Altered Expression Of Matrix Metalloproteinases Research Articles

Altered Expression of Matrix Metalloproteinases and Their Endogenous Inhibitors in a Human Isogenic Stem Cell Model of Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by a progressive movement disorder, psychiatric symptoms, and cognitive impairments. HD is caused by a CAG repeat expansion encoding a stretch of polyglutamine residues in the N-terminus of mutant huntingtin (mHTT) protein. Proteolytic processing of mHTT yields toxic fragments, which cause neurotoxicity and massive neuronal cell death predominantly in the striatum and cortex. Inhibition of mHTT cleavage reduces neuronal toxicity suggesting mHTT proteolysis contributes to HD pathogenesis. A previously conducted unbiased siRNA screen in our lab for known human proteases identified matrix metalloproteinases (MMPs) as modifiers of mHTT proteolysis and toxicity. To further study MMP activation in HD, isogenic HD, and control corrected (C116) neural stem cells (NSCs) prepared from HD patient-derived induced pluripotent stem cells were used to examine the role of MMPs and their endogenous inhibitors in this highly relevant model system. We found altered expression of MMP-2 and MMP-9 (gelatinases), MMP-3/10, and MMP-14, activity in HD-NSCs when compared to control C116-NSCs. Dysregulation in MMP activity was accompanied with concomitant changes in levels of endogenous inhibitors of MMPs, called tissue inhibitors of matrix metalloproteinases (TIMPs). Specifically, we observed decreased levels of TIMP-1 and TIMP-2 in HD-NSCs, suggesting part of the altered expression and activity of MMPs is due to lower abundance of these endogenous inhibitors. Immunofluorescence analysis revealed increased MMP/TIMP localization in the nucleus or aggregates of HD-NSCs, suggesting potential interaction with mHTT. TIMP-1 was found to associate with mHTT aggregates in discrete punctate structures in HD-NSCs. These events collectively contribute to increased neurotoxicity in HD. Previous characterization of these NSCs revealed transforming growth factor beta (TGF-β) pathway as the top dysregulated pathway in HD. TGF-β was significantly upregulated in HD-NSCs and addition of TGF-β to HD-NSCs was found to be neuroprotective. To determine if TGF-β regulated MMP and TIMP activity, C116- and HD-NSCs were exogenously treated with recombinant TGF-β. TIMP-1 levels were found to be elevated in response to TGF-β treatment, representing a potential mechanism through which elevated TGF-β levels confer neuroprotection in HD. Studying the mechanism of action of MMPs and TIMPs, and their interactions with mHTT in human isogenic patient-derived NSCs elucidates new mechanisms of HD neurotoxicity and will likely provide novel therapeutics for treatment of HD.

Altered proteolytic activity and expression of MMPs and aggrecanases and their inhibitors in Kashin-Beck disease.

Kashin-Beck disease (KBD) is a chronic, deforming endemic osteoarticular disease with altered metabolism of the cartilage matrix. Matrix metalloproteinases (MMPs), aggrecanases (ATAMTSs), and their inhibitors (TIMPs) play important roles in cartilage formation and matrix degradation. This study investigated these proteases and inhibitors in young KBD cartilage. The percentages of chondrocytes staining for MMP-1/-13 and MMP-generated DIPEN neoepitope, aggrecanase-generated ITEGE neoepitope in aggrecan in KBD patients were significantly higher than in controls. However, TIMP-1 was significantly less numerous than in controls in the superficial and middle zones of KBD samples, the percentage of chondrocytes staining for the TIMP-2 was significantly higher than in controls. Staining for MMP-1/-13 and, TIMP-1/-2 in KBD patients was prominent in the superficial zone and the middle zone of articular cartilage. Staining for ITEGE and DIPEN neoepitopes in KBD samples was prominent in the superficial zone and the middle zone of articular cartilage. The strongest staining for the MMP and aggrecanase-generated neoepitopes was adjacent to areas of chondronecrosis. These results indicated that KBD cartilage destruction depends on collagen- and aggrecan-degrading proteases such as collagenases (MMP-1/-13), as well as aggrecanases. Increased TIMP-2 level adjacent to necrotic areas suggest that attempted repair mechanism are also activated.

