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Biomedical subjects

C M Overall

Publications and source records attributed to C M Overall.

At least 37 records · Page 2Linked to original sources
PubMed1999

Assessment of a novel screening test for neutrophil collagenase activity in the diagnosis of periodontal diseases.

BACKGROUND: Increased levels of active neutrophil collagenase (MMP-8) in the gingival crevicular fluid (GCF) are associated with progressive periodontitis. The measurement of this enzyme in GCF could facilitate diagnosis. However, assays with sufficient sensitivity to detect collagenase in whole-mouth GCF currently use radiolabeled substrates and require several days to complete. To provide more rapid analyses of collagenase activity that are better adapted to clinical studies, we developed and validated a novel assay (soluble biotinylated-collagen assay: SBA) based on chemiluminescent detection of biotinylated collagen digestion products. METHODS: The concordance of the novel SBA assay with a radioactive collagen substrate assay was assessed by parallel analyses of enzyme from 35 neutrophil preparations and from 41 samples of GCF from periodontitis patients, followed by Pearson correlation analysis. To test whether the assay appropriately measured MMP-8 activity, enzyme activity was assessed after incubation with specific collagenase blockers. We examined the diagnostic utility of the SBA in cross-sectional and longitudinal analyses of 125 patients with adult periodontitis, 5 patients with early-onset periodontitis, 1 edentulous patient, and in 32 control patients without periodontitis. RESULTS: The assay detected <56 pg collagen degraded/hour/microl sample, which is comparable to the most sensitive radioactive assay. The total assay time was 22 hours and reproducibility on replicate measurements was high (r = 0.96). In direct comparisons of MMP-8 activity in GCF with enzyme from peripheral blood neutrophils using the SBA and radioactive assays, there was a high correlation (r = 0.97). As expected, EDTA and TIMP-1 and -2, known inhibitors of MMP-8, completely blocked enzyme activity with this assay. Cross-sectional and longitudinal analyses of GCF showed that MMP-8 activity was >18-fold higher in severe periodontitis than in stable periodontitis and decreased to <25% of pretreatment levels following therapy. Based on measurements of collagenase activity in different disease groups, we estimated a value of 80 nano units as a threshold for severe periodontitis. CONCLUSIONS: These results indicate that active MMP-8 is detected in GCF by a novel assay that is specific, simple, rapid, and reproducible and which may facilitate diagnostic discrimination between stable and progressive lesions.

Adult↗
PubMed1998

The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation.

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.

Antibodies↗
PubMed1998

Expression of matrix metalloproteinases (MMP-1 and -2) and their inhibitors (TIMP-1, -2 and -3) in oral lichen planus, dysplasia, squamous cell carcinoma and lymph node metastasis.

Although matrix metalloproteinases (MMPs) are among the potential key mediators of cancer invasion, their involvement in premalignant lesions and conditions is not clarified. Therefore, we studied, using in situ hybridization, immunohistochemistry and zymography the expression and distribution of MMP-1 and -2, and their tissue inhibitors (TIMPs -1, -2 and -3) in oral squamous cell carcinomas (SCC) and lymph node metastases as well as in oral lichen planus, epithelial dysplasias and normal buccal mucosa. In oral SCC and lymph node metastasis, MMP-1 mRNA was detected in fibroblastic cells of tumoral stroma. In two out of ten carcinomas studied, the peripheral cells of neoplastic islands were also positive. MMP-2 mRNA expression was noted in fibroblasts surrounding the carcinoma cells, and no signal in carcinoma cells was detected. A clear TIMP-3 mRNA expression was seen in stromal cells surrounding the neoplastic islands in all SCCs and lymph node metastases studied. TIMP-1 mRNA was detected in some stromal cells surrounding the neoplastic islands, whereas the mRNA expression for TIMP-2 was negligible. On the other hand, expression of MMPs and TIMPs was consistently low in oral epithelial dysplasias, lichen planus and normal mucosa. In certain epithelial dysplasias and lichen planus, MMP-1 and -2 mRNA expressions were detected in few fibroblasts under the basement membrane zone, but normal mucosa was completely negative. In SCC and lymph node metastasis, a detectable immunostaining for MMP-1 in stromal cells and in some carcinoma cells was observed. MMP-2 immunoreactivity was detected in the peripheral cell layer in neoplastic islands and in some fibroblast-like cells of tumoral stroma. Immunostaining for TIMP-3 was detected in stromal cells surrounding the neoplastic islands. A weak positive staining for TIMP-1 was located in tumoral stroma, whereas the immunostaining for TIMP-2 was negative. Using zymography, elevated levels of MMP-2 and MMP-9 were observed in carcinoma samples in comparison with lichen planus or normal oral mucosa. Our results indicate that the studied MMPs and TIMPs are clearly up-regulated during invasion in oral SCC. However, there was also a clear, although weak, up-regulation of the expression of the MMPs but not TIMPs in some of the lichen planus and dysplastic lesions.

