Heat shock of rabbit synovial fibroblasts increases expression of mRNAs for two metalloproteinases, collagenase and stromelysin

Two metalloproteinases, collagenase and stromelysin, are produced in large quantities by synovial fibroblasts in individuals with rheumatoid arthritis. These enzymes play a major role in the extensive destruction of connective tissue seen in this disease. In this study, we show that heat shock of monolayer cultures of rabbit synovial fibroblasts increases expression of mRNA for heat shock protein 70 (HSP-70), and for collagenase and stromelysin. We found that after heat shock for 1 h at 45 degrees C, the mRNA expression for HSP-70 peaks at 1 h and returns to control levels by 3 h. Collagenase and stromelysin mRNA expression is coordinate, reaching peak levels at 3 h and returning to control levels by 10 h. The increase in mRNA is paralleled by an increase in the corresponding protein in the culture medium. 3 h of heat shock at a lower temperature (42 degrees C) is also effective in inducing collagenase and stromelysin mRNAs. Concomitant treatment with phorbol myristate acetate (PMA; 10(-8) or 10(-9) M) and heat shock is not additive or synergistic. In addition, all-trans-retinoic acid, added just before heat shock, prevents the increase in mRNAs for collagenase and stromelysin. Our data suggest that heat shock may be an additional mechanism whereby collagenase and stromelysin are increased during rheumatoid arthritis and perhaps in other chronic inflammatory stress conditions.     

Biosynthesis of tissue inhibitor of metalloproteinases by human fibroblasts in culture. Stimulation by 12-O-tetradecanoylphorbol 13-acetate and interleukin 1 in parallel with collagenase

Biosynthesis of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) by human fibroblasts in culture has been characterized by functional assays, immunoprecipitation, and immunocytochemistry with a monospecific antiserum. As determined by radiolabeling with [35S]methionine, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the secreted form of TIMP had an Mr of 29,000, whereas the form associated with the cell layer had an Mr of 24,000. Unstimulated human lung fibroblasts (HFL-1) secreted TIMP at the rate of approximately 2 micrograms/10(6) cells/24 h, and normal foreskin fibroblasts (HS 27) and skin fibroblasts from a patient with Hurler's disease (GM 1391) secreted TIMP at 0.3 and 0.2 micrograms/10(6) cells/24 h, respectively. Secretion of TIMP was stimulated up to 10-fold by treating the cells with 20-100 ng/ml of 12-O-tetradecanoylphorbol 13-acetate or 10 units/ml of human interleukin 1. In the stimulated HFL-1 cells, TIMP accounted for 0.03-0.09% of the total [35S]methionine incorporated into protein, and 0.3-0.8% of the [35S]methionine in secreted protein. Although TIMP accounted for a relatively small proportion of total protein synthesis of the fibroblasts, greater than 80% of untreated and greater than 95% of stimulated fibroblasts synthesized TIMP, as determined by indirect immunofluorescence. The treatments of the human fibroblasts that increased TIMP secretion also induced synthesis and secretion of proenzyme forms of collagenase, indicating that degradative enzymes and their controlling inhibitors may be synthesized in parallel under certain conditions.     

Collagenase expression and endogenous activation in rabbit synovial fibroblasts stimulated by the calcium ionophore A23187

Collagenase is synthesized and secreted by stimulated rabbit fibroblasts as a proenzyme that must be proteolytically cleaved to yield catalytically active species. The calcium ionophore A23187 has provided new insights into the regulation of collagenase activation cascade by living cells. A23187, at concentrations of 10-40 ng/ml, induced expression of collagenase and stromelysin mRNA and the secretion of procollagenase of 57 and 53 kDa and prostromelysin of 51 kDa. Interestingly, it also stimulated activation of procollagenase to active forms of 47 and 43 kDa. The concentrations and treatment times required for induction of gene expression and activation indicated that they were independent events. Active collagenase constituted up to 16% of the total collagenase present in medium conditioned by A23187-treated cells. When grown on a collagen substrate, A23187-treated cells degraded collagen in a spatially localized manner. In cells treated with agents that induce procollagenase only, collagenase was localized in the perinuclear Golgi area; however, in A23187-treated cells, collagenase was located in widely dispersed granules, suggesting different intracellular pathways for collagenase before, during, and after activation. Addition of serine, thiol-, and metalloproteinase inhibitors with A23187 to rabbit fibroblasts inhibited conversion of procollagenase to its active form to varying degrees, suggesting that enzymes in these classes are involved in a cascade of proteolytic events leading to collagenase activation.     

Basic calcium phosphate crystals cause coordinate induction and secretion of collagenase and stromelysin.