Pacing-induced cardiomyopathy: pathophysiological insights through matrix metalloproteinases

Pacing-induced ventricular dysfunction and pacing-induced cardiomyopathy (PiCMP) are recognized complications of chronic right ventricular pacing. Alterations in myocardial perfusion and sympathetic innervation contribute to the development of pacing-induced heart disease. However, it is unlikely that these are the only processes involved. Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the collagenous extracellular matrix and play a central role in left ventricular remodelling during the development of heart failure. While the pathophysiological mechanisms and altered MMP expression that occur in chronic pressure overload, ischaemic and non-ischaemic dilated cardiomyopathy have been defined, those that occur in the clinicalsetting of pacing-induced ventricular dysfunction and PiCMP have not been reported. Here we review the clinical epidemiology of pacing-induced ventricular dysfunction and discuss how data derived from animal models provide insight into how changes in MMP expression and function contribute to the development of PiCMP. The review concludes by exploring pacing strategies that may be used to prevent pacing-induced ventricular dysfunction.

Differential Expression of Matrix Metalloproteinases (MMPs) in Lung Cancer Metastasis

Metastasis accounts for most cancer related deaths. The common metastasis models utilize intravenous injection of tumor cells, bypassing the egression of malignant cells. We developed a unique metastasis model by in vivo passaging the Lewis lung carcinoma (3LL) cells and maintaining them in vivo with 7–11 days of intermittent culture. When injected subcutaneously, parental 3LL cells have minimal metastasis, while in vivo passaged 3LL cells (p‐ 3LL) show robust metastasis. To determine the differences between metastatic p‐3LL versus non‐metastatic 3LL, we compared the gene expression profiles by microarray analysis of RNA from these cells as well as the primary tumors from each. The array analysis revealed alteration in the expression of numerous genes previously known to be associated with tumor metastasis. The data also revealed several novel markers associated with increased metastasis including enhanced expression of two matrix metalloproteinases (MMPs), MMP‐9 and MMP‐12, and the decreased expression of MMP‐2. Given the role of MMPs in the remodeling of the extracellular matrix and cell motility, we further investigated the altered RNA and protein expression of MMPs in p‐3LLs and their tumors by real time PCR and Western blot analysis. Together, these suggest that these three MMPs may play an important role in lung cancer metastasis.

Altered Expression of Matrix Metalloproteinases and Tight Junction Proteins in Rats following PEMF-Induced BBB Permeability Change

ObjectiveTo investigate the expression of occludin, ZO-1, MMP-2, and MMP-9 in cerebral microvasculature following Pulse Electromagnetic Field (PEMF) induced BBB permeability change. MethodsSprague-Dawley rats were randomized into PEMF and sham exposed groups (n=8). After exposure to PEMF at 0.5, 1, 3, 6, and 12 h, BBB permeability was measured by Evans-Blue extravasation. The expression of occludin, ZO-1, MMP-2, and MMP-9 were detected by real-time quantitative reverse transcriptase PCR and western blotting. MMP-2 and MMP-9 activity were detected by EnzChek gelatinase assay. ResultsCompared with the sham group, PEMF exposure led to increased permeability of the BBB to EB, which was prolonged after exposure. BBB permeability became progressively more severe, and recovered at 6 h. The gene and protein expression of occludin and ZO-1 were significantly decreased, while MMP-2 and MMP-9 expression were significantly increased after exposure to PEMF. All levels of expression recovered 12 h following PEMF. ConclusionChanges to BBB permeability were related to the alteration expression of tight junction proteins and matrix metalloproteinase after exposure to PEMF.

