Differential effects of interferon-gamma on metabolism of lipoprotein immune complexes mediated by specific human macrophage Fcgamma receptors.

The objectives were to determine whether there are differences in the mechanisms of lipoprotein metabolism associated with different FcgammaRs and how metabolism associated with FcgammaRs compares to that mediated by scavenger receptors (SRA). To analyze lipoprotein metabolism in a receptor-specific manner, bispecific antibodies were used to target low density lipoproteins (LDL) labeled with (125)I or [(3)H]cholesterol linoleate to FcgammaRI or FcgammaRIIA in human macrophages. Interferon-gamma (IFN-gamma), which stimulates expression of FcgammaRI while inhibiting expression of SRA, was used to help delineate differences in metabolism between each receptor. For each receptor, the total amount of lipoprotein degradation paralleled changes in receptor expression induced by IFN-gamma. In particular, while SRA-mediated degradation typically exceeded degradation mediated by FcgammaRI, in IFN-gamma-treated cells degradation associated with FcgammaRI and SRA was similar. Assay of [(3)H]cholesterol linoleate-labeled lipoproteins indicated that total uptake and hydrolysis of [(3)H]cholesterol linoleate was similar for each class of receptor, and inhibited by IFN-gamma. For FcgammaRI versus FcgammaRIIA, in the presence or absence of IFN-gamma, the [(3)H]cholesterol derived from FcgammaRIIA-mediated uptake was preferentially targeted for re-esterification to [(3)H]cholesterol oleate, in comparison to that resulting from hydrolysis of [(3)H]cholesterol linoleate incorporated by selective uptake. For SRA, the formation of [(3)H]cholesterol oleate, which was substantial in control cells, was significantly inhibited in the presence of IFN-gamma. We conclude that there may be differences in cholesterol trafficking with respect to lipoprotein immune complex metabolism mediated by different classes of FcgammaRs.

Transforming growth factor beta inhibitory element in the rabbit matrix metalloproteinase-1 (collagenase-1) gene functions as a repressor of constitutive transcription.

Transforming growth factor beta (TGF-beta) is a potent modulator of the extracellular matrix, enhancing collagen synthesis and regulating expression of several genes that encode the matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix. In this study, we explored the mechanisms whereby TGF-beta inhibits expression of the MMP-1 (collagenase 1) gene. We used transient transfection and gel mobility shift assays to characterize a TGF beta inhibitory element (TIE) at -249 bp in the rabbit MMP-1 promoter, which is also conserved at -246 bp in the human gene. This sequence shares homology to a previously identified TIE in the rat stromelysin-1 (MMP-3) promoter, where it is located at -709 bp. Mutational analyses and transient transfections indicate that MMP-1 TIE functions both as a constitutive repressor of MMP-1 gene expression and, in the presence of TGF-beta, as an antagonist of transcriptional induction by phorbol esters. c-Fos binds to the TIE in the rabbit MMP-1 promoter, along with other nuclear proteins, even in the absence of treatment with TGF-beta. However, the pattern of proteins binding to the TIE is altered in the presence of nuclear extracts from TGF-beta-treated cells, suggesting that TGF-beta leads to an alteration in protein/DNA interaction, with subsequent modulation of MMP-1 gene expression. We conclude that in the rabbit MMP-1 promoter, the TIE has dual functions as a repressor of basal transcription and as a mediator of the biologic effects of TGF-beta. Furthermore, these dual functions provide additional and subtle mechanisms for regulating MMP-1 gene expression under a variety of biological and pathological conditions.

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.

Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis.