Blotting, Western↗
PubMed1998

A novel organ culture method to study the function of human odontoblasts in vitro: gelatinase expression by odontoblasts is differentially regulated by TGF-beta1.

Odontoblasts cannot be cultured by traditional cell culture methods, thus restricting in vitro studies. Here we present an organ culture method for human odonto-blasts that utilizes the pulp chamber as a culture crucible. Crowns of human third molars were dissected, pulp was gently removed, and the odontoblasts attached to and in the walls of the pulp chambers were cultured in serum-free OPTI-MEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separately. Cell content and morphology were analyzed by SEM, and the removed pulps were examined by light microscopy. Proteins secreted into the medium with or without TGF-beta1 supplementation were metabolically labeled with [35S]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymography to analyze the effect of TGF-beta1 on gelatinase A and B expression. SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-free and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion until day 6 in both the odontoblast and pulp cultures, with no marked differences between TGF-beta1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B expression was always low and decreased with time in culture. However, gelatinase B levels were markedly increased upon TGF-beta1 treatment of cells and remained high to day 7. The results suggest that this method provides a novel technique for the study of human odontoblasts in vitro and that odontoblasts can be cultured even in serum-free conditions.

Cells, Cultured↗
PubMed1997

A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.

Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.

Animals↗
PubMed1997

Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2.

The binding properties of the newly described tissue inhibitor of metalloproteinases-4 (TIMP-4) to progelatinase A and to the COOH-terminal hemopexin-like domain (C domain) of the enzyme were examined. We present evidence for the first time of a specific, high affinity interaction between TIMP-4 and the C domain of human gelatinase A and show that TIMP-4 binds both progelatinase A and the C domain in a similar manner to that of TIMP-2. Saturable binding of recombinant C domain to TIMP-4 and to TIMP-2 but not to TIMP-1 was demonstrated using a microwell protein binding assay. The recombinant collagen binding domain of gelatinase A, comprised of the three fibronectin type II-like repeats, did not bind to TIMP-4, indicating that binding is mediated selectively by the C domain. Binding to TIMP-4 was of high affinity with an apparent Kd of 1.7 x 10(-7) M but slightly weaker than that to TIMP-2 (apparent Kd of 0.66 x 10(-7) M). Affinity chromatography confirmed the TIMP-4-C domain interaction and also showed that the complex could not be disrupted by 1 M NaCl or 10% dimethyl sulfoxide, thereby further demonstrating the tight binding. To verify the biological significance of this interaction, binding of full-length progelatinase A to TIMP-4 was investigated. TIMP-4 and TIMP-2 but not TIMP-1 bound specifically to purified TIMP-2-free human recombinant full-length progelatinase A and to full-length rat proenzyme from the conditioned culture medium of ROS 17/2.8 cells. Preincubation of the C domain with TIMP-2 was found to reduce subsequent binding to TIMP-4 in a concentration-dependent manner. Competition between TIMP-2 and TIMP-4 for a common or overlapping binding sites on the gelatinase A C domain may occur; alternatively TIMP-2 may prevent the binding of TIMP-4 by steric hindrance or induction of a conformational change in the C domain. We propose that the binding of progelatinase A to TIMP-4 represents a third TIMP-progelatinase interaction in addition to that of progelatinase A with TIMP-2 and progelatinase B with TIMP-1 described previously. This new phenomenon may be of important physiological significance in modulating the cell surface activation of progelatinase A.