The Role of Matrix Metalloproteinases in Diabetes Mellitus

Diabetes mellitus (DM) is a leading risk factor for cardiovascular disease that adversely affects multiple vascular components from early in its course. Current evidence implicates matrix metalloproteinases (MMPs) and their endogenous inhibitors in diverse pathways associated with the development and progression of diabetic microvascular complications. In diabetic nephropathy, altered MMPs expression contributes to extracellular matrix deposition and glomerular hypertrophy that eventually lead to proteinuria and renal insufficiency. In diabetic cardiomyopathy, MMPs participate in the breakdown of collagen and elastin, myocardial remodelling as well as the vulnerability of the coronary plaque. The development of diabetic peripheral arterial disease is mediated by the impaired angiogenesis caused by the activity of MMPs. Experimental data support an integral role of MMPs in cerebral circulation and stroke volume in diabetes. An excess of MMPs may contribute in poor diabetic wound healing. Future research should further clarify the role of MMPs within the pathophysiological substrate of diabetes, as well as potential therapeutic options.

Reduced NO signaling during pregnancy attenuates outward uterine artery remodeling by altering MMP expression and collagen and elastin deposition

Recent findings indicate that endothelial nitric oxide (NO) plays a key role in uterine artery outward circumferential remodeling during pregnancy. Although the underlying mechanisms are not known, they likely involve matrix metalloproteinases (MMPs). The goal of this study was to examine the linkage among NO inhibition, expansive remodeling, and MMP expression within the uterine vascular wall. Adult female rats were treated with N(G)-nitro-L-arginine methyl ester [L-NAME (LPLN)] beginning on day 10 of pregnancy and until death at day 20 and compared with age-matched controls [late pregnant (LP)]. Mean arterial pressure of LPLN rats was significantly higher than controls. LPLN fetal and placental weights were significantly reduced compared with controls. Main uterine arteries (mUA) were collected to determine dimensional properties (lumen area and wall thickness), collagen and elastin content, and levels of endothelial nitric oxide synthase (eNOS) and MMP expression. Circumferential remodeling was attenuated, as evidenced by significantly smaller lumen diameters. eNOS RNA and protein were significantly (>90%) decreased in the LPLN mUA compared with LP. Collagen and elastin contents were significantly increased in LPLN rats by ∼10 and 25%, respectively, compared with LP (P < 0.05). Both MMP-2 and tissue inhibitors of metalloproteinase-2 as assessed by immunofluorescence were lower in the endothelium (reduction of 60%) and adventitia (reduction of 50%) of LPLN compared with LP mUA. Membrane bound MMP-1 (MT1-MMP) as assessed by immunoblot was significantly decreased in LPLN. These data suggest a novel contribution of MMPs to gestational uterine vascular remodeling and substantiate the linkage between NO signaling and gestational remodeling of the uterine circulation via altered MMP, TIMP-2, and MT1-MMP expression and activity.

Expression of MMP-1 in histopathological different grades of oral squamous cell carcinoma and in normal buccal mucosa – an immunohistochemical study

Oral Cancer (OC) is the sixth most common cancer worldwide, while being the most common cancer among Indian males and the third most common cause of their deaths. 70-94% of all the malignancies arising within the oral cavity are oral squamous cell carcinomas (OSCC). Researches show that altered expression of matrix metalloproteinases (MMPs) may have a vital role in regulating the tumor microenvironment of head and neck carcinomas. To evaluate the expression of MMP-1 in histopathologically different grades of OSCC, to evaluate its expression in normal buccal mucosa (NBM), and to compare the expressions between these two groups, using the streptavidin horseradish peroxidase biotin labeled immuno-histochemistry (IHC) technique. 100% of OSCC showed cytoplasmic immune reactivity for MMP-1 in the epithelial and connective tissue cells. Their expression was elevated as the histopathological grade differed from well to poorly differentiated. 100% of epithelial cells and 80% of connective tissue of NBM expressed MMP-1.The immune reactivity was significantly over-expressed in OSCC in comparison to NBM. Evidence indicates that the elevated MMP-1 protein expression is associated with higher histopathological grade of OSCC. NBM express MMP-1 diffusely and weakly. MMP-1 immune reactivity increases as OC progresses from NBM to well to poorly differentiated OSCC.

