The molecular perspective: matrix metalloproteinase 2.
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Humans↗
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Myricetin inhibits matrix metalloproteinase 2 protein expression and enzyme activity in colorectal carcinoma cells.
Colorectal carcinoma is a leading cause of human mortality due to its high metastatic ability. Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, agents with the ability to inhibit MMP activity have potential in the treatment of colorectal carcinoma. In the present study, among 36 flavonoids examined, myricetin was found to be the most potent inhibitor of MMP-2 enzyme activity in COLO 205 cells (IC50 = 7.82 micromol/L). Myricetin inhibition of MMP-2 enzyme activity was also found in the human colorectal carcinoma cell lines COLO 320HSR, COLO 320DM, HT 29, and COLO 205-X (IC50 = 11.18, 11.56, 13.25, and 23.51 micromol/L, respectively). In contrast, no inhibitory effect of MMP-2 protein expression or enzyme activity was observed in myricitrin (myricetin-3-rhamnoside)-treated cells. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated COLO 205 cells, an increase in MMP-2 protein expression and enzyme activity, as well as of protein kinase C (PKC) alpha protein translocation, extracellular signal-regulated kinase (ERK) 1/2 protein phosphorylation, and c-Jun protein expression was observed. ERK inhibitor (PD98059) and PKC inhibitors (GF-109203X and H-7), but not p38 inhibitor (SB203580) or c-jun-NH2-kinase inhibitor (SP600125), significantly inhibited TPA-induced MMP-2 protein expression, with reduced ERK phosphorylation and c-Jun protein expression. Addition of myricetin but not myricitrin suppressed TPA-induced MMP-2 protein expression in COLO 205 cells by blocking the TPA-induced events, including translocation of PKCalpha from cytosol to membrane, phosphorylation of ERK1/2 protein, and induction of c-Jun protein expression. Addition of PD98059 or GF-109203X significantly enhanced the inhibitory effect of myricetin on MMP-2 enzyme activity induced by TPA. Furthermore, myricetin, but not myricitrin, suppressed TPA-induced invasion of COLO 205 cells in an in vitro invasion assay using Engelbreth-Holm-Swarm sarcoma tumor extract Matrigel-coated Transwells. Results of the present study indicate that myricetin significantly blocked both endogenous and TPA-induced MMP-2 enzyme activity by inhibiting its protein expression and enzyme activity. The blockade involved suppression of PKC translocation, ERK phosphorylation, and c-Jun protein expression.
Antineoplastic Agents↗
Targeted deletion of matrix metalloproteinase 2 ameliorates myocardial remodeling in mice with chronic pressure overload.
Matrix metalloproteinases (MMPs) play an important role in the extracellular matrix remodeling. Experimental and clinical studies have demonstrated that MMP 2 and 9 are upregulated in the dilated failing hearts and involved in the development and progression of myocardial remodeling. However, little is known about the role of MMPs in mediating adverse myocardial remodeling in response to chronic pressure overload (PO). We, thus, hypothesized that selective disruption of the MMP 2 gene could ameliorate PO-induced cardiac hypertrophy and dysfunction in mice. PO hypertrophy was induced by transverse aortic constriction (TAC) in male MMP 2 knockout (KO) mice (n=10) and sibling wild-type (WT) mice (n=9). At 6 weeks, myocardial MMP 2 zymographic activity was 2.4-fold increased in WT+TAC, and this increase was not observed in KO+TAC, with no significant alterations in other MMPs (MMP 1, 3, 8, and 9) or tissue inhibitors of MMPs (1, 2, 3, and 4). TAC resulted in a significant increase in left ventricular (LV) weight and LV end-diastolic pressure (EDP) with preserved systolic function. KO+TAC mice exerted significantly lower LV weight/body weight (4.2+/-0.2 versus 5.0+/-0.2 mg/g; P<0.01), lung weight/body weight (4.9+/-0.2 versus 6.2+/-0.4 mg/g; P<0.01), and LV end-diastolic pressure (4+/-1 versus 10+/-2 mm Hg; P<0.05) than WT+TAC mice despite comparable aortic pressure. KO+TAC mice had less myocyte hypertrophy (cross-sectional area; 322+/-14 versus 392+/-14 microm2; P<0.01) and interstitial fibrosis (collagen volume fraction; 3.3+/-0.5 versus 8.2+/-1.0%; P<0.01) than WT+TAC mice. MMP 2 plays an important role in PO-induced LV hypertrophy and dysfunction. The inhibition of MMP 2 activation may, therefore, be a useful therapeutic strategy to manage hypertensive heart disease.
