Tissue Inhibitor Of Metalloproteinase-1 Gene Research Articles

Temporal and spatial expression of tissue inhibitors of metalloproteinases during the natural ovulatory cycle of the mouse.

The role of tissue inhibitors of metalloproteinases (TIMPs) in ovarian function has primarily been documented by studies that utilize hormone-primed animals. In this study, our objectives were to elucidate the spatiotemporal expression of individual TIMP genes during the natural ovulatory cycle, and to correlate these with specific biological events. Two models of spontaneous ovulation used were the murine estrous cycle and the first ovulation postpartum. Ovaries were collected from mice at diestrus, estrus, and metestrus, or at early and late proestrus, and from pregnant females on Days 17 and 18 of gestation (D17, D18) and within 24 or 48 h postpartum (PP1, PP2). We observed that TIMP-1 mRNA was elevated at early proestrus and D18 and was maximal at late proestrus and PP1. The TIMP-3 pattern was distinct from that of TIMP-1, maximal expression occurring at early proestrus and D17 and D18. In both models, TIMP-2 mRNA remained constant and at very low levels throughout ovulation. In situ hybridization localized TIMP-1 mRNA to the corpus luteum at D18 and PP1, and to oocytes at specific stages of follicular development. Expression of TIMP-1 in granulosa and thecal cells was not observed at any stage. Demonstrating a distinct distribution, TIMP-3 mRNA was localized to oocytes, thecal and granulosa cells of small and large follicles, and corpora lutea only at D17. These data suggest specific hormonal regulation of individual TIMP gene expression in the ovary associated with distinct physiological functions. We propose that in the natural ovulatory process, TIMP-1 is probably a factor that regulates corpus luteum regression while TIMP-3 is important in maintaining the structural integrity of the corpus luteum.

Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in rabbit aortic neointima after selective de-endothelialization

Altered TIMP-1 synthesis in the arterial wall may be important for the balance between metalloproteinases and their inhibitors, and thus contribute to dysregulated extracellular matrix metabolism in atherosclerotic lesions. To examine this, we cloned the rabbit TIMP-1 gene from aortic neointima, developed in response to a balloon-catheter induced de-endothelialization. The apparent homology of cDNA with TIMP-1 genes from several sources suggested that it is a rabbit form of TIMP-1. We examined the recombinant rabbit TIMP-1 expression in Escherichia coli using the pTrxFus expression system and the synthesis of the resulting soluble protein was confirmed by immunostaining with anti-TIMP-1. The TIMP-1 concentration in normal and de-endothelialized rabbit aortas was compared using Northern blot, Western blot and mRNA in situ hybridization techniques. We observed a significant increase of TIMP-1 expression in neointimal SMCs at both nucleic acid and protein levels, suggesting a role of TIMP-1 in injury-induced atherogenesis.

Inhibition of metastasis in human gastric cancer cells transfected with tissue inhibitor of metalloproteinase 1 gene in nude mice

Tissue inhibitors of metalloproteinases (TIMPs) act as negative regulators of matrix metalloproteinases (MMPs) that degrade extracellular matrix. We evaluated the metastatic ability of the highly metastatic human gastric cell line KKLS, and that of cells transfected with exogenous TIMP-1 gene by the orthotopic transplantation model in nude mice. KKLS was derived from human gastric cancer. Expression of mRNA for tissue inhibitor of metalloproteinase-1 (TIMP-1) was almost undetectable in KKLS cells. KKLS cells were transfected with exogenous TIMP-1 gene by the Chen-Okay-ama method. Two clones (KTCLs) that expressed different levels of TIMP-1 and neomycin-resistant KKLS (K-neo) were obtained. The KKLS cells and these transfectants were orthotopically transplanted into nude mice (murine stomach) and metastasis in the murine liver was detected. As a method of detecting metastasis, we used a DNA fragment (human beta-globin gene) specific to human tumor cells that have metastasized into the murine liver by polymerase chain reaction (PCR). Differences in tumor growth in the murine stomach were not observed between KKLS cells, K-neo cells, and the two transfectants expressing the different TIMP-1 levels (low, KTCL-1; high, KTCL-14). The KKLS cells and K-neo cells had undergone liver metastasis, as shown by PCR amplification of the human beta-globin gene fragment from the murine liver samples, since Week 1 after transplantation and the metastasis had grown exponentially; however, although KTCL-1 cells and KTCL-14 cells had undergone liver metastasis since Week 2, the metastasis had not grown. The average intensities of the amplified gene fragments from K-neo cells, KTCL-1 cells, and KTCL-14 cells in Week 4 after transplantation were 100%, 45%, and 18%, respectively, of the parenteral KKLS cells. TIMP-1 was suggested to act as a negative regulator of the metastasis. The present data is thought to be especially important because the mice in this study underwent orthotopic transplantation with a metastatic model.