Animals↗
PubMed1997

The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding. Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components.

The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. Recombinant C domain (rC domain) (Gly417-Cys631) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted. As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and MatrigelTM were not bound by the rC domain. Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen. Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound. However, binding to heparin and fibronectin (Kd, 1.1 x 10(-6) M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (Kd values, approximately 4-6 x 10(-7) M). The Ca2+ ion, but not the potential structural Zn2+ ion, were found to be essential for maintaining the binding properties of the protein. The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion chelator 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn2+ ion in the rC domain. In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca2+ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important in maintaining the structure and function of the domain.

Binding Sites↗
PubMed1996

Expression of 72-kDa gelatinase (matrix metalloproteinase-2) in the developing mouse craniofacial complex.

Tissue remodelling is an important feature during embryogenesis. Although the matrix metalloproteinases are believed to participate in these processes, the relation between matrix metalloproteinases and tissue remodelling during craniofacial morphogenesis remains unclear. The purpose of the study was to look for the presence of enzymes involved in extracellular matrix degradation during craniofacial morphogenesis. Protein expression of the matrix metalloproteinase, 72-kDa gelatinase (matrix metalloproteinase-2, gelatinase A, 72-kDa type IV collagenase) was studied by gelatine zymography and by indirect immunofluorescence with conventional and confocal microscopy. In the anterior region of the developing mouse face, 72-kDa gelatinase was labelled mainly in the tips and peripheral regions of the nasal and facial prominences. Upon contact and fusion of the prominences, the staining was intensely localized to the zone of the fusion and the tips and peripheral regions of the nasal prominences and the maxilla. The labelling of 72-kDa gelatinase was also present in the peripheral regions of the mandible, second branchial arch, and the face around the developing eye. However, during lens vesicle formation, the staining of 72-kDa gelatinase was absent in the invaginated lens ectoderm. After the lens had completely detached from the surface ectoderm, the staining was resumed in the corneal epithelium and mesenchyme. Gelatine zymography was used to confirm the presence of active and latent 72-kDa gelatinase in the developing mouse craniofacial complex. Collectively, these data indicate that 72-kDa gelatinase may play a significant part in localized tissue remodelling during craniofacial morphogenesis and the aberrant expression or function of the enzyme could be involved in causing facial abnormalities.

Animals↗
PubMed1995

Extracellular matrix binding properties of recombinant fibronectin type II-like modules of human 72-kDa gelatinase/type IV collagenase. High affinity binding to native type I collagen but not native type IV collagen.