Animals↗
Expression of matrix metalloproteinases 2 and 9 in meningiomas associated with different degrees of brain invasiveness and edema.
OBJECT: Meningiomas display clinical characteristics that vary from very benign to clearly malignant with rapid invasive growth and metastasis. Benign meningiomas differ in their invasiveness and concomitant edema. This study was undertaken to analyze the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9, respectively) in meningiomas associated with different degrees of brain invasion and edema. METHODS: Tissue samples from 16 meningiomas were selected according to tumor invasiveness from a consecutive series of patients. Samples were analyzed for expression of both MMP-2 and MMP-9 by using in situ hybridization. The meningiomas consisted of three types: Group I, benign meningiomas that did not interfere with the arachnoid plane and exhibited no edema; Group II, benign meningiomas that invaded the arachnoid plane and caused edema; and Group III, aggressive and malignant meningiomas that caused edema and displayed brain invasion. In all 16 tumors analyzed, MMP-2 mRNA was identified. Levels of expression of MMP-2 mRNA were similar in all samples, and no correlation with increasing tumor invasiveness or associated edema could be detected. Expression of MMP-9 mRNA was identified in 14 of the 16 tumors, and a clear correlation with increasing tumor invasion into the brain was noted. CONCLUSIONS: Meningiomas express both MMP-2 and MMP-9. Tumor invasiveness, which ranged from minor with respect to the arachnoid membrane and progressed to frank brain invasion, correlated with the extent of MMP-9 expression. The findings indicate that MMP-9 expression and brain invasion are relevant mechanisms that must be interfered with in the treatment of aggressive and malignant meningiomas. No such correlation with MMP-2 was found.
Aged↗
Increased activity of matrix metalloproteinase 2 and 9 after hepatic radiofrequency ablation.
BACKGROUND: Radiofrequency (RF) ablation of hepatic metastases from colorectal cancer (CRC) is associated with a high rate of local and intrahepatic tumor recurrence. Matrix metalloproteinases (MMPs) play an important role in inflammation, tissue repair and tumor cell invasion and metastasis. MMP-2 and MMP-9 are associated with increased risk of recurrence and decreased survival in patients with colorectal cancer. The primary aim of the study was to determine if hepatic RF ablation increased MMP-2 and MMP-9 activity in the transition zone surrounding the coagulated hepatic tissue. MATERIALS AND METHODS: Twelve pigs were randomized to hepatic RF ablation with (n = 6) or without (n = 6) hepatic vascular occlusion (Pringle maneuver). Four days after ablation tissue specimens were collected from the transition zone surrounding coagulated hepatic tissue, and from normal hepatic parenchyma. MMP activity was quantified by gelatin zymography. Cellular localization of MMPs was determined by immunohistochemistry using antibodies against MMP-2, MMP-9, and the macrophage marker CD68. RESULTS: MMP-2 and MMP-9 activity was increased in the transition zone compared to normal hepatic parenchyma, with ratios of 3.0 (P = 0.005) and 2.6 (P = 0.001), respectively. Pringle maneuver did not influence MMP activity. MMP-2 and MMP-9 expression was localized to macrophages in the transition zone. CONCLUSIONS: Hepatic RF ablation is associated with increased expression of MMP-2 and MMP-9 in macrophages in the transition zone surrounding the coagulated hepatic parenchyma. These findings may contribute to the understanding of possible mechanisms for the high recurrence rates observed in patients after RF ablation of CRC hepatic metastases.
Animals↗
Urinary gelatinase activities (matrix metalloproteinases 2 and 9) in human bladder tumors.
The ability to degrade type IV collagen, the major component of the basement membrane, is unique to gelatinases A and B. These two matrix metalloproteinases (MMPs) are most often linked to the malignant phenotype of tumor cells, and their expression is elevated in several cases of human tumor aggressiveness and overall survival. By gelatin zymography, we verified MMP activity in the urine of patients with bladder cancer. Of these patients, 10 had well-, 8 had moderately and 7 had poorly differentiated bladder cancer. The urine of healthy volunteers with no evidence of disease was used for controls. Zymography showed five dominant gelatinolytic bands of 240, 220, 130, 92 and 72 kDa in tumor samples, whereas only traces of MMP were detected in the urine of healthy subjects. The majority of cancerous urine samples showed MMP-9 lytic activity but only a few contained MMP-2. Moreover, MMP-9 content is enhanced in the urine from patients with high-grade and advanced-stage bladder tumors. Finally, we determined the urinary levels of urinary bladder cancer (UBC), tissue polypeptide-specific antigen (TPS) and protein 22 of nuclear matrix (NMP22). The levels of TPS and NMP-22 were higher in G3 bladder cancer than in G1 and G2 neoplasias. The urinary values of these two biomarkers correlated with the increase in MMP-9 lytic activity in high-grade and advanced-stage bladder cancer.