Reduction-oxidation (redox) state regulation of extracellular matrix metalloproteinases and tissue inhibitors in cardiac normal and transformed fibroblast cells

Latent matrix metalloproteinases (MMPs) in normal myocardium are activated in end-stage heart failure. In vitro oxidized glutathione (GSSG) activates myocardial MMPs which contains a cysteine residue. In vivo GSSG induce the collagen lysis and cardiac dilatation. To assess whether thiol and non-thiol reducing agents have direct effect on the interstitial human heart fibroblast (HHF) proliferation and MMP expression, HHF and polyoma virus transformed fibroblast cells were cultured with or without the thiol-containing reduced (GSH) or oxidized (GSSG) glutathiones, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), and non-thiol ascorbic acid. After 100 micrograms/ml (approximately 0.3 mM) GSH or PDTC treatment the proliferative (synthetic) phenotype of transformed fibroblast cells was changed to quiescent (contractile) phenotype. Also, after GSH, PDTC, and ascorbic acid treatment the medium was then analyzed for MMP activity by zymography. The results indicate reduction in MMP expression in transformed fibroblast cells after GSH and PDTC treatments and no effect after ascorbic acid treatment. Based on reverse zymography, we observed the level of tissue inhibitor of metalloproteinase (TIMP) at a decreased level in transformed cells. The effect of the reducing agent at the gene transcription was measured by estimating mRNA (Northern blot analysis) of MMP and of TIMP in the cells that were cultured in medium in the presence and absence of GSH. These results indicate that GSH induces MMP-2 and MMP-1 expression in normal HHF and that GSH reduces MMP-2 and MMP-1 in transformed fibroblast cells. After the treatment, the TIMP-2 level was repressed in normal HHF and TIMP-2 level increased in transformed fibroblast cells. These events are dependent on the nuclear transcription factor activity on the collagenase promoter in normal HHF cells. On the other hand, in polyoma transform fibroblast cells these events are not dependent on this collagenase promoter. These results suggest that oxidative environment induces normal HHF cell proliferation, and the reducing agent decreases normal HHF cell proliferation by inducing MMP and repressing TIMP gene transcription. In transformed cells reducing agents inhibit MMP expression and increase TIMP levels, which suggests a role of antioxidants in preventing tumorigenesis.

The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities define the distinct TIMP gene family.

The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities define the distinct TIMP gene family.

Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts.

The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point in tissue remodeling. Previous studies have demonstrated elevated expression of the MMPs collagenase and stromelysin-1 by aged human diploid fibroblasts compared to early-passage cultures. We show here that aging cells display an altered response to transforming growth factor-beta 1 (TGF beta 1) that selectively affects MMP mRNA expression. In both young and old cells, phorbol myristoyl-13 acetate (PMA) induced the expression of transcripts of collagenase, stromelysin-1, gelatinase-B, TIMP-1, and TIMP-3. In young cells, TGF beta 1 reciprocally modulated PMA-induced MMP and TIMP gene expression leading to reduced levels of transcripts for the MMPs and augmented accumulation of TIMP-1 and TIMP-3 mRNAs. However, repressing effects of TGF beta 1 on collagenase, stromelysin-1, and gelatinase-B RNA expression were not apparent in old cells, though induction of the TIMP genes was unimpaired. By electrophoretic mobility shift analysis the nuclear transcription factors AP1 and serum response factor (SRF) showed reduced levels of DNA binding activities in old fibroblasts compared to young cells. A probe for the TGF beta-inhibitory element (TIE) gave equivalent levels of complexes with nuclear extracts from both types of cells, though of different mobilities. We conclude that the effects of TGF beta 1 on MMP and TIMP gene expression involve different cellular intermediaries, and suggest that altered composition or modification of TIE binding factors in aging cells may underlie the failure of TGF beta 1-mediated transcription repression. This mechanism may contribute to elevated constitutive expression of MMPs in old cells and to the connective tissue deterioration that accompanies the aging process.