Altered Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Matured Rat Adipocytes in Vitro

Obesity is recognized as a risk factor for delayed cutaneous wound healing. The authors hypothesized that the secretion of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) from subcutaneous adipose tissue correlates with disorder of the healing process in obese subjects. Findings from previous studies on the expression of MMPs and TIMPs in obese adipose tissue are inconsistent. Since these conflicting results could be due to the effect of several intrinsic factors, the authors conducted a simple in vitro experiment to clarify the change in profile of MMPs and TIMPs in excessively matured adipocytes. The authors cultured the induced adipocytes under conditions of high or low glucose and with or without insulin supplementation. Oil red O staining and its dye extraction assay revealed excessive lipid accumulation in high glucose and insulin-supplemented adipocytes. Additionally, there was altered expression of adipokines, similar to the change in adipose tissue in obese subjects. Under these conditions, the expression/activity of MMP8 was promoted and the expression of MMP3 and TIMP3 was inhibited. Further studies to determine the effect of other obesity-related factors, such as insulin resistance, on MMPs and TIMPs are required.

Presence of intestinal Mycobacterium avium subspecies paratuberculosis(MAP) DNA is not associated with altered MMP expression in ulcerative colitis

BackgroundMycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.MethodsColonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.ResultsMAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.ConclusionsThe presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

The Role of Matrix Metalloproteinases in the Pathogenesis of Endometriosis

Endometriosis is a benign, estrogen-dependent disease with an obscure etiology. Even the most widely accepted theory of retrograde menstruation cannot satisfactorily explain the development of endometriosis due to the many gaps in our understanding of its pathophysiology. Although most women have retrograde menstruation; only some develop endometriosis. Apart from simply being present in the peritoneal cavity, the endometrial cells are able to attach to, invade the peritoneum, and proliferate to create and maintain an endometriotic lesion. Matrix metalloproteinases (MMPs) are a group of enzymes that degrade the extracellular matrix. These enzymes participate in the histologic changes of the endometrium during the menstrual cycle with generally a higher expression during the menstrual and proliferative phase of the endometrium and a decreased expression during the secretory phase. As noted above, not only do these enzymes play a crucial factor in the cycling endometrium but the degradation of extracellular matrix is essential for the endometrial cells to invade the peritoneum and to develop an endometriotic lesion as well. The aim of this review is to describe the altered expression of MMPs in the development of endometriosis.

128. Altered Expression of Matrix Metalloproteinases in Chronic Descending Thoracic Aortic Dissection

128. Altered Expression of Matrix Metalloproteinases in Chronic Descending Thoracic Aortic Dissection

Matrix metalloproteinases: their potential role in the pathogenesis of diabetic nephropathy

Matrix metalloproteinases (MMPs), a family of proteinases including collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs, affect the breakdown and turnover of extracellular matrix (ECM). Moreover, they are major physiologic determinants of ECM degradation and turnover in the glomerulus. Renal hypertrophy and abnormal ECM deposition are hallmarks of diabetic nephropathy (DN), suggesting that altered MMP expression or activation contributes to renal injury in DN. Herein, we review and summarize recent information supporting a role for MMPs in the pathogenesis of DN. Specifically, studies describing dysregulated activity of MMPs and/or their tissue inhibitors in various experimental models of diabetes, including animal models of type 1 or type 2 diabetes, clinical investigations of human type 1 or type 2 diabetes, and kidney cell culture studies are reviewed.