Adenocarcinoma↗
Matrix metalloproteinases 2 and 9 are dispensable for pancreatic islet formation and function in vivo.
Pancreatic islet formation is a highly regulated process that is initiated at the end of gestation in rodents. Endocrine precursor cells first form within the epithelium of duct-like structures and then delaminate from the epithelium, migrate, and cluster during the early stages of islet formation. The molecular mechanisms that regulate endocrine cell migration and islet formation are not well understood. Cell culture studies suggest that matrix metalloproteinases (MMPs) 2 and 9 are required for islet formation. To address whether MMP2 and MMP9 function are essential for endocrine cell migration and islet formation in vivo, we analyzed pancreas development in MMP2/MMP9 double-deficient mice. Our results show that islet architecture and function are unperturbed in these knockout mice, demonstrating that both MMP2 and MMP9 functions are dispensable for islet formation and endocrine cell differentiation. Our studies also show that a number of other MMPs are expressed at the time islet formation is initiated. This observation suggests that other MMPs may substitute for MMP2 and MMP9 loss in pancreatic tissue. However, islet formation is unaffected in transgenic mice with modified tissue inhibitor of metalloproteinase-1 (TIMP1) levels, suggesting that MMP activity may contribute little to islet morphogenesis in vivo.
Animals↗
A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo.
Extracellular matrix breakdown as well as increased expression in cancer cells and tumor microvascular endothelial cells make matrix metalloproteinase 2 (MMP2) an attractive target for cancer treatment. By taking advantage of MMP2's properties, an MMP2 cleavable melittin/avidin conjugate was designed. Melittin alone is extremely toxic to cells and induces immediate cell lysis, but becomes inactive when coupled with avidin. The incorporation of the MMP2 target sequence into the peptide was used as a means for targeting tumor cells. In vitro, the melittin/avidin conjugate showed strong cytolytic activity against cancer cells with high MMP2 activity; DU 145 prostate cancer cells and SK-OV-3 ovarian cancer cells. The conjugate exhibited very little cytolytic activity against normal L-cells that displayed low MMP2 activity. These data demonstrate the MMP2 specificity of the melittin/avidin conjugate. In vivo, the size of tumors injected with the melittin/avidin conjugate was significantly smaller as compared to untreated tumors. Therefore, due to its tumor targeting capabilities as well as its cytolytic properties in vitro and in vivo, the melittin/avidin conjugate displays the potential for use in cancer therapy.
Amino Acid Sequence↗
Function of alpha3beta1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2).
Tumor cell migration through the three- dimensional extracellular matrix (ECM) environment is an important part of the metastatic process. We have analyzed a role played by the integrin-tetraspanin protein complexes in invasive migration by culturing MDA-MB-231 cells within Matrigel. Using time-lapse video recording, we demonstrated that the Matrigel-embedded cells remain round and exhibit only limited ability for migration by extending short, highly dynamic pseudopodia. The alpha3beta1-tetraspanin protein complexes were clustered on the thin microvilli-like protrusions extending from both the main cell body and pseudopodia. Ligation of the alpha3beta1-tetraspanin protein complexes with monoclonal antibodies specifically stimulates production of matrix metalloproteinase 2 (MMP-2) and induces formation of long invasive protrusions within Matrigel. Accordingly, treatment with the monoclonal antibodies to various tetraspanin proteins and to the alpha3 integrin subunit increases invasive potential of the MDA-MB-231 cells in the Matrigel-penetration assay. A specific inhibitor of phosphoinositide 3-kinase (PI3K), LY294002, negated the effect of the monoclonal antibodies on the morphology of the Matrigel-embedded cells and on production of MMP-2. Interestingly, broad-spectrum inhibitors of protein tyrosine kinases (genistein) and protein tyrosine phosphatases (orthovanadate), and actin filament stabilizing compound (jasplakinolide), also block protrusive activity of the Matrigel-embedded cells but have no effect on the production of MMP-2. These results indicate that alpha3beta1-tetraspanin protein complexes may control invasive migration of tumor cells by using at least two PI3K-dependent signaling mechanisms: through rearrangement of the actin cytoskeleton and by modulating the MMP-2 production.