Effect of retinoic acid in combination with platelet-derived growth factor-BB or transforming growth factor-β on tissue inhibitor of metalloproteinases and collagenase secretion from human skin and synovial fibroblasts

This report shows for the first time that platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta (TGF-beta) can interact in a synergistic manner with retinoic acid to stimulate the production of tissue inhibitor of metalloproteinases (TIMP) from human skin and synovial fibroblasts. When cells are treated with 1, 10, and 100 ng/ml of either of these growth factors in combination with 10(-5) M retinoic acid, this results in a dose-dependent synergistic induction of TIMP protein secretion which is greater than the additive effect of the agents by up to fourfold. These responses can be inhibited by the presence of specific neutralising antibodies to the growth factors, demonstrating that they are not the result of an experimental artefact such as contamination with bacterial endotoxin. The mechanisms of these synergistic responses may involve the induction of receptors for retinoic acid, PDGF, or TGF-beta or may result from synergistic effects on TIMP gene transcription. We have also found that retinoic acid potently down-regulates PDGF-BB-stimulated collagenase in both types of fibroblast and that the effect of PDGF-BB alone on collagenase secretion from skin fibroblasts is biphasic. Finally, this study reports that retinoic acid and TGF-beta do not act in an additive fashion to inhibit the production of collagenase from skin fibroblasts.

Synergistic Transcriptional Activation of the Tissue Inhibitor of Metalloproteinases-1 Promoter via Functional Interaction of AP-1 and Ets-1 Transcription Factors

The tissue inhibitor of metalloproteinases-1 (TIMP-1) is an inhibitor of the extracellular matrix-degrading metalloproteinases. We characterized response elements that control TIMP-1 gene expression. One contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. Adjacent to this is a binding site for Ets domain proteins. Although c-Ets-1 alone did not activate transcription from this element, it enhanced transcription synergistically with AP-1 either in the context of the natural promoter or when the sequence was linked upstream of a heterologous promoter. Furthermore, a complex of c-Jun and c-Fos interacted with c-Ets-1 in vitro. These results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation. Collectively, our results indicate that TIMP-1 expression is controlled by several DNA response elements that respond to variations in the level and activity of AP-1 and Ets transcriptional regulatory proteins.

Structure of the human TIMP-3 gene and its cell cycle-regulated promoter.

The gene encoding tissue inhibitor of metalloproteinases-3 (TIMP-3) is regulated during development, mitogenic stimulation and normal cell cycle progression. The TIMP-3 gene is structurally altered or deregulated in certain diseases of the eye and in tumour cells. A detailed knowledge of the TIMP-3 gene and its regulatory elements is therefore of paramount importance to understand its role in development, cell cycle progression and disease. In this study, we present the complete structure of the human TIMP-3 gene. We show that TIMP-3 is a TATA-less gene, which initiates transcription at one major site, is composed of five exons and four introns spanning a region of approximately 30 kb, and gives rise to three distinct mRNAs, presumably due to the usage of alternative polyadenylation signals. Using somatic cell hybrids the TIMP-3 locus was mapped to chromosomal location 22q13.1 We also show that the TIMP-3 5' flanking region is sufficient to confer both high basal level expression in growing cells and cell cycle regulation in serum-stimulated cells. While the first 112 bases of the promoter, which harbour multiple Sp1 sites, were found to suffice for high basal level activity, the adjacent region spanning positions -463 and -112 was found to be a major determinant of serum inducibility. These results provide an important basis for further investigations addressing the role of TIMP-3 in physiological processes and pathological conditions.

The Gene Structure of Tissue Inhibitor of Metalloproteinases (TIMP)-3 and Its Inhibitory Activities Define the Distinct TIMP Gene Family

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a critical role in extracellular matrix homeostasis. We have previously cloned human and mouse TIMP-3 cDNAs and mapped their chromosomal loci (Apte, S. S., Mattei, M-G., and Olsen, B. R. (1994) Genomics 19, 86-90; Apte, S. S., Hayashi, K., Seldin, M. F., Mattei, M-G., Hayashi, M., and Olsen, B. R. (1994) Dev. Dynam. 200, 177-197); the identification of TIMP3 mutations in Sorsby's fundus dystrophy has underscored the functional importance of TIMP-3. We now report that TIMP-3 is encoded by five exons spanning over 30 kilobase pairs of mouse genomic DNA. In the attribution of protein domains to specific exons, as well as exon structures, the Timp-3 and Timp-1 genes are similar, confirming the common evolutionary origin of the TIMPs and defining a distinct gene family. We have expressed human and mouse TIMP-3 in mouse NSO myeloma cells. In each case, an N-glycosylated 27-kDa protein was generated, that, like TIMP-1 and TIMP-2, inhibited collagenase-1, stromelysin-1, and gelatinases A and B. TIMP-3 and TIMP-1 inhibition were quantitatively similar, implying that all TIMPs are equally efficient in MMP inhibition. Instead, differential regulation of the TIMP genes or divergent C-terminal protein sequences may underlie distinct biological functions for each TIMP.

