Biomedical subjects

J Lohi

Publications and source records attributed to J Lohi.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1997

Expression of type IV collagen alpha1(IV)-alpha6(IV) polypeptides in normal and developing human kidney and in renal cell carcinomas and oncocytomas.

Type IV collagen trimer is a major component of basement membranes (BMs). It is composed of polypeptides named alpha1(IV)-alpha6(IV) chains. Chains alpha1,2(IV) are widely expressed in BMs while alpha3(IV)-alpha6(IV) are more restricted in human tissues. We have now studied by immunohistochemical means the distribution of collagen IV chains in fetal and adult human kidney, in oncocytomas, in renal cell carcinomas (RCCs) and their metastases and in experimental xenografts of human tumors. alpha1,2(IV) chains were found in all BMs of fetal and adult kidney as well as of renal tumors, while alpha3(IV)-alpha6(IV) chains were found in BMs of distal segments of developing and mature tubules. alpha3(IV)-alpha5(IV) chains were seen also in BMs of developing fetal glomeruli after the capillary loop stage. Most of the RCCs and their metastases showed occasional expression of alpha3(IV)-alpha6(IV) with papillary variants showing only expression of alpha5(IV) chain. There was a distinct expression of alpha3(IV)-alpha5(IV) chains in BMs of 3 oncocytomas. In 2 of them a variable expression of the alpha6(IV) chain was seen. In 3 of 4 xenografts, immunoreactivity for human-specific monoclonal antibody (MAb) for alpha1,2(IV) was seen in the BM-like structures. No alpha3-alpha6(IV) was seen in any of the xenografts, while polyclonal antiserum for type IV collagen presented immunoreactivity in BMs of all xenografts. Our results show that oncocytomas and most of the RCCs express scarce variants of type IV collagen containing alpha3(IV)-alpha6(IV) chains. In experimental xenograft tumors, both implanted RCC cells and host stromal cells have a capacity to produce type IV collagen.

Adenoma, Oxyphilic↗

PubMed1997

Changes in the distribution of integrins and their basement membrane ligands during development of human thyroid follicular epithelium.

Interactions between cells and basement membrane components are crucial for the regulation of epithelial cell differentiation and polarization. We have studied by immunohistochemical methods the distribution of integrin adhesion proteins and some of their basement membrane ligands in foetal (16-19 weeks) and adult thyroid follicular epithelia. A diffuse immunoreactivity for only alpha 3, alpha v and beta 1 integrins was found in foetal follicular epithelium, whereas in adult follicular epithelium these integrins were expressed basally in a polarized manner. Additionally, beta 3 integrin was seen in a more basolaterally confined manner in adult follicular epithelium. Among basement membrane components, laminin alpha 1, beta 1, gamma 1 and beta 2 chains were found in epithelial basement membranes of the foetal thyroid gland, suggestive of the presence of laminins-1 and -3. In contrast, the basement membranes of adult follicular epithelium presented a much weaker immunoreactivity for the laminin beta 2 chain. Furthermore, immunoreactivity for the laminin alpha 2 chain was occasionally seen in adult thyroid glands, apparently confined to myofibroblasts. Immunoreactivity for type IV collagen alpha 1 and alpha 2 (IV) chains was found in follicular basement membranes of foetal as well as adult thyroid gland. The results suggest that during maturation of foetal thyroid follicular epithelium a distinct polarization of integrins takes place. In mature thyroid follicular epithelium, the presumable adhesion-mediating integrin complexes are alpha 3 beta 1, alpha v beta 1 and/or alpha v beta 3 mediating adhesion to laminin-1 (alpha 1-beta 1-gamma 1) and type IV collagen trimer alpha 1(2) alpha 2 (IV).

Adult↗

PubMed1997

Distinct changes in the laminin composition of basement membranes in human seminiferous tubules during development and degeneration.

