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Biomedical subjects

J Keski-Oja

Publications and source records attributed to J Keski-Oja.

At least 73 records · Page 4Linked to original sources
PubMed1992

Transforming growth factors-beta as regulators of cellular growth and phenotype.

An increasing number of cDNAs and polypeptides have been ascribed to the transforming growth factor-beta (TGF-beta) superfamily. It has become evident that this evolutionally well-conserved gene family has representatives in many organisms, from invertebrates to mammalians. As new members are being identified, the knowledge of their biological significance is derived not only from studies of their effects in various in vitro models, but also of their organismal expression during different stages of embryogenesis and morphogenesis. The major described functions of the TGF-beta and like proteins seem to be the regulation of cell proliferation, phenotype, and differentiation. Part of these effects may be attributed to changes in the adhesive properties of the cells and integrity of their extracellular matrices modulated by TGF-beta. Identification of the new members of the TGF-beta family, their biological effects, cell-surface receptors, and cell or tissue-specific expression will be invaluable in trying to understand the purpose of many seemingly alike multiregulators.

Animals↗
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↗
PubMed1990

TGF-beta 1 modulation of urokinase and PAI-1 expression in human bronchial epithelial cells.

Normal human bronchial epithelial (NHBE) cells respond to signals initiated by the binding of transforming growth factor-beta type 1 (TGF-beta 1) to its surface receptors by activating pathways that result in terminal squamous differentiation. By use of both normal and SV40 T-antigen-immortalized cells, it was found that treatment with TGF-beta 1 transiently increases mRNA levels for urokinase (uPA) and plasminogen activator inhibitor type 1 (PAI-1) approximately 5- and 50-fold, respectively, within 4 h. In NHBE cells, PAI-1 protein is increased by TGF-beta 1 in both extracellular matrix and medium. The net effect of TGF-beta 1 on plasminogen activator activity in the medium was a 50% reduction as measured by a caseinolytic assay. A T-antigen-immortalized bronchial epithelial cell line that does not undergo squamous differentiation in response to TGF-beta 1 but binds this growth factor did not respond to TGF-beta 1 by modulation of either uPA or PAI-1 expression. Comparison of human bronchial epithelial, pleural mesothelial, and lung fibroblastic cell strains indicated that the epithelial cells have a constitutively higher ratio of uPA to PAI-1 mRNA expression. These data suggest that modulation of pericellular proteolysis in bronchial epithelial cells in response to TGF-beta 1 represents a significant biological change in their pericellular environment. The induction of uPA and PAI-1 expression in human bronchial epithelial cells may be related to the ability of the cell to undergo squamous differentiation in response to TGF-beta 1. These observations identify specific changes in gene expression that may serve as markers for the differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchi↗
PubMed1990

Characterization and purification of a secreted plasminogen activator inhibitor (PAI-1) induced by transforming growth factor-beta 1 in normal rat kidney (NRK) cells: decreased PAI-1 expression in transformed NRK cells.

Type 1 transforming growth factor-beta (TGF beta 1) was found to be a potent inducer of the secretion of a 49,000 mol wt protein by normal rat kidney (NRK) cells. This protein was related to type 1 plasminogen activator inhibitor (PAI-1) on the basis of molecular mass, activity in the presence of sodium dodecyl sulfate, immunoprecipitation by antibodies to PAI-1, and N-terminal sequence analysis. PAI-1 levels in the conditioned medium of NRK cells were increased 5- to 11-fold when cells were incubated with picomolar concentrations of TGF beta 1 for 24 h, reaching a concentration of approximately 0.3 microgram/ml. The secreted PAI-1 was deposited in the NRK extracellular matrix as well as released into the culture medium. A spontaneously transformed NRK cell line was found to secrete 3-4 times less PAI-1, in the absence or presence of TGF beta 1, compared to the parent cell line, while PAI-1 secretion in Kirsten sarcoma virus-transformed NRK cells was almost completely abrogated. A novel purification procedure was established, which results in the isolation of highly active and detergent-free TGF beta 1-induced PAI-1.

Amino Acid Sequence↗
PubMed1989

Growth stimulation, altered regulation of epidermal growth factor receptors, and autocrine transformation of spontaneously transformed normal rat kidney cells by transforming growth factor beta.