Matrix metalloproteinase and tissue inhibitor expression in atherosclerotic and nonatherosclerotic thoracic aortic aneurysms

The altered expression of matrix metalloproteinases and their inhibitors influences the formation of atherosclerotic abdominal aortic aneurysms. Their association with thoracic aneurysms is less clear. This study describes the expression of metalloproteinases and their inhibitors in atherosclerotic and nonatherosclerotic thoracic aneurysms, and compares these with age-matched controls. Matrix metalloproteinase-2 and 9 activity were measured by antibody capture, and tissue inhibitor-1 and 2 levels were measured by enzyme-linked immunosorbent assay in 24 patients with atherosclerotic aneurysms and in 63 patients with nonatherosclerotic aneurysms. Gene expression was assessed with reverse transcriptase polymerase chain reaction. The results were compared with 17 controls. Data are in nanograms per milligram of protein. Matrix metalloproteinase-2 activity was greater in controls than in the atherosclerotic and nonatherosclerotic groups (80 +/- 67 vs 49 +/- 50 and 35 +/- 44, P = .002). Matrix metalloproteinase-9 activity was greater in the atherosclerotic group than in the nonatherosclerotic group and controls (11.7 +/- 15.7 vs 2.5 +/- 2.2 and 1.7 +/- 1.9, P = .001). Tissue inhibitor-1 and 2 levels were greater in controls than in either aneurysm group (tissue inhibitor of metalloproteinase-1: 376 +/- 192 vs 234 +/- 233 and 174 +/- 148, P = .003; tissue inhibitor of metalloproteinase-2: 143 +/- 74 vs 14 +/- 13 and 27 +/- 43, P < .001). Atherosclerotic aneurysms expressed more matrix metalloproteinase mRNA than controls. The metalloproteinase/tissue inhibitor phenotype of atherosclerotic thoracic aneurysms is similar to that of abdominal aneurysms. The diminished expression of metalloproteinases and tissue inhibitors in nonatherosclerotic thoracic aneurysms relative to aged controls may represent a loss of smooth muscle cells.

Altered Expression of Matrix Metalloproteinases Within Mineralizing Bone Cells In Vitro in the Presence of Fluoride

Fluoride is known to alter mineralization within bone, although the mechanism for its action is unclear. An important stage in the formation of mineralized tissues is the remodeling of the osteoid, facilitating mineral deposition. Using a bone mineralizing culture system derived from rat femur washes, this study investigated the influence of fluoride on MMP expression at a developmental stage relating to the onset of mineralization. Bone cells cultured in the absence of fluoride synthesized an active form of a 45-kD MMP, which was immunoreactive with an antibody to human MMP-1 (although full characterization of this MMP was not achieved), trace levels of an MMP immunoreactive with anti-MMP-3, and a 66-kD proteolytic species. Incubation in 10 &#109 7 and 10 &#109 5 M fluoride resulted in a decrease in expression of the 45-kD MMP, sharp increases in the expression of MMP-3, and the appearance of a band at 110 kD, which showed immunoreactivity for MMP-9. The influence of fluoride on MMP expression is likely to influence the composition of the remodeling matrix and subsequent mineralization and offers a potential mechanism by which fluoride alters mineralization.

Altered Expression of Matrix Metalloproteinases Within Mineralizing Bone Cells In Vitro in the Presence of Fluoride

Fluoride is known to alter mineralization within bone, although the mechanism for its action is unclear. An important stage in the formation of mineralized tissues is the remodeling of the osteoid, facilitating mineral deposition. Using a bone mineralizing culture system derived from rat femur washes, this study investigated the influence of fluoride on MMP expression at a developmental stage relating to the onset of mineralization. Bone cells cultured in the absence of fluoride synthesized an active form of a 45-kD MMP, which was immunoreactive with an antibody to human MMP-1 (although full characterization of this MMP was not achieved), trace levels of an MMP immunoreactive with anti-MMP-3, and a 66-kD proteolytic species. Incubation in 10 m 7 and 10 m 5 M fluoride resulted in a decrease in expression of the 45-kD MMP, sharp increases in the expression of MMP-3, and the appearance of a band at 110 kD, which showed immunoreactivity for MMP-9. The influence of fluoride on MMP expression is likely to influence the composition of the remodeling matrix and subsequent mineralization and offers a potential mechanism by which fluoride alters mineralization.

