five insect cells. They were then purified by cation-exchange followed by gel-filtration chromatography. DeltaTM was able to cleave denatured type I collagen and fibronectin and activate MMP-2/gelatinase-A, while E240ADeltaTM had only low proteolytic activity against denatured collagen I. The current expression and purification protocol should prove useful for the production of large amounts of enzymatically active soluble MT1-MMP.

Amino Acid Substitution↗
PubMed2000

Basement membrane laminin-5 is deposited in colorectal adenomas and carcinomas and serves as a ligand for alpha3beta1 integrin.

Interplay between laminin-5 (Ln-5) and its integrin (Int) receptors alpha2beta1, alpha3beta1 and alpha6beta4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immunoreactivity for chains of Ln-5 (alpha3-beta3-gamma2) and Ln-10 (alpha5-beta1-gamma1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln-5 appeared to accumulate along the invading edges of carcinomas, while Ln-10 was mostly absent. Immunoreactivity for Ln al chain, a component of Lns-1 and -3, was not seen in adenomas or carcinomas. Immunoreactivity for alpha2, alpha6, beta1 and beta4 Ints was found in all tumors and that for alpha3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln-5. Immunoblotting of carcinoma tissues showed that the gamma2 chain of Ln-5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln-5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function-blocking MAbs to alpha3 and beta1 Int subunits, but not those to Int alpha2 or alpha6 subunits, significantly inhibited the adhesion of cells to Ln-5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln-5, produced by carcinoma cells, is a major BM component and the cells use the alpha3beta1 Int complex for adhesion to Ln-5.

Adenoma↗
PubMed1999

Renal cell carcinomas and pancreatic adenocarcinomas produce nidogen in vitro and in vivo.

The production of nidogen by four renal cell carcinoma (RCC) and three pancreatic adenocarcinoma (PAc) cell lines has been studied in cell culture and in xenografted tumours in nude mice. In RCC cells, immunoreactivity for nidogen was seen only after exposure to monensin to induce cytoplasmic accumulation of secretory proteins. In PAc cells, immunoreaction was also detectable in control cells. Immunoblotting of control and monensin-exposed cells and immunoprecipitation of culture media of radioactively labelled cells demonstrated the production of nidogen polypeptide of Mr ca. 150000 by six of the seven cell lines. Basement membranes (BMs) and stroma of the xenografted tumours derived from these six cell lines demonstrated immunoreactivity for both human and mouse nidogen, as revealed with species-specific antibodies. The ability of the cells to produce nidogen in vitro and deposit in vivo was positively correlated with high histological grade of the xenografted tumours, although the small number of cell lines studied calls for further studies to confirm this. The distribution of nidogen in human RCC and PAc specimens was also studied by immunohistochemistry. There was strong immunoreactivity for nidogen in tumour stroma, BM of carcinoma cell nests, and endothelial basal lamina, but no conclusions could be drawn regarding histological grade and immunostaining patterns, because stromal production could not be ruled out. The results show that nidogen is produced by human carcinoma cells both in vitro and in vivo.

Adenocarcinoma↗
PubMed1999

Hemidesmosomal molecular changes in dermatitis herpetiformis; decreased expression of BP230 and plectin/HD1 in uninvolved skin.

Recent BP230-knockout experiments with subsequent blistering and recently identified plectin/HD1 mutations in epidermolysis bullosa simplex patients suggest that defective expression of BP230 and plectin/HD1 may predispose to blister formation in human skin. We have studied the expression of the epithelial adhesion complex as well as the basement membrane and anchoring fibril antigens in uninvolved dermatitis herpetiformis skin to find out if alterations can be detected in these structures predisposing to the blister formation typical of the disease. Ten uninvolved dermatitis herpetiformis skin specimens, which all showed clear granular deposits of IgA under the basement membrane in direct immunofluorescence and five normal skin specimens, were studied by indirect immunofluorescence technique. Six uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for BP230 and four uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for plectin/HD1. All five skin controls showed strong immunoreactions for BP230 and plectin/HD1. Other hemidesmosomal proteins including BP180 and integrin alpha6beta4, as well as basement membrane proteins laminin-5, laminin-1, nidogen and type IV collagen, and the anchoring fibril protein type VII collagen showed a normal strong expression. Our results suggest that alterations in BP230 and plectin/HD1 may contribute or predispose to blister formation in dermatitis herpetiformis skin.

Autoantigens↗
PubMed1999

Expression of collagenases-1 and -3 and their inhibitors TIMP-1 and -3 correlates with the level of invasion in malignant melanomas.

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 MT1-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.

Adult↗
PubMed1998

Proteolytic processing of membrane-type-1 matrix metalloproteinase is associated with gelatinase A activation at the cell surface.