The tumorigenic NRK-PT14 cell line requires exogenous epidermal growth factor (EGF), but has lost the requirement for transforming growth factor beta (TGF-beta) for anchorage-independent growth, compared to normal rat kidney (NRK) cells. Development of an optimized serum-free medium for the growth of these cells revealed that NRK-PT14 cells also exhibit a qualitatively altered sensitivity to exogenous type 1 TGF-beta, compared to NRK cells. EGF-induced serum-free monolayer growth of NRK-PT14 cells was stimulated 2-fold by TGF-beta under conditions where growth of NRK cells was inhibited by 67%. TGF-beta only stimulated the growth of NRK-PT14 cells when EGF was present and when EGF was added before TGF-beta. In addition, the stimulation of EGF-induced NRK-PT14 cell growth by TGF-beta was associated with a specific, reversible loss of the high-affinity subpopulation of EGF receptors from the surface of these cells. Treatment of NRK cells with TGF-beta resulted in an increase in this EGF receptor population. Finally, EGF-induced anchorage-independent growth of NRK-PT14 cells was shown to be dependent on secreted TGF-beta, demonstrating an autocrine role for TGF-beta in the transformed phenotype of these cells. Autocrine transformation of NRK-PT14 cells by TGF-beta may result directly from the acquisition of an altered (positive) sensitivity to this growth factor.

Animals↗
PubMed1989

Active and latent forms of transforming growth factor beta activity in synovial effusions.

We have evaluated the possible involvement of TGF-beta in rheumatoid arthritis by assay of 16 cell-free synovial fluids for the presence of its active and "latent" forms. Evidence has been obtained for TGF-beta-like activity in synovial effusions by four criteria: (a) TGF-beta receptor competition, (b) soft-agar colony formation of AKR-2B and NRK-49F indicator cells, (c) immunological neutralization of the biological activity, and (d) biochemical activation of a latent form.

Antigen-Antibody Reactions↗
PubMed1989

The cellular response to induction of the p21 c-Ha-ras oncoprotein includes stimulation of jun gene expression.

We have studied the effects of c-Ha-ras oncogene in mouse NIH 3T3 fibroblasts by DNA transfection and analysis of gene expression at the mRNA and protein level in a heat- and heavy metal-inducible model system. The human c-Ha-ras proto-oncogene and oncogene were cloned under the hsp70 heat-shock promoter. Clonal lines of cells with negligible basal expression of the hsp-c-Ha-ras oncogene construct were chosen on the basis of the inducibility of p21c-Ha-ras protein and several transformation parameters. We demonstrate that the expression of ornithine decarboxylase (ODC) mRNA is enhanced approximately 4-6 h after the induction of the p21c-Ha-ras oncoprotein. This increase was reversible upon cessation of c-Ha-ras mRNA and protein synthesis, while constitutively elevated ODC was characteristic for stably c-Ha-ras-transformed cells. The high-level expression of ODC in ras-transformed cells was insensitive to tumour promoter stimulation. A similar mRNA induction by c-Ha-rasVal-12 was also observed for two other serum- and tumour promoter-regulated genes associated with the transformed phenotype: transin (stromelysin) and the glucose transporter. This prompted us to examine also potential changes in the expression of the serum- and tumour promoter-induced transcription factor genes junB and c-jun after induction of the hsp--c-Ha-ras construct. The junB mRNA was enhanced approximately 10-fold and the c-jun oncogene mRNA to a lesser degree in the hsp--c-Ha-ras-transfected cells after zinc activation of the hsp70 promoter. These effects were not seen in similarly treated control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Transforming growth factor-beta does not alter interleukin-1 expression in cultured human macrophages.

Transforming growth factor-beta (TGF beta) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI-1) by cultured cells. TGF beta appears also to have various immunoregulatory effects, suppressing both T- and B-cell activities. It has been proposed that it might increase the expression of interleukin-1 (IL-1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGF beta in IL-1 production we have now quantitated the effect of this factor on the production of biologically active IL-1 as well as IL-1 beta mRNA expression. The effect of TGF beta on IL-1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL-1 activity and mRNA levels were rapidly elevated by LPS but not by TGF beta. Culture fluids from monocytes treated with TGF beta alone or with TGF beta plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGF beta-treated cultures showed no activity in the molecular weight area of IL-1 (approx. 15 kD), while the supernatants from TGF beta plus LPS-induced cells contained IL-1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1989

Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation.

Transforming growth factor beta (TGF beta) is a multifunctional polypeptide that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF beta signal transduction in cells is unknown. We report here that an early effect of TGF beta is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF beta, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF beta, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TGF beta. The increase in jun mRNA occurred with picomolar TGF beta concentrations within 1 h of TGF beta stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-line levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF beta, evidently in a protein synthesis-independent fashion. The junB response to TGF beta was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF beta may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF beta.

