Transforming growth factor-beta suppresses the invasiveness of human fibrosarcoma cells in vitro by increasing expression of tissue inhibitor of metalloprotease

We have investigated the effects of TGF-beta on the ability of the human fibrosarcoma cell line, HT1080, to invade a reconstituted basement membrane (Matrigel) in vitro. Exposure of HT1080 cells to TGF-beta (1-10ng/ml) caused a dose-dependent inhibition of HT1080 cell invasion. Unexpectedly, TGF-beta (10ng/ml) significantly enhanced (10-fold) the mRNA expression of the 68-72kDa latent type IV collagenase. Zymogram analysis revealed a 7-fold increase in the 68-72kDa latent type IV collagenase concomitant with an increase in the activated form (62kDa). TGF-beta induced the 92kDa type IV collagenase to a lesser degree. HT1080 cells exposed to TGF-beta also produced more tissue inhibitor of metalloprotease (TIMP) at both the mRNA (10-fold) and protein levels (5-fold). Although TGF-beta induced both type IV collagenases and TIMP, the net collagenolytic activity in the conditioned media after invasion assay was reduced in the presence of TGF-beta. The data suggest that the inhibition of invasiveness is due, at least in part, to the increased TIMP expression. These data suggest that TGF-beta may play a role in tumor cell invasion by increasing the expression of TIMP.     

Discoordinate expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases-1 in rheumatoid human synovial fibroblasts. Synergistic effects of interleukin-1 and tumor necrosis factor-alpha on stromelysin expression.

Primary and passaged human synovial fibroblasts isolated from rheumatoid pannus were treated with recombinant interleukin-1 (IL-1) alpha or beta, tumor necrosis factor-alpha (TNF), or phorbol myristate acetate (PMA) to determine the effects of these stimuli on the relative expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases (TIMP). The steady-state mRNA levels for these genes and glyceraldehyde-3-phosphate dehydrogenase were determined on Northern blots. Immunoblot analyses of the conditioned media using monoclonal antibodies generated against recombinant human stromelysin, collagenase, or TIMP showed that protein levels reflected the corresponding steady-state mRNA levels. The results revealed that 1) stromelysin and collagenase were not always coordinately expressed; 2) IL-1 was more potent than TNF or PMA in the induction of stromelysin expression; 3) neither IL-1 nor TNF significantly affected TIMP expression; 4) PMA induced both metalloproteinase and TIMP expression; and 5) the combination of IL-1 plus TNF had a synergistic effect on stromelysin expression. Dose response and time course experiments demonstrated that the synergistic effect of IL-1 plus TNF occurred at saturating concentrations of each cytokine and lasted for 7 days. In summary, the ability of IL-1 and TNF to preferentially induce stromelysin and collagenase expression, versus TIMP, may define a pivotal role for these cytokines in the pathogenesis of rheumatoid arthritis.     

Induction and stimulation of 92-kDa gelatinase/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor alpha

Production of a 92-kDa gelatinase/type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) was investigated with human sarcoma cell lines. Among the cytokines and growth factors examined, only human recombinant tumor necrosis factor alpha (TNF alpha) induced and stimulated the proteinase with concomitant increase in TIMP expression, but matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) expression was unchanged. These data suggest that gene expression of the two metalloproteinases is regulated in a different fashion and TNF alpha may be important to allow cancer cells to be more invasive and metastatic.     

Transforming growth factor-beta 1 stimulates glucose uptake and the expression of glucose transporter mRNA in quiescent Swiss mouse 3T3 cells

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates the proliferation and differentiation of various types of animal cells. TGF-beta 1 stimulated glucose uptake and the expression of a brain-type glucose transporter (GLUT1) mRNA in quiescent mouse 3T3 cells. TGF-beta 1 also synergistically stimulated these activities when given together with calf serum, phorbol ester, fibroblast growth factor, or epidermal growth factor. The increases in glucose uptake and the GLUT1 mRNA level were induced by picomolar concentrations of TGF-beta 1 within 3 h of stimulation, reached a peak between 6 and 9 h, and then decreased gradually to basal levels before an increase in DNA synthesis. The stimulation of GLUT1 mRNA expression was completely abolished by actinomycin D, but was not affected by cycloheximide, suggesting that new protein synthesis was not required for the expression of GLUT1 mRNA. TGF-beta 1 had little mitogenic activity and did not affect serum-induced DNA synthesis in quiescent 3T3 cells. However, it stimulated DNA synthesis synergistically when given with fibroblast growth factor, epidermal growth factor, phorbol ester, or insulin. These results suggest that TGF-beta 1 mediates the stimulation of glucose uptake, GLUT1 mRNA expression, and DNA synthesis via a pathway(s) and cellular components distinct from those for other growth factors. The possible role of the TGF-beta 1-induced stimulation of glucose transport activity in the control of mouse fibroblast proliferation is also discussed.     

Modulation of the expression of matrix metalloproteinase and tissue inhibitors of metalloproteinases by TGF-beta1 and IGF-1 in primary human articular and bovine nasal chondrocytes stimulated with TNF-alpha.

Tumour necrosis factor-alpha (TNF-alpha) was able to promote collagen breakdown from bovine cartilage in explant culture. This release was dependent upon matrix metalloproteinases (MMPs) and could be prevented by transforming growth factor-beta1 (TGF-beta1) or insulin-like growth factor-1. Both growth factors reduced the expression and secretion of collagenase enzymes, and TGF-beta1 induced tissue inhibitor of metalloproteinase production. This study shows for the first time that these anabolic growth factors can protect cartilage against TNF-alpha-induced destruction.     

Epidermal growth factor (EGF)-nonresponsive variants of normal rat kidney cell line: response to EGF and transforming growth factor-beta

Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) (or TGF-alpha). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-beta treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-beta, whereas that of N-3 was significantly enhanced by TGF-beta alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-beta mRNA, but there appeared little expression of TGF-beta mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-beta enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.     

Regulation of vascular smooth muscle cell integrin expression by transforming growth factor beta1 and by platelet-derived growth factor-BB.

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.     

Inhibition of myogenic differentiation by fibroblast growth factor or type beta transforming growth factor does not require persistent c-myc expression

Skeletal muscle differentiation is accompanied by accumulation of the mRNA encoding the muscle isoenzyme of creatine kinase (MCK) and can be suppressed by serum components, fibroblast growth factor (FGF), or type beta transforming growth factor (TGF beta). Using the nonfusing myogenic cell line, BC3H1, the potential involvement of c-myc in growth factor-dependent inhibition of myogenesis was examined. Withdrawal of undifferentiated myoblasts from the cell cycle in medium with 0.5% serum was associated with a precipitous decline in expression of c-myc mRNA followed by induction of MCK mRNA. In 0.5% serum containing TGF beta, c-myc mRNA declined to a level identical to that in differentiated cells; however, MCK mRNA was not expressed. Exposure of quiescent differentiated cells to FGF or TGF beta caused disappearance of muscle-specific gene products and was accompanied by only transient low level induction of c-myc mRNA. These data indicate that persistent c-myc expression is not required for growth factor-mediated inhibition of myogenic differentiation.