We studied the distribution of laminin (Ln) chains and their integrin (Int) receptors in normal developing and adult and in atrophied human testes by using immunohistochemistry. Immunostaining for EHS Ln and type IV collagen was used to identify basement membranes (BMs). In the BM of seminiferous epithelium of fetal testis, a panel of monoclonal antibodies showed immunoreactivity for Ln alpha 1-, alpha 2-, beta 1-, beta 2- and gamma 1-chains, suggestive of the presence of Lns 1 to 3. In BM of adult seminiferous epithelium with active spermatogenesis, immunoreactivity for Ln beta 2- and gamma 1-chains was found but not for Ln alpha-chains, suggesting a complex of Ln chains not compatible with any known trimers. Instead, with polyclonal Ln antiserum and monoclonal antibody to type IV collagen, a distinct BM-like reactivity was seen. In atrophied testes, prominent immunoreactivities for Ln chains, compatible with Lns 1 to 3, were seen in the thickened BM of seminiferous tubules, hence suggestive of reappearance of fetal Lns. Among the subunits of Ln-binding Int receptors in fetal seminiferous tubules, a strong immunoreactivity for Int beta 1- and Int alpha 6-subunits was seen throughout the seminiferous epithelium, other Int subunits being found in interstitial cells. In the adult and atrophied testes, immunoreactivities for Int beta 1- and Int alpha 6-subunits were seen to be confined to the basal aspect of the seminiferous epithelium whereas immunoreactivities for Int alpha 1-, alpha 2-, alpha 3- and beta 4-subunits were seen in the myoid cells. The results show that both maturation and degenerative changes of human testes are accompanied by distinct changes in the Ln expression of BM of seminiferous epithelium, which appears to accompany epithelial differentiation of the Sertoli cells. Furthermore, they suggest the presence of a novel Ln trimer in BM of adult human seminiferous tubules.

Adult↗

PubMed1996

Expression of laminin in renal-cell carcinomas, renal-cell carcinoma cell lines and xenografts in nude mice.

We studied the expression of laminin (Ln) chains (alpha1-alpha3, beta1-beta3, gamma1) in human renal-cell carcinomas (RCC), papillary renal neoplasms (PRN) and oncocytomas, in RCC cell lines and their xenografts. In RCCs the basement membranes (BM) showed immunoreactivity for chains of Ln-1 (alpha1-beta1-gamma1). Only in well-differentiated RCCs could vessel BMs be distinguished from those of carcinoma cell islets. RCCs and oncocytomas also exhibited an abundant immunoreactivity for Ln beta2 chain in both vessel and tumor cell BMs, while Ln alpha2 chain was not seen in any renal tumors. In distinction from RCCs, PRNs presented a strong BM immunoreactivity for Ln alpha3 and beta3 chains and for Ln-5, as well as lack of Ln beta2 chain. A more variable reactivity for Ln-5 was seen in oncocytomas. As PRNs and oncocytomas have been suggested to originate from collecting ducts, it is notable that in normal human kidney, we could detect immunoreactivity for Ln-5 and its chains only in BM of the tubules of the loop of Henle. In immunoprecipitation experiments, an abundant production of Ln-1, but not of Ln-5, was seen in cultured RCC cells, while in xenografts of the same cells BM-confined immunoreactivity for both Ln-1 and Ln-5 was seen. Ln beta2 chain was produced by 2 of the 4 RCC cell lines in culture but was found only in 1 of the xenografted tumors.

Adenoma, Oxyphilic↗

PubMed1996

Regulation of membrane-type matrix metalloproteinase-1 expression by growth factors and phorbol 12-myristate 13-acetate.