Animals↗
PubMed1988

Regulation of mRNAs for type-1 plasminogen activator inhibitor, fibronectin, and type I procollagen by transforming growth factor-beta. Divergent responses in lung fibroblasts and carcinoma cells.

Transforming growth factor-beta (TGF beta) is a growth modulator which stimulates the growth of fibroblasts but acts as a strong growth inhibitor for cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins and of proteases and protease inhibitors by cultured cells. One important protease inhibitor whose production is affected rapidly by TGF beta is the type-1 plasminogen activator inhibitor (PAI-1). To investigate the relationships between PAI-1 and the extracellular matrix, we analyzed the regulation by TGF beta of PAI-1, fibronectin, and type I procollagen in malignant human lung carcinoma cells (A549) and in human lung fibroblasts (WI-38). The expression of the respective genes was examined by polypeptide analyses and by measurements of the steady-state levels of the corresponding mRNAs by Northern hybridization. The mRNA levels for PAI-1 were elevated rapidly by TGF beta in both cell lines. This induction occurred in the presence of cycloheximide and thus was not dependent on protein synthesis. In fact, the effects of TGF beta and cycloheximide on PAI-1 mRNA were additive. In contrast, the induction of fibronectin, beta-actin, and type I procollagen (synthesized only in WI-38 cells) was abrogated by cycloheximide. In general, the effects of TGF beta on the steady-state levels of mRNAs for fibronectin, actin, and type I procollagen mRNA were similar in the two cell types. However, the production of PAI-1 mRNA in response to TGF beta differed in the two cell types, being transient (peak within 5 h) in the carcinoma cells and more persistent in fibroblasts. Thus, the major difference in TGF beta regulation of PAI-1 mRNA between lung fibroblasts and carcinoma cells was the duration of the effect. These results, together with previous data, suggest that PAI-1 and plasminogen activators may be regulated oppositely by TGF beta. We therefore propose a model for TGF beta in the regulation of extracellular proteolytic activity.

Actins↗
PubMed1988

Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-beta.

Transforming growth factor-beta (TGF beta) is a regulator of cellular proliferation which can alter the proteolytic activity of cultured cells by enhancing the secretion of endothelial type plasminogen activator inhibitor and affecting the secretion of plasminogen activators (PAs) in cultured fibroblastic cells. We used the TGF beta-responsive malignant human lung adenocarcinoma cell line A549 to study the relationships between the known TGF beta-induced growth inhibition and the effects of TGF beta on the secretion of PA activity by A549 cells. PA activity was quantitated by caseinolysis assays, and characterized by urokinase mRNA analysis, immunoprecipitation, and zymography assays. PA-inhibitor production was observed in autoradiograms of SDS-polyacrylamide gels and reverse zymography assays. It was found that TGF beta enhanced the production of PA activity by these cells, in accordance with an enhancement of urokinase mRNA levels. A concomitant stimulation of type 1 PA-inhibitor production was also observed in A549 cells in response to TGF beta. In contrast to the observations of A549 cells, TGF beta caused a decrease in the expression of both urokinase and the tissue-type PA mRNA in human embryonic WI-38 lung fibroblasts indicating opposite regulation of the expression of PAs in these cells. The results suggest that TGF beta may play a role in the regulation of the invasive, proteolytically active phenotype of certain lung carcinoma cells.

Cell Line↗
PubMed1988

Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium.

Transforming growth factor-beta (TGF beta) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGF beta were studied using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model. Active TGF beta was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGF beta was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGF beta as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. In an effort to define more physiological means of TGF beta activation, the effects of some proteases were tested. Plasmin and cathepsin D were found to generate 25-kD bands corresponding to the active form of TGF beta as shown by immunoprecipitation analysis of radiolabeled cell-conditioned medium. Plasmin treatment of the medium resulted in activity that was quantitatively similar to that of mild acid treatment as measured by radioreceptor and soft agar assays. In addition, the plasmin-generated activity was inhibited by anti-TGF beta antibodies. Sequential treatments of AKR-MCA cell-conditioned medium with mild acid followed by plasmin or plasmin followed by mild acid gave activation comparable to either treatment alone. The data suggest that conditioned medium may contain at least two different pools of latent TGF beta. One pool is resistant to mild acid and/or plasmin and requires strong acid or alkali treatment for activation. A second pool is activated by mild pH change and/or plasmin. Activation of this form of latent TGF beta may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGF beta-binding protein complex.

Animals↗