72-kDa gelatinase/type IV collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens (gelatin). These proteolytic processes are required for pathologic tissue destruction and physiologic tissue remodeling. To investigate the molecular determinants of substrate specificity of this enzyme, a 21-kDa domain of 72-kDa gelatinase, consisting of three tandem fibronectin type II-like modules, was expressed in Escherichia coli. Similar to full-length 72-kDa gelatinase and the type II modules in fibronectin, the recombinant (r) fibronectin-like domain of this proteinase bound denatured type I collagen with an apparent Kd in the micromolar range. This domain, designated the collagen-binding domain (rCBD123), possesses at least two collagen-binding sites that can each be simultaneously occupied. rCBD123 also avidly bound elastin and denatured types IV and V collagens, but neither native types IV and V collagens nor fibronectin, all of which are substrates of the enzyme. Although 72-kDa gelatinase is involved in basement membrane degradation, rCBD123 also did not bind reconstituted basement membrane, laminin, or SPARC. Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBD123 also displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme. Since rCBD123 showed reduced binding to pepsin-cleaved type I collagen, either or both of the collagen telopeptide ends contain recognition sites for the 72-kDa gelatinase fibronectin-like domain. This was confirmed by the avid binding of rCBD123 to the alpha 1(I) collagen cyanogen bromide fragment CB2 from the NH2-terminal telopeptide. rCBD123 also bound alpha 1(I)-CB7, which encompasses the fibronectin-binding site, and to alpha 1(I)-CB8, a fragment not bound by fibronectin. Thus, type I collagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and fully exposed upon unfolding of the triple helix. The potential of the fibronectin-like collagen binding domain of 72-kDa gelatinase to bind extracellular matrix proteins may facilitate enzyme localization in connective tissue matrices.

Base Sequence↗
PubMed1995

Repression of tissue inhibitor of matrix metalloproteinase expression by all-trans-retinoic acid in rat bone cell populations: comparison with transforming growth factor-beta 1.

Retinoids and transforming growth factor-beta 1 (TGF-beta 1) reduce the transcriptional activation of matrix metalloproteinases (MMPs) and increase the expression of the specific tissue inhibitor of MMPs (TIMP-1) in fibroblasts. In contrast, all-trans-retinoic acid (retinoic acid) increases MMP expression in osteoblasts. Therefore, the mechanistic aspects of TIMP-1 regulation by retinoic acid in primary cultures of rat calvarial bone cell populations were studied and compared with those of TGF-beta 1 to determine if modulation of TIMP-1 would augment MMP expression. Retinoic acid was found to reduce TIMP-1 mRNA levels after 24 and 72 hr of culture by up to 60% in a dose-dependent manner. Maximal inhibition occurred at 10(-6) M retinoic acid with half maximal repression at approximately 5 x 10(-8) M. To determine the half life of TIMP-1 mRNA, the specific RNA polymerase II inhibitor DRB was added to cultures and the chase RNA analyzed by slot blots. TIMP-1 mRNA had a half life of approximately 14 hr and this was unaltered by retinoic acid treatment, suggesting that retinoic acid exerts its effects on TIMP-1 transcriptionally. When retinoic acid was added to cycloheximide-treated cultures TIMP-1 mRNA levels were reduced at 5 hr compared with controls. This showed that ongoing protein synthesis was not required to mediate the retinoic acid repression of TIMP-1 mRNA levels and supports the evidence that retinoic acid acts at the transcriptional level to reduce TIMP-1 expression. In contrast, TGF-beta 1 increased TIMP-1 mRNA levels by 3.5-fold at 24 hr to > 10-fold at 72 hr without alterations in mRNA stability indicating that transforming growth factor (TGF)-beta 1 also acts at the transcriptional level to upregulate TIMP-1 expression in bone cells. Thus, these studies have revealed that TIMP-1 regulation by retinoic acid is different in osteoblasts from other cells and that retinoic acid has the property of generating resorptive and formative cell phenotypes in a tissue-specific manner. In bone, reduced TIMP-1 expression would favor bone matrix degradation and bone resorption that is a characteristic action of retinoids.

Animals↗
PubMed1995

Papillon-Lefèvre syndrome associated early onset periodontitis: a review and case study.