Human fibroblasts and HT-1080 fibrosarcoma cells express membrane-type-1 matrix metalloproteinase (MT1-MMP), the cell surface activator of gelatinase A, in separate forms of 63 kDa, 60 kDa and in some cases 43 kDa. In the present work the interrelationships between MT1-MMP processing and gelatinase A activation were analysed using HT-1080 fibrosarcoma cells as a model. It was found that MT1-MMP was synthesized as a 63 kDa protein, which was constitutively processed to a 60 kDa active enzyme with N-terminal Tyr112, as shown by immunoprecipitation, immunoblotting and sequence analyses. Co-immunoprecipitation results indicated that only the active 60 kDa form of MT1-MMP bound gelatinase A at the cell surface. Both the activation of pro-MT1-MMP and the membrane binding of the tissue inhibitor of metalloproteinases type 2 (TIMP-2) and gelatinase A, and subsequent activation of gelatinase A, were inhibited by calcium ionophores. Although the active MT1-MMP was required for cell surface binding and activation of gelatinase A, it was inefficient in activating gelatinase A in fibroblasts or in control HT-1080 cells alone. Low expression levels of TIMP-2 and rapid synthesis of MT1-MMP were found to be critical for gelatinase A activation. In HT-1080 cells, MT1-MMP was further processed to an inactive, 43 kDa cell surface form when overexpressed, or when the cells were treated with PMA. Under these conditions, the activated gelatinase A was detected in the culture medium, in cell membrane extracts and in MT1-MMP-containing complexes. These results indicate that proteolytic processing (activation and degradation/inactivation) of MT1-MMP and MT1-MMP/TIMP-2 relationships at the cell surface are important regulatory levels in the control of gelatinolytic activity.

Amino Acid Sequence↗
PubMed1998

Neoexpression of the epithelial adhesion complex antigens in thyroid tumours is associated with proliferation and squamous differentiation markers.

Integrin dimer alpha 6 beta 4 is a transmembrane component of an epithelial cell adhesion complex that consists of hemidesmosomes (HDs), basement membrane (BM)-associated laminin-5 (Ln-5), and anchoring filaments/type VII collagen, all of which are absent from normal thyroid follicular epithelium. In the present study, the expression of epithelial cell adhesion complex antigens in thyroid tumours was investigated using immunohistochemistry. In addition to integrin subunits alpha 6 and beta 4, immunoreactivity was found for all chains of Ln-5, alpha 3, beta 3 and gamma 2, type VII collagen and hemidesmosomal antigen, HD1, in most thyroid carcinomas associated with tumour anaplasia and papillary growth pattern and located at the border of parenchymal cells and connective tissue or blood vessel walls. In addition, a more restricted expression of bullous pemphigoid antigens 180 and 230 (BP180 and BP230), constituents of HDs, was found in some papillary and anaplastic carcinomas and atypical adenomas. Adhesion complex antigens were located to regions of cells which were immunoreactive for cytokeratin (ck)-5 and proliferating cell nuclear antigen Ki-67. The results suggest that in thyroid carcinomas, the emergence of adhesion complex antigens is associated with squamous differentiation and high proliferative activity.

Antigens, Neoplasm↗
PubMed1998

Extracellular matrix in renal cell carcinomas.

Extracellular matrix (ECM) may be divided into interstitial matrix and the basement membrane (BM). ECM influences a variety of epithelial cell behaviours, including proliferation, differentiation, and morphogenesis, maybe most widely studied in kidney morphogenesis. In carcinomas, including renal cell carcinomas (RCCs), these properties and interactions of cells with interstitial matrix and BM are disturbed. As a carcinoma with a tendency to spread to distant sites, RCC is an interesting target for the study of epithelial-stromal interactions. Among interstitial collagens, type VI collagen appears to be widely distributed in RCCs. Also EDA-fibronectin (EDA-Fn) as well as tenascin-C (Tn) are important stromal components especially in poorly differentiated carcinomas. BMs of RCC islets and those of tumor blood vessel endothelia may merge in poorly differentiated carcinomas. As a dynamic component of BMs, laminins (Ln) are important in kidney development and RCC progression. Type IV collagen and nidogen, other components of BMs in RCCs, are produced by stromal as well as epithelial cells. ECM proteins may function in RCC progression by binding and regulating the activity of growth factors e.g. transforming growth factor beta 1 and basic fibroblast growth factor. Also the expression of cell surface receptors for ECM is disturbed in RCCs. At least alpha v integrin (Int) and CD44 emerge in renal epithelial cells during malignant transformation. Papillary renal neoplasms differ from RCCs by cell adhesion receptor expression and BM composition as well as by ECM avascularity and capacity to bind growth factors, thus suggesting a distinct property for this renal tumor.

Animals↗