Overexpression of membrane-type matrix metalloproteinase (MT-MMP-1) results in the activation of both endogenous and exogenous 72-kDa gelatinase. To understand the effects of MT-MMP-1 on 72-kDa gelatinase activation, we analyzed its expression in human fibroblasts and HT-1080 fibrosarcoma cells. Both cell types expressed the MT-MMP-1 mRNA constitutively at a considerable level and treatment of cells with PMA enhanced the expression about 2-3-fold. Concanavalin A treatment increased MT-MMP-1 mRNA levels in fibroblasts about 4-fold. Induction of MT-MMP-1 by phorbol 12-myristate 13-acetate (PMA) required protein synthesis as shown by cycloheximide inhibition. The induction was also inhibited by dexamethasone. Analysis of MT-MMP-1 mRNA stability using actinomycin D indicated that the half-life was rather long and not affected by PMA, suggesting transcriptional regulation. Only HT-1080 cells had significant 72-kDa gelatinase processing activity after treatment with PMA or concanavalin A, while fibroblasts were virtually negative. Immunoblotting analysis of fibroblast lysates indicated that MT-MMP-1 was present mainly in a 60-kDa form. PMA and concanavalin A caused 2-4-fold increases in its protein levels, while in HT-1080 cells PMA, concanavalin A, or overexpression of MT-MMP-1 did not significantly enhance the level of the 60-kDa protein. Instead, an immunoreactive, proteolytically processed 43-kDa form was observed, and its appearance correlated to 72-kDa gelatinase processing activity. Thus 72-kDa gelatinase activation, while enhanced by MT-MMP-1 expression, needs additional co-operating factors.

Base Sequence↗

PubMed1996

Laminin chains in the basement membranes of human thymus.

In recent studies, the alpha 2 chain of laminin (Ln) has been suggested to be the only laminin alpha chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16-to 18-week thymus presented a bright immunoreactivity for Ln alpha 1, alpha 3, beta 1, beta 3 and gamma 1 chains but not for alpha 2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln beta 2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin alpha 1, alpha 3, beta 1, beta 3 and gamma 1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin alpha 2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunoreactivity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and beta 4 integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.

Animals↗

PubMed1996

Laminins, tenascin and type VII collagen in colorectal mucosa.

The distribution of different laminin polypeptides, type VII collagen and tenascin has been studied in adult and foetal colorectal mucosa by using the indirect immunofluorescence technique. Immunoreactivity for laminin alpha 1 chain was located to basement membranes of epithelia, muscularis mucosae, and blood vessels, respectively in different segments of adult colon and rectum. Laminin beta 1 and gamma 1 chains were additionally expressed in lamina propria. Laminin alpha 2 chain was also found in lamina propria around the pericryptal fibroblasts. Immunoreactivity for laminin beta 2 chain was restricted to basement membranes in the muscularis mucosae and arteries. Laminin alpha 3 and beta 3 chains, suggestive for laminin-5, were confined especially to surface epithelial basement membranes. Immunoreactivity for type VII collagen was confined to basement membrane of surface epithelium in a punctate manner, while that for tenascin was seen slightly more broadly in the basement membrane zone and also in the muscular layer. The distribution of laminin chains in 16-week-foetal colon mostly resembled that of corresponding adult tissue, although immunoreactivities for laminin alpha 2 and beta 2 chains were lacking. Type VII collagen and the high molecular weight isoform of tenascin were also absent from the foetal colon. The results show that the basement membrane of the surface epithelium of colon and rectum express the components of epithelial adhesion complex, laminin-5 (alpha 3-beta 3-gamma 2) and type VII collagen, resembling in this respect small intestine and stomach while laminin-2 (alpha 2-beta 1-gamma 1) appears to be associated with pericryptal fibroblasts, and laminin-1 (alpha 1-beta 1-gamma 1) widely in most basement membranes.

Adult↗

PubMed1995

Tenascin and fibronectin isoforms in human renal cell carcinomas, renal cell carcinoma cell lines and xenografts in nude mice.