Cancer mortality usually results from the tumor invading the local environment and metastasizing to vital organs, e.g. liver, lung, and brain. Degradation of the extracellular matrix is, therefore, the sine qua non of tumor cell invasion. this degradation is mediated mainly by MMPs, and thus, inhibition of MMP synthesis is a target for anticancer agents. Tumor cells must traverse both the basement membrane (type IV collagen) and the interstitial stroma (type I collagen). Therefore, we used scanning electron microscopy to examine the invasive behavior of several aggressive tumor cell lines, A2058 melanoma cells, and SCC and FaDu squamous cell carcinomas through these matrices; and we monitored the ability of all-trans retinoic acid and several RAR-specific ligands to block invasion. We demonstrate that several retinoids, which are specific RAR alpha, beta, or gamma agonists/antagonists, selectively inhibited MMP synthesis in the three tumor cell lines. However, there was not a common pattern of MMP inhibition by a particular retinoid. For instance, a RAR alpha antagonist suppressed MMP-1 and MMP-2 synthesis in the melanoma cell line, but not in the FaDu or SCC-25 cells. On the other hand, synthesis of MMP-1 and MMP-9 by the FaDu cells was affected hardly at all, while a RAR gamma antagonist reduced the levels of MMP-2. Only all-trans retinoic acid reduced MMP-1 synthesis in these cells. We postulate that the differences may be related to a differential pattern of RAR expression in each of these cells, and that the RARs expressed by each cell line may not be targets of these RAR specific compounds. All-trans retinoic acid is a pan ligand, binding to all three RARs and, therefore, may modulate gene expression more generally. We conclude that the power of these new ligands lies in their specificity, which can be directed towards modulating expression of certain RARs and, thus, of certain MMPs. By blocking MMP synthesis, retinoids may be effective in cancer therapy by decreasing tumor invasiveness.

A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription.

Matrix metalloproteinases (MMPs) facilitate cellular invasion by degrading the extracellular matrix, and their regulation is partially dependent on transcription. Binding sites for members of the Ets family of transcription factors are present within MMP promoters and are potent positive regulators. We report a single nucleotide polymorphism at -1607 bp in the MMP-1 promoter, where an additional guanine (G) creates an Ets binding site, 5'-GGA-3'. This polymorphism displays significantly higher transcription in normal fibroblasts and in melanoma cells than the 1 G polymorphism, and it binds substantially more nuclear extract and recombinant ETS-1. Analysis of control DNAs from the Center d'Etude du Polymorphisme Humain pedigrees reveals that this polymorphism is not a mutation, with a frequency of the 2 G polymorphism at 30%. In contrast, in eight tumor cell lines, this frequency increased to 62.5% (P < 0.0001). Thus, this MMP-1 polymorphism contributes to increased transcription, and cells expressing the 2 G polymorphism may provide a mechanism for more aggressive matrix degradation, thereby facilitating cancer progression.

The acute phase reactant serum amyloid A (SAA3) is a novel substrate for degradation by the metalloproteinases collagenase and stromelysin.

We found that the matrix metalloproteinases collagenase (MMP-1) and stromelysin (MMP-3) each has the ability to degrade a novel substrate, serum amyloid A (SAA3). SAA3 is a product of rabbit synovial fibroblasts stimulated with phorbol esters or interleukin-1, and it acts in an autocrine or paracrine manner to induce collagenase in both rabbit and human fibroblasts. Recombinant rabbit fibroblast procollagenase and human fibroblast prostromelysin were produced by baby hamster kidney (BHK) cells stably transfected with these genes, and latent enzyme was activated with aminophenylmercuric acetate (APMA). The Km for both enzymes was approximately 10 microM, and the Vmax for collagenase was approximately 6 pmol/minute/100 ng enzyme, while that for stromelysin was about 3-fold faster. Treatment of SAA3 with either enzyme generated a fragment of approx. 6 kDa that has the same amino terminus as the parent molecule, but this fragment was rapidly degraded. We have been unable to isolate C-terminal fragments, suggesting that the mature protein is cleaved at multiple sites and/or that the initial cleavage fragment is readily digested. The amino acid composition of the 6 kDa fragment suggests that the 14 kDa protein is cleaved at residues 50-57, a hydrophobic region that is conserved between rabbit SAA3 and human SAA1. We conclude that the ability of collagenase and stromelysin to degrade SAA3 broadens the repertoire of substrates for these matrix degrading enzymes, and we speculate that the presence of a feedback mechanism that can subvert the autocrine/paracrine stimulation of matrix-degrading enzymes may play a role in limiting matrix degradation during inflammatory conditions.

Serum amyloid A (SAA3) produced by rabbit synovial fibroblasts treated with phorbol esters or interleukin 1 induces synthesis of collagenase and is neutralized with specific antiserum.