A case of Papillon-Lefèvre syndrome that has been managed successfully for six years is reported. Papillon-Lefèvre syndrome is a rare form of early onset periodontitis that occurs at a rate of 1-3 per million. Diagnostic features include palmar-plantar hyperkeratosis and rapid periodontal destruction. Although the etiology of this syndrome is unknown, current theories on the nature of the underlying defect fall into three main categories: anatomical, bacterial and host response. Historically, Papillon-Lefèvre syndrome was thought to lead to the inevitable loss of both the primary and permanent dentitions. However, a recently proposed treatment involving antibiotic coverage, extraction of the primary dentition and a period of edentulism has been shown to be effective in maintaining the permanent dentition. Since treatment may begin prior to the eruption of the permanent dentition, early recognition of Papillon-Lefèvre syndrome is critical. Any young patient who exhibits palmar hyperkeratosis should be examined carefully for periodontal breakdown. Since the number of cases available for study is limited, referral of such individuals to University dental clinics may allow for a more specific analysis of immune or bacterial factors that may lead to a better understanding of this disease.

Aggregatibacter actinomycetemcomitans↗
PubMed1993

Specific alterations in the expression of alpha 3 beta 1 and alpha 6 beta 4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane.

Highly invasive cell subpopulations from a human prostate carcinoma cell line, PC-3, were selected for by allowing the parental PC-3 cells to invade through reconstituted basement membrane, Matrigel. These cells were collected, cultured and then selected further by repeated invasion through the in vitro invasion chamber. The invasive subpopulations (I-PC3 (2) and (3)) were found to be approximately 15-fold more invasive in vitro than the parental cells, had a distinct rounded morphology in culture, and proliferated more rapidly than the parental cells. When injected either subcutaneously or intraperitoneally into immunocompromised SCID mice, the I-PC3 cells were found to form tumors at the primary sites and to be highly invasive and metastatic. In contrast, the parental PC-3 cells formed tumors at the site of inoculation in these mice but failed to invade or metastasize. The I-PC3 cells attached equally as well as PC-3 cells to fibronectin, laminin, collagen type IV and vitronectin, but unlike the parental PC-3 cells these invasive variants failed to spread on any of these substrates. On Matrigel, the PC-3 cells became highly organized, whereas the I-PC3 cells remained rounded, clumped together and penetrated into the Matrigel. Biochemical analysis of the expression of adhesion proteins and integrins demonstrated that whereas the parental cells synthesized and secreted substantial amounts of fibronectin, the I-PC3 cell variants did not secrete any fibronectin. Although both PC-3 and I-PC3 cells expressed equivalent levels of cell surface alpha v beta 3, alpha 2 beta 1 and alpha 5 beta 1 integrins, the expression of the alpha 3 beta 1 integrin, which is expressed at very high levels on the parental PC-3 cells, was drastically reduced on the invasive I-PC3 cells. This decrease in expression of alpha 3 occurred also at the level of mRNA expression. Finally, whereas the PC-3 cells express alpha 6 beta 1, in the invasive I-PC3 cells the alpha 6 subunit was associated mostly with the beta 4 subunit. Since the alpha 6 beta 4 integrin is analogous to the A9 tumor antigen which is associated with aggressive human squamous cell carcinomas, the apparent overexpression of alpha 6 beta 4 may also participate in the aggressive behavior of these variant prostate carcinoma cells. Alterations in the expression of the alpha 3 beta 1 and alpha 6 beta 4 integrins may thus allow these cells to become more invasive, and lead to an increased propensity for metastasis.

Antigens, Surface↗
PubMed1992

Oncogenic consequences of down-modulating TIMP expression in 3T3 cells with antisense RNA.