The Tissue Inhibitor of Metalloproteinases-3 Gene in Breast Carcinoma: Identification of Multiple Polyadenylation Sites and a Stromal Pattern of Expression

Tissue inhibitor of metalloproteinases-3 (TIMP3) is the third member of the TIMP family of proteins, believed to play a significant role in controlling extracellular matrix remodeling. Differential screening of a human breast carcinoma cDNA library using substracted and PCR-amplified cDNA probes identified a 4.6-kb TIMP3 cDNA, which was used for further cDNA library screenings, Northern blot hybridizations, and the synthesis of riboprobes for in situ RNA hybridization analyses. The 4.6-kb full-length TIMP3 cDNA contains 3.7 kb of 3'-untranslated sequence. Additional TIMP3 cDNAs subsequently identified were colinear with the original sequence, but revealed use of four different polyadenylation signals within the 3'-untranslated region, which accounted for the 4.6-, 2.7-, 2.5-, and 2.1-kb TIMP3 transcripts noted in this and in previous studies. In situ RNA hybridizations demonstrated that in breast carcinoma the TIMP3 gene was predominantly expressed by fibroblastic cells within the tumor stroma adjacent to cancer cells. TIMP3 transcripts were also strongly detected in fibroblastic decidual cells of pregnant endometrium. Modulating the length of the 3'-untranslated region may represent a mechanism by which TIMP3 gene expression is controlled in tissues. The strong expression of the TIMP3 gene by fibroblastic cells in breast carcinoma supports the importance of tumor stroma as a source of factors influencing human carcinoma growth and progression.

SORSBY??S FUNDUS DYSTROPHY

The authors studied the variable expressivity of the phenotype and molecular genetics of a South African family with the fundus appearance of Sorsby's fundus dystrophy. Seventeen members of a single family underwent examination. Five of these were affected. Clinical details of five other affected family members were obtained from their ophthalmologists. Patients were questioned about age of onset, history of night blindness, and color defects. Examination included fundoscopy, color testing, color photographs, and fluorescein angiography. Blood for molecular genetic analysis was taken from 23 family members. The age of onset of symptoms was 29 to 56 years, and the rate of progression was variable. Sudden decreased vision was associated with subretinal choroidal neovascularization. Slower visual decline was associated with central chorioretinal atrophy. Progressive retinal pigment epithelial atrophy and peripheral pigment clumping occurred in elderly relatives who lost ambulatory vision. DNA studies show linkage to the long arm of chromosome 22 and a point mutation in the tissue inhibitor of metalloproteinases-3 gene. This South African family presents with a dominantly inherited fundus dystrophy with clinical and molecular features compatible with Sorsby's fundus dystrophy.

Identification of the interleukin-6/oncostatin M response element in the rat tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter.

The rat tissue inhibitor of metalloproteinase 1 (TIMP-1) gene is expressed in rat hepatocytes, and this expression is up-regulated by interleukin 6 (IL-6). We report here the cloning of the 5' flanking region of the rat TIMP-1 gene and identification of an IL-6/oncostatin M (OSM) response element at -64 to -36 which functions in hepatic cells. Within this element we have identified two functional binding sites for transcription factors AP-1 (activatory protein-1) and STAT (signal transducer and activator of transcription). IL-6/OSM stimulation induces binding of a protein, identified as STAT3, to the IL-6/OSM response element, while binding of the AP-1 protein was constitutive. Binding sites for both AP-1 and STAT3 are necessary for full responsiveness of the TIMP-1 promoter to IL-6/OSM, as shown by deletion and mutation analysis. Furthermore, the entire IL-6/OSM response element conferred responsiveness onto a heterologous promoter, whereas this has not been observed when AP-1 and STAT elements were separately tested.

Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby's fundus dystrophy

The hereditary macular dystrophies are progressive degenerations of the central retina and contribute significantly to irreversible visual loss in developed countries. Among these disorders, Sorsby's fundus dystrophy (SFD), an autosomal dominant condition, provides an excellent mendelian model for the study of the genetically complex age-related macular degeneration (AMD), the most common maculopathy in the elderly. Recently, we mapped the SFD locus to 22q13-qter. This same region contains the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which is known to play a pivotal role in extracellular matrix remodeling. We have now identified point mutations in the TIMP3 gene in affected members of two SFD pedigrees. These mutations are predicted to disrupt the tertiary structure and thus the functional properties of the mature protein.

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 microgram/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-alpha, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.

Mechanisms of methotrexate action in rheumatoid arthritis. Selective decrease in synovial collagenase gene expression.

To measure the effect of methotrexate (MTX) treatment in rheumatoid arthritis (RA) on the expression of synovial collagenase, stromelysin, and tissue inhibitor of metalloproteinase 1 (TIMP-1) gene expression in a prospective study. Serial percutaneous synovial biopsies (pretreatment and after 3-4 months) were performed on the knees of 8 patients (7 with RA, 1 with seronegative arthritis) who were beginning oral MTX therapy. Synovial gene expression was determined by quantitative in situ hybridization using computer-assisted image analysis. After therapy, patients had decreased joint counts, morning stiffness, and erythrocyte sedimentation rates. Synovial inflammation in the biopsy tissues was slightly decreased after therapy. In situ hybridization on pretreatment and posttreatment frozen sections was performed to quantify synovial messenger RNA (mRNA) levels. Collagenase gene expression significantly decreased after MTX therapy (P = 0.006) even though cell density in the region was unchanged. TIMP-1 and stromelysin mRNA levels were not changed by MTX therapy. To study the mechanism of MTX action in vitro, MTX-treated and control fibroblast-like synoviocytes were stimulated with interleukin-1 beta (IL-1 beta). MTX did not alter collagenase or TIMP-1 mRNA levels after IL-1 exposure. MTX therapy decreases collagenase gene expression but not TIMP-1 or stromelysin gene expression in the synovium. This action is probably an indirect effect due to an alteration in the synovial cytokine milieu, rather than a direct effect on gene expression.

Quantitative detection of TIMP-3 promoter hypermethylation and its prognostic significance in esophageal squamous cell carcinoma

Tissue inhibitor of metalloproteinase-3 (TIMP-3) has an inhibitory effect on tumor development, growth and metastasis due to its interaction with matrix metalloproteinases (MMPs). We investigated the prognostic significance of TIMP-3 gene promoter methylation in esophageal squamous cell carcinoma (ESCC). TIMP-3 methylation was analyzed by MethyLight, a quantitative and methylation-specific PCR method. Hypermethylation was found in 9/51 (18%) surgically resected ESCC samples and was associated with reduced disease-free (p=0.0039) and overall survival (p=0.0047). Upon multivariate analysis, it was found to be an independent prognostic parameter for poor survival and was associated with earlier recurrence after surgery (p=0.0238), especially via pleural dissemination (p=0.001). Expression levels for TIMP-3 protein and for several MMPs were analyzed by Western blot analysis in 20 pairs of ESCC and adjacent normal tissue. The expression of MMP-2, -7 and -9 proteins in tumor tissue was significantly higher than in corresponding normal mucosa (p=0.0051, 0.0064 and 0.0004, respectively). In contrast, the expression of TIMP-3 protein in tumor tissue was significantly lower than in matched normal mucosa (p=0.0152). Tumors with TIMP-3 hypermethylation expressed lower levels of TIMP-3 protein compared to those without hypermethylation (p=0.0357). These results demonstrate that TIMP-3 hypermethylation is associated with lower TIMP-3 protein expression in ESCC and with poor patient survival due to a high frequency of recurrence by pleural dissemination.

Human glomeruli express TIMP-1 mRNA and TIMP-2 protein and mRNA.

Alterations in the balance between synthesis and degradation of extracellular matrix may result in glomerulosclerosis. The interaction between metalloproteinases and their inhibitors presumably modulates the rate of glomerular matrix degradation. We examined the gene expression of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in human glomeruli and TIMP-2 protein in tissue sections. Kidney tissue was obtained from adults undergoing nephrectomy for renal tumor (n = 9) or biopsy for nephrosis and renal failure (n = 1). Glomeruli were microdissected and subjected to reverse transcription. TIMP cDNAs were quantitated by competitive polymerase chain reaction assays. Five nephrectomy specimens had normal glomeruli and four had diffuse glomerulosclerosis. TIMP-1 and TIMP-2 cDNA levels, detected in glomeruli from all patients, were increased fourfold and threefold, respectively, in patients with glomerulosclerosis. The elevated TIMP cDNA levels could not be attributed to an increased number of glomerular cells. TIMP-2 protein was detected within normal and sclerotic glomeruli. In conclusion, both TIMP genes were expressed in normal glomeruli, and their level of expression was increased in glomerulosclerosis associated with renal carcinoma, suggesting that expression of these inhibitors may correlate with the development of sclerosis.