Actins↗
Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts.
Indiscriminate invasion upon the endometrium by normal trophoblasts is strictly regulated unlike that by choriocarcinoma cells. In this study, we focused on the activity of matrix metalloproteinase (MMP)-2 and MMP-9 as parameters of invasion in normal human placenta. In situ hybridization (ISH), immunohistochemical staining (IH) and film in situ zymography (FIZ) were performed to identify cells having MMP-2 or MMP-9 expression and activity. Purified cytotrophoblasts (CTs) were used to examine the expression and activity of MMP-2 and MMP-9, and their invasive ability. In first trimester placental tissue, the MMP-2 expression was observed in extravillous trophoblasts (EVTs), and MMP-9 mainly in villous cytotrophoblasts (VCTs). FIZ revealed marked gelatinase activity in the EVTs which MMP-2 expression was observed in. In full-term placental tissue, the MMP-2 expressions was observed in the EVTs similar to that in first trimester, whereas the gelatinase activity in these cells was decreased or completely lost. Using purified CTs, the gelatinase activity was marked in early CTs, but not term CTs. Invasive ability of early CTs was inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 and MMP-2 antibody in a dose dependent manner. These suggests that the invasive ability of trophoblasts may be regulated by the enzyme activity of gelatinases, especially MMP-2.
Adult↗
Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities.
Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.
Animals↗
Matrix metalloproteinases 2 and 9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura.
Matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and -9), play a significant role in tumour invasion and angiogenesis. The expression and activities of MMPs have not been characterised in malignant mesothelioma (MM) tumour samples. In a prospective study, gelatinase activity was evaluated in homogenised supernatants of snap frozen MM (n=35), inflamed pleura (IP, n=12) and uninflammed pleura (UP, n=14) tissue specimens by semiquantitative gelatin zymography. Matrix metalloproteinases were correlated with clinicopathological factors and with survival using Kaplan-Meier and Cox proportional hazard models. In MM, pro- and active MMP-2 levels were significantly greater than for MMP-9 (P=0.006, P<0.001). Active MMP-2 was significantly greater in MM than in UP (P=0.04). MMP-2 activity was equivalent between IP and MM, but both pro- and active MMP-9 activities were greater in IP (P=0.02, P=0.009). While there were trends towards poor survival with increasing total and pro-MMP-2 activity (P=0.08) in univariate analysis, they were both independent poor prognostic factors in multivariate analysis in conjunction with weight loss (pro-MMP-2 P=0.03, total MMP-2 P=0.04). Total and pro-MMP-2 also contributed to the Cancer and Leukemia Group B prognostic groups. MMP-9 activities were not prognostic. Matrix metalloproteinases, and in particular MMP-2, the most abundant gelatinase, may play an important role in MM tumour growth and metastasis. Agents that reduce MMP synthesis and/or activity may have a role to play in the management of MM.
Adult↗
Developmental expression of matrix metalloproteinases 2 and 9 and their potential role in the histogenesis of the cerebellar cortex.
The development of the cerebellar cortex depends on intrinsic genetic programs and orchestrated cell-cell/cell-matrix interactions. Matrix metalloproteinases (MMPs) are proteolytic enzymes that play an important role in these interactions. MMP-2 and MMP-9 are involved in diverse neuronal functions including migration, process extension, and synaptic plasticity. We investigated the spatiotemporal pattern of expression/activity of MMP-2/MMP-9 in the developing cerebellum and their role in the histogenesis of the cerebellar cortex. The levels of transcripts of MMP-2/MMP-9 were measured with real-time quantitative polymerase chain reaction. An initial decrease in MMP-2/MMP-9 transcripts was observed between postnatal days 3 (PD3) and PD6, and the mRNA levels remained relatively constant thereafter. Zymographic analysis revealed that the expression/activity of MMP-2/MMP-9 persisted longer than their transcripts; the downregulation occurred around PD9, suggesting a mechanism of translational or post-translational regulation. The gelatinase activity was localized in the external granule layer (EGL) and the internal granule layer during PD3-PD12. The immunoreactivity of MMP-2 was mainly localized in the EGL, the Bergmann glial fibers, and the Purkinje cell layer (PCL), whereas MMP-9 immunoreactivity was detected intensively in the PCL and the extracellular space of the molecular layer. Expression of MMP-9 was relatively weak in the EGL. The immunoreactivity of MMP-2/MMP-9 became undetectable after PD21. A similar expression pattern of MMP-2/MMP-9 was observed in organotypic cerebellar slice cultures. Exposure of organotypic slices to a specific MMP-2/MMP-9 inhibitor significantly increased the thickness of the EGL and concurrently decreased the number of migrating granule neurons in the molecular layer. Thus, MMP-2 and MMP-9 play a role in the postnatal cerebellar morphogenesis.