The role of chondrocyte senescence in osteoarthritis.

Replicative senescence occurs when normal somatic cells stop dividing. Senescent cells remain viable, but show alterations in phenotype, e.g. altered expression of matrix metalloproteinases (MMPs); these enzymes are known to be involved in cartilage destruction. It is assumed that cells deplete their replicative potential during aging, and age is a major risk factor for osteoarthritis (OA). Therefore, we hypothesized that chondrocytes in aging or diseased cartilage become senescent with associated phenotypic changes contributing to development or progression of OA. Articular cartilage was obtained from OA patients undergoing arthroplasty, with 'normal' cartilage from trauma surgery for hip fracture. Senescent cells were identified using the senescence-associated beta-galactosidase (SA-beta-gal) marker. Telomere length was assessed using Southern blot. MMP expression was measured at the mRNA level using Taqman RT-PCR. No SA-beta-gal staining was observed in control cartilage regardless of patient age. In contrast, SA-beta-gal staining was observed in damaged OA cartilage adjacent to the lesion. Cultured chondrocytes isolated from sites near a lesion contained a greater percentage of SA-beta-gal positive cells than cultures isolated from distal sites or normal cartilage. Mean telomere length was shorter in cells near the lesion compared to distal sites in the same joint; thus the former population has undergone cell division. The expression of collagenases MMP-1, -8 and -13 and tissue inhibitor of metalloproteinases (TIMP)-1 was altered in OA cartilage, but no difference was detected between lesion and distal sites in the same joint (i.e. no correlation was found between senescent cells and proteinase/ inhibitor expression).

Human odontoblast culture method: the expression of collagen and matrix metalloproteinases (MMPs).

Studies on mature human odontoblasts have suffered for the lack of in vitro models. We recently introduced a human odontoblast and pulp tissue organ culture method, in which the odontoblasts are cultured in the pulp chamber after removal of the pulp tissue, and the pulp tissue can be cultured separately (Tjäderhane et al., 1998a). With this method, we have studied the effects of growth factors on the expression of collagen and extracellular matrix (ECM)-degrading enzymes, matrix metalloproteinases (MMPs), in mature human odontoblasts. TGF-beta 1 was selected because of its ability to regulate the response of the dentin-pulp complex to external irritation. The effect of TGF-beta 1 (10 ng/mL) on pro alpha 1(I) collagen mRNA was analyzed by quantitative PCR, and type I procollagen propeptide (PINP) was analyzed from conditioned culture media with RIA. Odontoblast media were also assayed for respective type III procollagen propeptide (PIIINP). TGF-beta had a negligible effect on collagen mRNA expression or protein synthesis, indicating that TGF-beta alone does not markedly induce dentin matrix formation per se in the human dentin-pulp complex (Palosaari et al., 2001). However, TGF-beta 1 seems to regulate MMP expression in mature human odontoblasts differentially. A strong down-regulation of MMP-8 (Palosaari et al., 2000), a modest down-regulation of MMP-20 (Tjäderhane et al., 2000), and considerable up-regulation of MMP-9, with no apparent effect on MMP-2 expression (Tjäderhane et al., 1998b), indicate that growth factors may affect the matrix synthesis by controlling the expression and activity of MMPs instead of collagen synthesis. The altered expression of MMPs may result in altered ECM formation, which in turn may contribute to the formation of atubular reparative dentin.

Transforming growth factor beta1 acts as an inducer of matrix metalloproteinase expression and activity in human bone-metastasizing cancer cells.