Synovial fluid basic calcium phosphate crystals (BCP) are often found in severely degenerated joints. Crystalline BCP is a growth factor stimulating fibroblast mitogenesis and acting as a competence factor similar to platelet-derived growth factor. In human fibroblasts (HF), the synthesis of collagenase and stromelysin is coordinately induced after stimulation with a variety of cytokines and growth factors. We sought to determine whether BCP, like other growth factors, might induce proteases that would damage articular tissue. Northern blot analysis of mRNA for collagenase and stromelysin in HF stimulated with BCP was performed. Secreted enzymes were analyzed by immunoblot using a monoclonal antibody to collagenase and by immunoprecipitation using a polyclonal antibody to stromelysin. Stromelysin activity was confirmed using casein substrate gels. A significant, dose-dependent accumulation of collagenase and stromelysin message was evident after 4 h and continued for at least 24 h in BCP-stimulated cultures. Forty-nine and 54 kD proteins immunoreacting with collagenase antibody were identified in the conditioned media (CM) from BCP-stimulated cultures while 50 and 55 kD proteins were identified by immunoprecipitation with stromelysin antibody. Collagenase activity was increased significantly in the CM from BCP treated cells; casein substrate gels showed casein degrading bands at molecular weights consistent with stromelysin. BCP stimulates coordinate induction of collagenase and stromelysin which may mediate the joint destruction associated with these crystals.     

SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway

SPARC (osteonectin/BM40) is a secreted protein that modifies the interaction of cells with extracellular matrix (ECM). When we added SPARC to cultured rabbit synovial fibroblasts and analyzed the secreted proteins, we observed an increase in the expression of three metalloproteinases--collagenase, stromelysin, and the 92-kD gelatinase-- that together can degrade both interstitial and basement membrane matrices. We further characterized the regulation of one of these metalloproteinases, collagenase, and showed that both collagenase mRNA and protein are upregulated in fibroblasts treated with SPARC. Experiments with synthetic SPARC peptides indicated that a region in the neutral alpha-helical domain III of the SPARC molecule, which previously had no described function, was involved in the regulation of collagenase expression by SPARC. A sequence in the carboxyl-terminal Ca(2+)-binding domain IV exhibited similar activity, but to a lesser extent. SPARC induced collagenase expression in cells plated on collagen types I, II, III, and V, and vitronectin, but not on collagen type IV. SPARC also increased collagenase expression in fibroblasts plated on ECM produced by smooth muscle cells, but not in fibroblasts plated on a basement membrane-like ECM from Engelbreth-Holm-Swarm sarcoma. Collagenase was induced within 4 h in cells treated with phorbol diesters or plated on fibronectin fragments, but was induced after 8 h in cells treated with SPARC. A number of proteins were transiently secreted by SPARC-treated cells within 6 h of treatment. Conditioned medium that was harvested from cultures 7 h after the addition of SPARC, and depleted of residual SPARC, induced collagenase expression in untreated fibroblasts; thus, part of the regulation of collagenase expression by SPARC appears to be indirect and proceeds through a secreted intermediate. Because the interactions of cells with ECM play an important role in regulation of cell behavior and tissue morphogenesis, these results suggest that molecules like SPARC are important in modulating tissue remodeling and cell-ECM interactions.     

Uncoordinate regulation of collagenase,stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: Selective enhancement of collagenase gene expression in human dermal fibroblasts in culture

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E₂ (PGE₂) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01–1.0 μg/ml) of PGE₂ led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE₂ had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE₂ did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE₂ by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-α, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.     

Coregulation of collagenase and collagenase inhibitor production by phorbol myristate acetate in human skin fibroblasts

Phorbol myristate acetate (PMA), a tumor promotor known to stimulate collagenase production in fibroblasts and endothelial cells, was examined with regard to its ability to regulate the expression of the collagenase inhibitor secreted by human skin fibroblasts. Confluent human skin fibroblasts were incubated with concentrations of PMA ranging from 10(-11) to 10(-7) M, and the conditioned medium was analyzed by enzyme-linked immunosorbent assay for both immunoreactive collagenase and collagenase inhibitor. PMA stimulated the production of both collagenase and collagenase inhibitor in several cell lines to maximal rates that were very similar, 300 to 350 vs 230 to 330 pmol 10 micrograms DNA-1 48 h-1, respectively. Due to differences in the basal levels of expression of these proteins, such rates reflected a two- to sevenfold stimulation in collagenase production, in comparison to a more uniform two- to threefold enhancement in inhibitor synthesis. Production of inhibitor was 50% of maximal at 7 X 10(-9) M and maximal at 10(-7) M phorbol. This concentration-dependent effect was very similar to that observed for collagenase expression. Total protein synthesis by the phorbol-conditioned cells, as studied by incorporation of [3H]leucine into newly synthesized protein, was not significantly increased, nor was cellular DNA content. The onset of the effect of PMA on inhibitor production occurred between 4 and 8 h, was maximal by 8 h, and continued undiminished for at least another 64 h. After the first 8 h, inhibitor production continued at a roughly constant rate of approximately 10 pmol 10 micrograms DNA-1 h-1. Interestingly, following the removal of phorbol from culture medium, such fibroblasts continued to produce increased quantities of inhibitor protein for at least 72 h. Metabolic labeling studies in which fibroblasts were exposed to [3H]leucine followed by immunoprecipitation using inhibitor-specific antibody suggested that stimulation of inhibitor production by PMA was mediated via an increased synthesis of new inhibitor protein. Therefore, in response to the tumor promoter, PMA collagenase and collagenase inhibitor expression by human skin fibroblasts appear to be coregulated.     

Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression

We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.     

Collagenase is a major gene product of induced rabbit synovial fibroblasts

We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.     

Cholesterol flux between cells and high density lipoprotein. Lack of relationship to specific binding of the lipoprotein to the cell surface

The bidirectional flux of unesterified cholesterol between cells and high density lipoprotein (HDL) was studied in relationship to the binding of HDL to cells. At 100 micrograms at HDL protein/ml, the rate constant for cholesterol efflux from rat Fu5AH hepatoma cells is 3 X 10(-3)/min (t1/2 for efflux of 3.9 h), whereas efflux from GM3468 human fibroblasts is 0.075/4 h (equivalent to a t1/2 for efflux of 37 h). The relatively slow efflux of cholesterol from fibroblasts in comparison to rat hepatoma cells was observed previously with micellar and vesicular phospholipid-containing acceptors, which promote efflux by a mechanism involving the diffusion of cholesterol in the aqueous phase between the plasma membrane and the acceptor particles. When plotted against the logarithm of HDL concentration, the isotherms for efflux are centered at 300 and 100 micrograms of HDL protein/ml with the hepatoma cells and fibroblasts, respectively. These concentrations are 8-150 times greater than the corresponding values for Kd of specific HDL binding (2 and 12 micrograms of protein/ml, for hepatoma cells and fibroblasts, respectively). The treatment of HDL with tetranitromethane reduces the lipoprotein's affinity for specific cell-surface binding sites by 80-90%. However, at HDL concentrations of 5-60 micrograms of protein/ml, this treatment does not significantly inhibit cholesterol efflux from hepatoma cells, and inhibits efflux from fibroblasts an average of about 15%. Over the same range of concentrations, nitration alters influx by amounts less than 30% in the two cell types. These effects on flux do not parallel the reduced affinity of nitrated HDL for specific cell-surface binding sites. In summary, the present results do not support the concept that cholesterol transfer is facilitated by the specific cell-surface binding of HDL, but are consistent with the aqueous diffusion model of cholesterol transfer between cells and lipoproteins.     

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.     

Gene expression during 3T3-L1 adipocyte differentiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation.

The mouse 3T3-L1 fibroblastic cell line rapidly differentiates to an adipocyte phenotype when post-confluent cells are treated for 48 h in fetal calf serum-containing medium supplemented with 1 microM dexamethasone (D), 0.5 mM methylisobutylxanthine (M) and 10 micrograms/ml insulin (I). D and I act synergistically to commit the cells to differentiate 24-48 h after initiating treatment, and this is blocked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. In order to identify cellular proteins involved in the differentiation process we analyzed differentiating 3T3-L1 cells using two-dimensional electrophoresis on large format gels. We observed changes in over 300 proteins during differentiation (over 100 within 5 h of initiating differentiation) and many of these are also changed at the level of mRNA (by analysis of in vitro translation products). About 75% of the initial changes were maximally induced by treatment with a combination of M and I, while no more than 10 proteins and their corresponding mRNAs were maximally induced by D within 3.5 h. Another 10 proteins were synergistically regulated by the combination of all three agents (DMI) within 3.5 h. Additional species were induced at later times. Five of these were synergistically induced by treatments that lead to differentiation, were first expressed at elevated levels during commitment and remained elevated in fully differentiated adipocytes. One or more of these proteins could well have a functional role in the commitment to and/or expression of the adipocyte differentiation program.     

DNA breaks and repair in the mouse leukemia L1210 cells exposed to three different types of interstrand DNA cross-linkers

DNA breaks and repair in mouse leukemia L1210 cells treated with 3 different types of cross-linkers, mitomycin C (MMC), 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) and SN-07 (a macromolecular antibiotic), were studied. Measured in D37 values, MMC gave the highest number of cross-links per lethal 'hit' directly after the 1-h treatment in the alkaline elution assay, followed by ACNU and SN-07. A good dose-response increase in induced interstrand DNA cross-linking frequency was observed in cells treated with 2.5-10 micrograms/ml MMC and with 10-100 micrograms/ml ACNU for 1 h with and without 24-h post-incubation. After 6-h post-incubation, the highest frequency of cross-linking was observed in cells treated with 2.5 micrograms/ml MMC and 30 micrograms/ml ACNU, while cross-link production continued in the cells treated with SN-07 for 12-h post-incubation. No significant increase in DNA breaks was observed in cells treated with MMC throughout 24-h post-incubation. The highest frequency of single-strand DNA breaks in cells treated with ACNU was observed immediately after the treatment and they disappeared after 6-h post-incubation. After 24-h post-incubation, a marked enhancement of the DNA breaks was observed in cells treated with SN-07 and the cells contained double-strand DNA breaks also. RNA synthesis was not affected in the cells treated with 10 micrograms/ml MMC and slightly inhibited to 70% of control in those treated with 100 micrograms/ml ACNU, while DNA synthesis in both cells was significantly inhibited after 24-h post-incubation. By contrast, both RNA and DNA synthesis were completely inhibited in cells treated with 8.0 micrograms/ml SN-07.