Animals↗
Effect of piroxicam on matrix metalloproteinase 2 and apoptosis.
We examined the effect of a nonsteroidal anti-inflammatory drug (NSAID), piroxicam, on apoptosis and matrix metalloproteinase 2 (MMP-2) activity compared with diclofenac and dexamethasone. The fibrosarcoma (WEHI-164) cell line was used to assess tolerability, MMP-2 activity and apoptosis. Piroxicam, dexamethasone and diclofenac were used at concentrations of 10-200 microg/ml in triplicate and 2-fold dilutions. MMP-2 activity was assessed using zymography. For assessment of apoptosis, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method was used. The results of this study show that piroxicam is able to diminish MMP-2 activity and induce apoptosis under in vitro conditions. Piroxicam also showed high tolerability compared with diclofenac and dexamethasone. In conclusion, piroxicam is able to induce apoptosis and suppress MMP-2 activity.
Animals↗
Increased level of matrix metalloproteinases 2 and 9 in the ripening process of the human cervix.
The human uterine cervix is a fibrous organ with a high connective tissue content. An extensive remodeling of the connective tissue prior to parturition, i.e., cervical ripening, requires the presence of proteolytic enzymes. The exact mechanism of cervical ripening has not been clarified. We evaluated in vivo distribution and expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in the human cervix at term pregnancy and immediately after parturition compared with the nonpregnant state. Cervical biopsies were obtained from term pregnant, postpartum, and nonpregnant women. MMP-2 and MMP-9 proteins were localized by immunohistochemistry. Messenger RNA levels of MMP-2 and MMP-9 were evaluated by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR) using an invariable internal standard. The mRNA levels of MMP-2 and MMP-9 were increased in the cervix at term pregnancy and postpartum compared with the nonpregnant state. Cervical stromal fibroblasts and smooth muscle cells were identified as main sources of MMP-2, whereas the MMP-9 protein was observed exclusively in invading leukocytes. These data indicate the involvement of MMP-2 and MMP-9 in the cervical ripening process.
Adult↗
Matrix metalloproteinases 2 and 9 in oral squamous cell carcinomas: manifestation and localization of their activity.
PURPOSE: The process of invasion and metastasis is closely related to the prognosis of oral squamous cell carcinoma (OSCC). Matrix metalloproteinases (MMPs) are a group of enzymes characterized by their ability to degrade extracellular matrix proteins and contribute to the tumor invasion and metastasis. Especially MMP-2 and MMP-9 are known to be related to destruction of basement membrane as collagenases. This study focused on protein expression of MMP-2 and MMP-9 and their extracellular matrix degradation activity in OSCCs. METHODS: Freshly frozen samples from 31 OSCC patients were analyzed for the localization and activity of MMP-2 and MMP-9. Serial frozen sections were used by routine hematoxylin and eosin staining, immunohistochemistry for MMP-2 and MMP-9, and film in situ zymography (FIZ) for gelatinolytic activity. We also evaluated the activity of MMP-2 and MMP-9 by zymography using the same samples as frozen sections. The activated form/proform ratio of MMPs in zymography was evaluated using an image scanner. RESULTS: In MMP-2 the proportion in T3 and T4 clinical stage groups was significantly higher than that in T1 and T2. The proportion in lymph node metastasis cases (N+) was also significantly higher than that in non-lymph node metastasis cases (N-). In contrast to MMP-2, the activated form/proform ratio of MMP-9 was very low, suggesting that MMP-9 is not activated in the matrix degradation of OSCC, although both MMP-2 and MMP-9 protein expression are presented in tumor cells. FIZ revealed MMP in both tumor cells and stromal cells of 70% of the N+ cases and of 47.6% of the N- cases. CONCLUSIONS: These results indicate that two types of proform and activated form matrix metalloproteinases, MMP-2 and MMP-9, are present in human OSCC, and that the activated MMP-2 could be a main enzymatic activity of gelatinolysis in OSCC. Interaction of tumor cells and stromal cells seems to play an important role in the invasion and metastasis of human OSCC. Combination analysis of zymography and FIZ is a useful method to detect activity and localization of MMPs in human OSCC.