We have used recombinant DNA technology to engineer a set of murine 3T3 cell lines that vary in the extent to which they express TIMP, tissue inhibitor of metalloproteinases, and we have found that both invasiveness and tumorigenic potential are conferred when TIMP production is impaired. These cell clones were produced by transfecting immortal Swiss 3T3 cells with plasmid constructs capable of expressing antisense TIMP RNA under the control of the mouse metallothionein promoter (Khokha & Denhardt, AntiCancer Res. 7: 653-660, 1987). The ability of these cells to invade the amnion membrane in vitro was dependent upon metalloproteinase expression (Yagel et al., JNCI 81: 768-775, 1989). Cells expressing TIMP at a reduced level acquired the ability to form tumors in nude mice (Khokha et al. SCIENCE 243: 947-950, 1989). These results suggest not only that TIMP controls the invasive character of the immortal 3T3 cell, but also that it determines cellular tumorigenic potential in the mouse. We presume that these phenotypes are conferred as a consequence of a net increase in extracellular matrix metalloproteinase activity resulting from the reduced quantities of TIMP secreted. The lag in formation of tumors by the cells down-modulated for TIMP production suggests that further changes in gene expression may be necessary. In support of this hypothesis, recent experiments indicate that the expression of genes encoding one or more matrix metalloproteinases is increased in cell lines derived from tumors that have developed from the engineered cells. Thus, in the immortal 3T3 cell line, TIMP has the properties of a tumor-suppressor gene, or anti-oncogene.

3T3 Cells↗
PubMed1992

Matrix metalloproteinases in periodontal tissue remodelling.

Inflammation of the periodontium leads to connective tissue degradation and eventual tooth loss. The regulation of matrix metalloproteinases (MMPs) has been studied to determine their role in these processes and also during tissue remodelling. Analysis of gingival crevicular fluid has revealed the presence of collagenase and gelatinase that, in the acute stages of periodontal disease, are derived predominantly from polymorphonuclear leukocytes. These MMPs appear to be intimately associated with tissue destruction since the levels of the active forms of these enzymes obtained from either crevicular fluid or mouthrinse samples correlate with tissue destruction and, therefore, provide a sensitive means of demonstrating disease activity. Transforming growth factor-beta, an important regulator of connective tissue remodelling, has been implicated in the rapid remodelling of periodontal tissues. TGF-beta promotes tissue matrix formation by stimulating both the synthesis of matrix proteins (collagen, fibronectin and SPARC) and proteinase inhibitors (TIMP, PAI-1) and by decreasing the synthesis of MMPs, but not the 72 kDa-gelatinase. Nuclear run-on analyses have shown that TGF-beta reduces collagenase and stromelysin synthesis by suppressing gene transcription without altering mRNA stabilities. In contrast, the transcription of the gelatinase and TIMP genes was increased by TGF-beta, which also increased gelatinase mRNA stability. Remodelling of alveolar bone involves interaction between osteoblasts and osteoclasts. Osteoblasts, under the influence of osteotropic hormones (vit D3, PTH and retinoic acid), produce MMPs which appear to function in the removal of soft tissue that precludes access of osteoclasts to the mineralized tissue surface. Rat osteoblastic cells produce MMPs with activity on native collagen, native collagen 3/4-fragments and gelatin and, in addition, two forms of TIMP activity. The 3/4-collagen endopeptidase, purified to apparent homogeneity, also has significant collagenase and gelatinase activities and an amino terminal sequence almost identical to human 72 kDa-gelatinase. The production of this enzyme was stimulated by TGF-beta, which suppresses bone resorption, and by osteotropic hormones which stimulate bone resorption, supporting a bifunctional role for the gelatinase in connective tissue remodelling. Although there is strong evidence for the involvement of MMPs in the resorption of bone and in the inflammation-mediated destruction of periodontal tissues, the role of MMPs in the remodelling of mature soft connective tissues remains equivocal.

Alveolar Bone Loss↗
PubMed1991

Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression.