Inhibition of N-linked oligosaccharide processing in tumor cells is associated with enhanced tissue inhibitor of metalloproteinases (TIMP) gene expression.

Previous studies on tumor-cell glycosylation mutants and drugs which inhibit oligosaccharide processing suggest that expression of sialylated and highly branched complex-type N-linked oligosaccharides is required for efficient tumor-cell metastasis. These observations prompted the present investigation, in order to determine whether loss of sialylated and highly branched complex-type oligosaccharide in cellular glycoproteins might affect the expression of genes, particularly of genes which can influence the malignant phenotypes. Swainsonine, an inhibitor of Golgi alpha-mannosidase II, has previously been shown to inhibit invasion in vitro and reduces solid tumor in vivo. Metastatic sub-lines of the SP1 murine mammary carcinoma cells cultured in the presence of swainsonine for 48 hr showed approximately 3-fold enhancement of TIMP mRNA levels, while urokinase (uPA) transcripts remained unchanged. To determine whether swainsonine's effect on TIMP mRNA levels was related to inhibition of oligosaccharide processing, we examined somatic glycosylation mutants with processing defects which attenuate metastatic potential. The Golgi UDP-Gal transport defect in murine MDAY-D2 lymphoma cells, Chinese hamster ovary cells (CHO) and human MeWo melanoma cells (i.e., D35W25, Lec8, 3S5 cell lines, respectively) was associated with increased TIMP mRNA levels. A revertant of Lec8 showed a return to the wild-type levels of TIMP mRNA, consistent with a causal relationship between the glycosylation mutation and TIMP gene expression. Similarly, CHO and MDAY-D2 mutants defective in GlcNAc-TV (i.e., Lec4 and KBL-1 respectively), which also reduces metastatic potential, showed increases in TIMP transcript levels. Nuclear run-on assays showed that transcription of the TIMP gene was increased in cells where N-linked oligosaccharide processing was inhibited either by swainsonine or by a glycosylation mutation. The results suggest that cell-specific patterns of glycoprotein glycosylation in human, murine and hamster cell lines affects the transcription of select genes, including TIMP, which may influence the invasive phenotype.

Involvement of AP1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-1) transcription

Transcription of tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted protein that regulates the activities of the metalloproteinases, collagenase and stromelysin, is activated by serum growth factors. Transient transfection experiments have revealed several regions of cis-acting regulatory sequences involved in the response of the murine TIMP-1 gene to serum. One area is in the vicinity of the promoter, consisting of a non-consensus binding site (5′-TGAGTAA-3′ at −59/−53) for transcription factor AP1 and an adjacent 24 bp region of dyad symmetry that contains a PEA3-binding site. A second is an upstream region (−1020 to −780) that acts as an enhancer when linked to a heterologous promoter, and contains a consensus AP1 binding site (at −803/−797). Gel retardation assays revealed differences between nuclear factors in mouse C3H10T1/2 cells that bound to the TIMP(−59/−53)AP1 site and a consensus collagenase TRE (TPA-response element). The TIMP(−59/−53)AP1 site is a promiscuous motif that binds c-Fos/c-Jun AP1 translated in vitro and is an effective competitor for binding of nuclear AP1 factors to the consensus TRE, but in addition it binds factors that do not associate with the consensus TRE. The TIMP(−59/−53)AP1 motif and the dyad symmetry region stimulated expression from a thymidine kinase promoter in an additive fashion, and competition experiments showed that excess copies of these factor binding sites reduced expression from a reporter plasmid driven by the TIMP-1 promoter. Our data show that binding sites for AP1 and PEA3 transcription factors are involved in the regulation of TIMP-1 transcription, which suggests that the coordinated induction of TIMP-1, collagenase and stromelysin may be achieved through the actions of a shared set of nuclear transcription factors. However, the properties of the TIMP-1(−59/−53)AP1 motif likely reflect an additional type of transcriptional regulation restricted to TIMP-1.