We report that nucleic acid sequence analysis of a full-length cDNA clone for a rabbit serum amyloid A (SAA)-like protein has identified this protein as more closely related to SAA3 than to SAA1. SAA3 induced collagenase synthesis in rabbit synovial fibroblasts, and immune IgG raised against this SAA protein abrogated the induction. Using antisera to immunoprecipitate biosynthetically labeled 3H-SAA and 3H-collagenase from culture medium, we compared the levels of SAA and collagenase synthesized by cultures of rabbit fibroblasts at early passage (passages 3-6) with those synthesized by late passage cells (passage 16). Comparatively high levels of both proteins were produced constitutively by fibroblasts at low passage. With increasing passage, levels of both proteins drop so that by passage 16, constitutive production of SAA and collagenase was only approximately 15-20% that of passage 3 cells. Cells at low passage could be readily stimulated with phorbol myristate acetate (PMA) or interleukin 1 (IL-1) to synthesize increased amounts of both SAA and collagenase. In passage 5 cells treated with PMA, we detected increased SAA mRNA by 1.5 h and collagenase mRNA by 5 h. However, older passage cells were more refractory to stimulation and required longer induction times. We suggest that SAA3 may be expressed by fibroblasts at sites of acute inflammation or injury, and that elevated levels of SAA3 may signify "activated" fibroblasts which are already producing increased amounts of collagenase constitutively and which are predisposed to further stimulation.

Rabbit procollagenase synthesized and secreted by a high-yield mammalian expression vector requires stromelysin (matrix metalloproteinase-3) for maximal activation.

A chimeric gene composed of the mouse metallothionein promoter linked to the 5' end of the 9.1-kilobase pair rabbit procollagenase (matrix metalloproteinase-1) gene was stably transfected into baby hamster kidney (BHK) cells. Like the native protein, the recombinant procollagenase synthesized and secreted by these cells was the product of a 2.1-kilobase pair transcript which was translated into a procollagenase protein of 57 kDa, with a small amount of protein that co-migrated with the glycosylated form of the native protein at 61 kDa. The BHK cells expressed levels of recombinant procollagenase equal to or exceeding those of rabbit synovial fibroblasts stimulated with phorbol myristate acetate, where procollagenase mRNA may comprise 2% of the mRNA population. Although minimal (approximately 10%) collagenolysis was seen when the zymogen was activated with trypsin or an organ-omercurial compound, the expression of full collagenolytic activity of the recombinant protein depended on the presence of stromelysin (matrix metalloproteinase-3). Purified recombinant collagenase displayed a specific activity of 8,400 units/mg of enzyme (1 unit degraded 1 microgram of collagen/minute at 37 degrees C) when fully activated, which was accomplished by the specific cleavage of the Gln80-Phe81 bond of procollagenase by stromelysin. We conclude that 1) these stably transfected BHK cells represent a high yield source of recombinant mammalian procollagenase, 2) activation of procollagenase depends on the presence of stromelysin, and 3) recombinant procollagenase from this high yield source may be useful in future studies to elucidate the detailed mechanism(s) involved in the activation of this enzyme.

Autocrine induction of collagenase by serum amyloid A-like and beta 2-microglobulin-like proteins.

Two autocrine proteins of 14 and 12 kilodaltons that induce the synthesis of rabbit fibroblast collagenase were identified. The proteins were purified from serum-free culture medium taken from rabbit synovial fibroblasts stimulated with phorbol myristate acetate. The amino-terminal sequences of the 14- and 12-kilodalton species were approximately 60 to 80 percent homologous with serum amyloid A and beta 2 microglobulin, respectively. The polyacrylamide gel-eluted proteins retained the ability to induce collagenase synthesis in rabbit and human fibroblasts. These autocrine proteins may provide a means to modulate collagenase synthesis in normal remodeling as well as in inflammation and disease states.

Autocrine control of collagenase synthesis by synovial fibroblasts.

Fibroblasts respond to exogenous stimuli, such as Interleukin 1, phorbol esters, or crystals of monosodium urate monohydrate, by synthesizing and secreting large quantities of collagenase. Here we show that addition of exogenous stimuli results in the production of an autologous protein that is, itself, capable of inducing collagenase. This autocrine has been partially purified. Activity resides in a protein(s) with a pl of 5 or 8 and with Mr of approximately 15K. Conversely, conditioned medium taken from unstimulated cultures contains an inhibitor of collagenase synthesis. This protein, which has a Mr approximately 20-25k by HPLC gel filtration antagonizes collagenase synthesis induced by phorbol esters, exogenous parallel 1, and the autologous inducer. We conclude that synovial fibroblasts regulate collagenase synthesis via an autocrine mechanism that includes the synthesis of both an inducer and inhibitor. Both proteins are active at nanomolar amounts and may function as polypeptide hormones in regulating collagenase synthesis and, hence, connective tissue remodeling.