The temporal aspects and mechanisms of the regulation of the matrix metalloproteinase (MMP) 72-kDa gelatinase/type IV collagenase (MMP-2) by transforming growth factor-beta 1 (TGF-beta 1) were investigated in early passage human gingival fibroblasts and compared with the regulation of the genes for collagenase (MMP-1) and TIMP, the tissue inhibitor of MMPs. Northern hybridization analyses revealed that 1.0 ng/ml TGF-beta 1 increased the abundance of MMP-2 mRNA/cell approximately 1.5-fold at 24 h, an increase similar to that observed in the level of [35S]methionine pulse-labeled MMP-2 at 24 h (1.9-fold). At 48 and 72 h, the increase in MMP-2 mRNA abundance remained elevated by 1.5-2.2-fold on a per cell basis whereas TIMP mRNA levels were elevated by up to 3.3-fold. In contrast, the relative levels of collagenase mRNA were reduced by 66-75%. The changes in the MMP-2, collagenase, and TIMP mRNA concentrations in response to TGF-beta 1 were blocked by cycloheximide indicating that protein synthesis was required to mediate the effects of TGF-beta 1 on these mRNA levels. TGF-beta 1 was also found to increase the half-life of the MMP-2 mRNA from approximately 46 to approximately 150 h but did not alter the stability of TIMP mRNA (t1/2 approximately 60 h). Nuclear run-off transcription assays revealed that MMP-2 gene transcription was increased approximately 5-fold 7 h following TGF-beta 1-treatment but returned to control levels by 24 h. In comparison, increased TIMP gene transcription was only detectable after 24 h whereas collagenase gene transcription, although low in control cells, was undetectable at 24 h. Gene transcription, mRNA levels, and message stability of the genes for the extracellular matrix proteins type I collagen and fibronectin were also increased by TGF-beta 1. Thus, the similarity in the control of MMP-2, alpha 1 (I) procollagen, and fibronectin expression at the transcriptional and post-transcriptional levels indicates that these genes may share regulatory elements. In comparison, TGF-beta 1 reduced the level of collagenase mRNA and increased the level of TIMP mRNA as a result of altered transcriptional activities, through pathways that required protein synthesis, and without changes in mRNA stability.

Blotting, Northern↗
PubMed1991

Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor beta. Comparison of transcriptional and post-transcriptional control with fibronectin and type I collagen.

Transforming growth factor beta (TGF-beta) and secreted protein acidic rich cysteine (SPARC) have been associated with the rapid remodeling of connective tissues that occurs in wound healing and developmental processes. To study the temporal and mechanistic aspects of TGF-beta-regulated extracellular-protein gene expression in human fibroblasts, confluent cells were pulse labeled for 30 min with [35S]methionine at various times following the single addition of 1.0 ng/ml TGF-beta. After a 4-h chase period, specific radiolabeled media proteins were isolated by either immunoprecipitation or affinity chromatography and quantitated. Stimulation of SPARC synthesis was first apparent 5 h after addition of TGF-beta, reached a maximum (3.5-fold increase) at 24 h and persisted for at least 96 h. A similar temporal response to TGF-beta was observed for the extracellular matrix proteins collagen and fibronectin. In contrast, TGF-beta induced a strong (greater than sixfold increase at 9 h after addition of TGF-beta), but transient stimulation of the synthesis of endothelial-type plasminogen activator inhibitor. Northern blot analysis showed that SPARC mRNA levels were increased by TGF-beta in parallel with increase in SPARC synthesis; a maximum 3.9-fold increase in SPARC mRNA being reached at 24 h. Similarly, the levels of both collagen and fibronectin mRNA were increased by TGF-beta treatment. In each case the stimulation of mRNA was blocked by the presence of the translation inhibitor, cycloheximide. Stability of SPARC mRNA (half-life of approximately 50 h) was not significantly altered by TGF-beta. In contrast, the stability of collagen and fibronectin mRNA were both increased in the presence of TGF-beta; the increased stability being pronounced in less dense cells. In addition to effects on stability, transcription of the collagen and fibronectin genes was increased 7 h after TGF-beta addition, but returned to control levels by 24 h. However, transcription of the SPARC gene was unaffected by TGF-beta at both time points and, together with the stability data, indicates that TGF-beta regulates SPARC expression via a nuclear post-transcriptional mechanism. Differential regulation of gene expression by TGF-beta in a precise temporal pattern via transcriptional and post-transcriptional pathways may be an important aspect of the response of fibroblast cells in a wound environment.

Animals↗