Adult↗
Bone sialoprotein, matrix metalloproteinase 2, and alpha(v)beta3 integrin in osteotropic cancer cell invasion.
BACKGROUND: Bone sialoprotein (BSP) interacts separately with both matrix metalloproteinase 2 (MMP-2) and integrin alpha(v)beta3 and is overexpressed in many metastatic tumors. Its role in tumor biology, however, remains unclear. We investigated whether BSP enhances cancer cell invasiveness by forming a trimolecular complex with MMP-2 and cell-surface integrin alpha(v)beta3. METHODS: Invasiveness of breast, prostate, lung, and thyroid tumor cell lines was measured with a modified Boyden chamber assay. Binding and co-localization of BSP, MMP-2, and integrin alpha(v)beta3 were investigated with immunoprecipitation and in situ hybridization. All statistical tests were two-sided. RESULTS: Treatment with BSP increased invasiveness of many breast, prostate, lung, and thyroid cancer cells through Matrigel in a dose-dependent manner. BSP at 50 nM increased the invasiveness of SW-579 thyroid cancer cells (95.2 units, 95% confidence interval [CI] = 90.4 to 100 units) by approximately 10-fold compared with that of untreated control SW-579 cells (9.1 units, 95% CI = 5.7 to 12.5 units) (P<.001). Addition of an inactive mutated BSP, in which BSP's integrin-binding RGD tripeptide was altered, or addition of integrin alpha(v)beta3-blocking antibodies resulted in invasiveness equivalent to that of untreated cells. Inhibiting cellular MMP-2 activity with chemical inhibitors or a specific antibody also blocked BSP-enhanced invasiveness. Osteopontin and dentin matrix protein 1, proteins related to BSP that also bind integrin alpha(v)beta3 and form complexes with other MMPs (but not MMP-2), did not enhance invasiveness. Immunoprecipitation showed that a complex containing BSP, integrin alpha(v)beta3, and MMP-2 formed in vitro. Addition of BSP increased the amount of MMP-2 bound by cells in an integrin-dependent fashion. Co-expression of BSP, integrin alpha(v)beta3, and MMP-2 in papillary thyroid carcinoma cells was shown by in situ hybridization. CONCLUSION: Cancer cells appear to become more invasive when BSP forms a cell-surface trimolecular complex by linking MMP-2 to integrin alpha(v)beta3.
Bone Neoplasms↗
Extracellular matrix metalloproteinase 2 levels are regulated by the low density lipoprotein-related scavenger receptor and thrombospondin 2.
We have recently shown that the adhesive defect observed in dermal fibroblasts derived from thrombospondin 2 (TSP2)-null mice results from an increase in matrix metalloproteinase 2 (MMP2) levels (Yang, Z., Kyriakides, T. R., and Bornstein, P. (2000) Mol. Biol. Cell 11, 3353-3364). Adhesion was restored by replacement of TSP2 and by inhibitors of MMP2 activity. In pursuing the observation that TSP2 and MMP2 interact, we now demonstrate that this interaction is required for optimal clearance of extracellular MMP2 by fibroblasts. Since TSP2 is known to be endocytosed by the scavenger receptor, low density lipoprotein receptor-related protein (LRP), we determined whether interference with LRP function affected fibroblast adhesion and/or extracellular MMP2 levels. Addition of heparin, which competes for the binding of TSP2 to LRP coreceptor proteoglycans, inhibited adhesion of control but not TSP2-null cells, and a blocking antibody to LRP as well as the LRP inhibitor, receptor-associated protein, also inhibited adhesion and increased MMP2 levels only in control fibroblasts. TSP2 did not inhibit active MMP2 directly and did not inhibit the activation of pro-MMP2. Finally, the internalization of 125I-MMP2 was reduced in TSP2-null compared with control fibroblasts. We propose that clearance of MMP2-TSP2 complexes by LRP is an important mechanism for the regulation of extracellular MMP2 levels in fibroblasts, and perhaps in other cells. Thus, some features of the phenotype of TSP2-null mice, such as abnormal collagen fibrillogenesis, accelerated wound healing, and increased angiogenesis, could result in part from increased MMP2 activity.
Animals↗