We studied the expression of tenascin (Tn) and isoforms of fibronectin (Fn) in human renal cell carcinomas (RCC) and oncocytomas, in RCC cell lines and in their s.c. implanted xenografts in nude mice. In well-differentiated RCCs and oncocytomas extra-domain A (EDA)-Fn and Tn immunoreactivities were confined to the basement membranes and blood vessels, while in less-differentiated RCCs they were also widely seen in the stroma, correlating with the morphological differentiation of the tumor. Expression of EDB-Fn and oncofetal (onc)-Fn was very scarce in most of the RCCs and oncocytomas. Western blotting results demonstrated the predominance of the M(r) 190,000 Tn subunit in most RCCs. Among the 4 RCC cell lines, 3 showed Tn in the extracellular matrix. As xenografts, they formed moderately or poorly differentiated tumors, with abundant Tn. Three of the RCC cell lines also showed secretion of EDA-Fn and 2 of them secretion of onc-Fn and EDB-Fn into the culture medium, while in xenografts there was a strong expression of all Fn isoforms. In xenografts, the expression of Tn closely recapitulated that seen in clinical tumors and in the cell lines in vitro, while the expression of Fn isoforms in cultured cells and their xenografts was highly discordant.

Animals↗

PubMed1995

Calcium ionophores decrease pericellular gelatinolytic activity via inhibition of 92-kDa gelatinase expression and decrease of 72-kDa gelatinase activation.

To understand the roles of intracellular calcium levels on gelatinase/type IV collagenase expression, we analyzed the effects of calcium ionophores on the expression of 92- and 72-kDa gelatinases (MMP-9 and MMP-2) in human fibrosarcoma cells (HT-1080). Calcium ionophores ionomycin and A23187 reduced the levels of pericellular gelatinolytic activity in both untreated and phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-alpha (TNF alpha)-stimulated cells as determined by degradation of radiolabeled gelatin. Gelatin zymography and immunoblotting revealed a dose-dependent decrease in the levels of secreted 92-kDa gelatinase, which was paralleled by a decrease of its mRNA. Treatment of cells with thapsigargin caused similar decreases of 92-kDa gelatinase mRNA and protein. The decrease of 92-kDa gelatinase expression was due to lower transcription rate as determined by transfection assays with 92-kDa gelatinase/luciferase construct. The expression of 72-kDa gelatinase was only slightly decreased by ionophores. Treatment of HT-1080 cells with PMA, TNF alpha, or concanavalin A resulted in the conversion of 72-kDa gelatinase proenzyme to its presumed 64- and 62-kDa active forms as determined by gelatin zymography and immunoblotting. Simultaneous treatment with the ionophores or thapsigargin resulted in inhibition of PMA-induced gelatinase activation. The expression of membrane-type matrix metalloproteinase, a potential activator of 72-kDa gelatinase, was not affected by ionophores. The results indicate that calcium ionophores decrease gelatinolysis by repressing both the expression of 92-kDa gelatinase and the activation of the 72-kDa gelatinase.

Calcimycin↗

PubMed1995

Human mast cell chymase and leukocyte elastase release latent transforming growth factor-beta 1 from the extracellular matrix of cultured human epithelial and endothelial cells.

Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-beta 1 (TGF-beta 1) to extracellular matrices and its release and activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-beta 1, its propeptide (beta 1-latency-associated protein), and latent TGF-beta-binding protein and incorporated latent TGF-beta 1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-beta 1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-beta 1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized M(r) 25,000 TGF-beta 1 but did not dissociate high M(r) latent TGF-beta 1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-beta 1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-beta from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.

Amnion↗

PubMed1994

Cyclosporin A enhances cytokine and phorbol ester-induced fibroblast collagenase expression.