Bone metastases are a common complication in prostate and breast cancer patients. It leads to extensive morbidity and eventually mortality. Matrix metalloproteinases (MMPs) are known to be involved in the metastatic process. MMP activity can be down-regulated by transforming growth factor beta1 (TGF-beta1), a growth-modulating factor, found in high concentrations in the bone. TGF-beta1 acts through the TGF-beta1 inhibitory element (TIE) element, a cis-acting element found in the promoter region of most MMP genes, with the exception of MMP-2. We used three human cell lines relevant for bone metastases, namely prostate adenocarcinoma PC-3, breast adenocarcinoma MDA-MB-231, and adenocarcinoma cells of unknown origin, Hs696, and one human osteosarcoma cell line, SAOS-2, and showed that in these cell lines TGF-beta1 partially lost its repressing action on MMP expression. TGF-beta1 was able to induce MMP-9 activity and protein expression in all three bone-metastatic tumour cell types, whereas MMP-9 protein levels were repressed in SAOS-2 cells. In PC-3 cells, TGF-beta1 repressed MMP-1 expression, whereas in MDA-MB-231 and SAOS-2 cells, an increase in the expression of MMP-1 protein was detected. Additionally, an increase in MMP-3 expression was observed in Hs696 cells. Expression and activity of the tissue inhibitors of matrix metalloproteinases, TIMP-1 and TIMP-2, were found increased in both PC-3 and MDA-MB-231 cells. With respect to cell proliferation, TGF-beta1 was able to induce a dose-dependent growth inhibition of up to 50% in primary human mammary epithelial cells. However, in none of the tumour cell lines was TGF-beta1 able to suppress growth substantially. Data presented in this paper support the hypothesis that TGF-beta1 can potentially disrupt the balance existing between osteoclast- and osteoblast-derived MMP activity by inducing altered expression of matrix metalloproteinases and their tissue inhibitors derived from bone-metastasizing cancer cells. This could eventually lead to skeletal destruction in patients with advanced metastatic disease.

Collagenase-1, stromelysin-1 and 92 kDa gelatinase are associated with tumor necrosis factor-alpha induced morphological change of human endothelial cells in vitro.

Recently, we have shown that the tumor necrosis factor-alpha (TNF-alpha)-induced morphological change of EA.hy 926 human endothelial cells is associated with a decrease in the net synthesis of two proteoglycans (PGs), biglycan and syndecan-1, both of which have been suggested to play a role in cell adhesion. Here we have examined whether this phenotypic modulation of EA.hy 926 cells also involves altered expression of matrix metalloproteinases (MMPs) or their tissue inhibitors (TIMPs). We demonstrate that, when forming cobblestone-like monolayer cultures, these cells express and synthesize collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and 72 kDa (MMP-2) and 92 kDa (MMP-9) gelatinases, all of which have previously been found in either normal or pathological human vascular wall. EA.hy 926 cells also express membrane-typel MMP (MT1-MMP), but not matrilysin (MMP-7) and collagenase-3 (MMP-13). As regards TIMPs, we show that these cells express TIMP-1 and TIMP-2, but not TIMP-3 or TIMP-4. Exposure of the cells to TNF-alpha changed the cell morphology from a polygonal shape into a spindle shape and also increased the mRNA levels of MMP-1, MMP-3 and MMP-9, but slightly decreased the MMP-2 mRNA level. No change at the mRNA level of MT1-MMP was observed. Similarly to unstimulated cultures, no mRNA for MMP-7 or MMP-13 was detected in the TNF-alpha treated cultures. TNF-alpha had no effect on the TIMP-1 and TIMP-2 mRNA levels and did not induce TIMP-3 or TIMP-4 expression. Gelatin zymography and Western blot analysis revealed that the increase observed at the mRNA level of MMP-3 and MMP-9 was similar to that of their net protein level; furthermore, the active form of MMP-1 was induced. Our results indicate that the TNF-alpha-induced morphological change of EA.hy 926 cells is associated not only with specific changes in the expression of PGs by the cells, but also with specific changes in the expression of MMPs.