Cyclosporin A is successfully used in the treatment of scleroderma, a condition with excessive deposition of collagen in the dermis. Cultured human dermal fibroblasts were used as a model to study the effects of cyclosporin A on metalloproteinase expression and activity. Fibroblasts were treated with collagenase inducing agents, phorbol 12-myristate 13-acetate (PMA), cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and the calcium ionophore A23187 in the presence of cyclosporin A under serum-free conditions, and alterations in metalloproteinase expression were studied by Northern hybridization and immunoblotting analyses, and assays for collagenolytic activity. Induction of collagenase expression by PMA and cytokines was enhanced severalfold by 1-10 microM cyclosporin A. Treatment of cells with cyclosporin A alone caused only a minor increase in collagenase mRNA levels. The secretion of immunoreactive collagenase protein and the level of p-aminophenylmercuric acetate activatable collagenase activity were increased by PMA and further enhanced by cyclosporin A. The expression of the other metalloproteinases stromelysin-1, 92-kD gelatinase, and 72-kD gelatinase or metalloproteinase inhibitor TIMP-1 were not affected by cyclosporin A. Time dependence analysis of the expression of the mRNAs for c-jun and junB indicated that the induction of these genes persisted significantly longer in cells treated with both PMA and cyclosporin A than in cells treated with PMA alone. Enhanced induction of collagenase mRNA may thus result from prolonged AP-1 activity. The results indicate that cyclosporin A potently enhances the expression of collagenase in dermal fibroblasts.

Blotting, Northern↗

PubMed1994

Okadaic acid-elicited transcriptional activation of collagenase gene expression in HT-1080 fibrosarcoma cells is mediated by JunB.

Okadaic acid (OA) is a novel, non-phorbol ester-type tumor promoter, which is a specific inhibitor of protein phosphatases 1 and 2A. Treatment of human fibrosarcoma HT-1080 cells with OA resulted in induction of collagenase and stromelysin-1 mRNA levels, while mRNA levels for tissue inhibitor of metalloproteinases-1 were enhanced to a lesser extent. Induction of collagenase and stromelysin-1 mRNA levels was dependent on protein synthesis. Exposure of HT-1080 cells to OA resulted in marked and persistent induction of junB, junD, and c-fos mRNA levels up to 24 h, while c-jun mRNA levels were only slightly elevated. In transiently transfected HT-1080 cells, OA-elicited activation of a 3.8-kilobase collagenase promoter/reporter gene construct was entirely dependent on junB expression, as determined by cotransfection with a junB antisense expression construct. Overexpression of JunB in HT-1080 cells enhanced collagenase promoter activity by 10-fold, and OA augmented trans-activation of collagenase promoter by c-Jun and JunB. The results indicate that induction of collagenase gene expression by OA is mediated by enhanced JunB expression, as well as enhanced trans-activating capacity of AP-1 complexes containing c-Jun and JunB. These results also suggest that selective overexpression of junB may enhance invasive and metastatic potential of neoplastic cells.

Collagenases↗

PubMed1992

Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin.

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.

Cell Line↗

PubMed1992

Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator.

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.

Cell Line↗

PubMed1991

Complete structure of the human gene for 92-kDa type IV collagenase. Divergent regulation of expression for the 92- and 72-kilodalton enzyme genes in HT-1080 cells.

The complete structure of the human gene for 92-kDa type IV collagenase was determined. Two overlapping genomic clones spanning 26 kilobases (kb) of genomic DNA were shown to contain the entire 7.7-kb structural gene together with 15 and 3.5 kb of 5'-end and 3'-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene. Exons 5, 6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in exon 9 which also codes for sequences with homology to the other metalloproteinases. The initiation site for transcription was determined by primer extension analysis. Sequencing analysis of 599 base pairs of the 5'-end flanking region showed that the promoter does not have a TATA motif, but a TTAAA sequence at position -29 to -25. A CAAT motif was not observed but there was one GC box. Two putative 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response elements, that might serve as binding sites for the transcription factor AP-1 and a consensus sequence of a transforming growth factor beta 1 (TGF-beta 1) inhibitory element were found in the promoter region. Gelatinase assay of enzyme secreted by cultured human fibrosarcoma cells (HT-1080) revealed only low levels of 92-kDa type IV collagenase activity, whereas considerable activity of the 72-kDa enzyme was present. Northern hybridization analysis confirmed these findings. Treatment of the HT-1080 cells with TPA resulted in induction of the secretion of 92-kDa type IV collagenase activity. This induction could not be significantly inhibited by concomitant incubation with TGF-beta 1. TPA and TGF-beta 1 did not markedly affect the activities of the 72-kDa enzyme. The activities of the secreted 92- and 72-kDa enzymes by HT-1080 cells correlated with the amounts of mRNA as estimated by Northern analyses.

Amino Acid Sequence↗

PubMed1991

Enhancement of vitronectin expression in human HepG2 hepatoma cells by transforming growth factor-beta 1.

Liver cells are considered the principal source of plasma vitronectin. The human hepatoma cell line HepG2 produces vitronectin into its culture medium. In the current work we have analyzed the regulation of vitronectin by transforming growth factor-beta 1 (TGF beta 1) in this hepatoma cell line by Northern hybridization, polypeptide and immunoprecipitation analyses and compared the response to another TGF beta-regulated gene, plasminogen activator inhibitor (PAI-1). Rabbit antibodies raised against human plasma-derived vitronectin were used in immunodetection. Polypeptide and immunoprecipitation analyses of the medium and cells, as well as immunoblotting analysis of the cells and their extracellular matrices, indicated enhanced TGF beta 1-induced production and extracellular deposition of vitronectin. Accordingly, TGF beta 1 enhanced the expression of vitronectin mRNA at picomolar concentrations (2-20 ng/ml) as shown by Northern hybridization analysis. Comparison of the temporal TGF beta induction profiles of vitronectin and PAI-1 mRNAs showed that vitronectin was induced more slowly but the vitronectin mRNAs persisted longer. In addition, platelet-derived and epidermal growth factors had an effect on vitronectin expression, but it was of lower magnitude. TGF beta 1 enhanced the expression of PAI-1 but, unlike previous reports, epidermal growth factor did not have any notable effect on PAI-1 in these cells. The results indicate that TGF beta 1 is an efficient regulator of the production of vitronectin by HepG2 cells and that PAI-1 and vitronectin are not coordinately regulated. In addition, with affinity purified antibodies to vitronectin receptor, we observed strong enhancement of the alpha subunit of the receptor in response to TGF beta 1. These effects of TGF beta are probably involved in various processes of the liver where matrix induction and controlled pericellular proteolysis is needed, as in tissue repair.

Blood Proteins↗

PubMed1991

Growth factors in the regulation of plasminogen-plasmin system in tumor cells.

Growth regulatory polypeptides, which act in an autocrine or paracrine fashion, are increasingly implicated in the control of pericellular proteolysis. Representatives of major growth factor families, like EGF, PDGF, IGF, FGF, IL, and TGF beta, and in addition, TNFs have effects both on cell proliferation and proteolytic events. Some of them participate in the control of proteolytic events by affecting pericellular PA activity. These factors regulate the synthesis, secretion, and activity of both PAs and their inhibitors in a cell and factor-specific manner. Interestingly, most of these affect simultaneously the secretion of both PAs and their inhibitors, sometimes concomitantly. In addition to PAs, growth factors modulate secretion of collagenases, transin, and stromelysin, and their respective inhibitors, TIMP. The balance of pericellular proteolytic activity is regulated according to the nature and interaction of various growth factors. Pericellular proteolysis can be modulated by growth factors at different levels. Several growth factors are able to regulate the amount and composition of the extracellular matrices. This, in turn, may affect the interactions of certain growth factors with the pericellular matrix structures. Altered structure of the matrix due to excessive proteolytic activity may thus limit the amount and activity of matrix-associated growth factors. Several growth factors exist in latent forms, and activation of these growth factors often requires proteolytic processing. A regulatory loop is thus formed where active growth factors affect the secretion of proteolytic enzymes and thus the concentrations of active ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Endopeptidases↗