Abl kinases are required for invadopodia formation and chemokine-induced invasion

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1α, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.     

Characterization of matrix metalloproteinase expressed by human embryonic kidney cells

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3−MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.     

Evidence for a cooperative role of gelatinase A and membrane type-1 matrix metalloproteinase during Xenopus laevis development

Matrix metalloproteinases (MMPs) are a large family of extracellular or membrane-bound proteases. Their ability to cleave extracellular matrix (ECM) proteins has implicated a role in ECM remodeling to affect cell fate and behavior during development and in pathogenesis. We have shown previously that membrane-type 1 (MT1)-MMP [corrected] is coexpressed temporally and spatially with the MMP gelatinase A (GelA) in all cell types of the intestine and tail where GelA is expressed during Xenopus laevis metamorphosis, suggesting a cooperative role of these MMPs in development. Here, we show that Xenopus GelA and MT1-MMP interact with each other in vivo and that overexpression of MT1-MMP and GelA together in Xenopus embryos leads to the activation of pro-GelA. We further show that both MMPs are expressed during Xenopus embryogenesis, although MT1-MMP gene is expressed earlier than the GelA gene. To investigate whether the embryonic MMPs play a role in development, we have studied whether precocious expression of these MMPs alters development. Our results show that overexpression of both MMPs causes developmental abnormalities and embryonic death by a mechanism that requires the catalytic activity of the MMPs. More importantly, we show that coexpression of wild type MT1-MMP and GelA leads to a cooperative effect on embryonic development and that this cooperative effect is abolished when the catalytic activity of either MMP is eliminated through a point mutation in the catalytic domain. Thus, our studies support a cooperative role of these MMPs in embryonic development, likely through the activation of pro-GelA by MT1-MMP.     

TGF-beta induces formation of F-actin cores and matrix degradation in human breast cancer cells via distinct signaling pathways

Transforming growth factor beta regulates many biological processes including cell motility and invasion. Podosomes are specialized F-actin rich structures found in normal cells, such as osteoclasts and macrophages. Tumor cells often form related structures called invadopodia that are thought to promote invasion and metastasis. Here we show that human breast cancer cells organize F-actin rich structures in response to transforming growth factor beta that colocalize with areas of extracellular matrix degradation. We further show that organizing the complex of proteins needed to form these structures requires signaling through phosphatidylinositide 3-kinase and Src kinase, while activating the proteases involved in degradation of extracellular matrix requires extracellular signal-regulated kinase signaling, and that each of these pathways is activated by transforming growth factor beta in CA1D human breast cancer cells.     

Control of matrix metalloproteinase catalytic activity

As their name implies, MMPs were first described as proteases that degrade extracellular matrix proteins, such as collagens, elastin, proteoglycans, and laminins. However, studies of MMP function in vivo have revealed that these proteinases act on a variety of extracellular protein substrates, often to activate latent forms of effector proteins, such as antimicrobial peptides and cytokines, or to alter protein function, such as shedding of cell-surface proteins. Because their substrates are diverse, MMPs are involved in variety of homeostatic functions, such as bone remodeling, wound healing, and several aspects of immunity. However, MMPs are also involved in a number of pathological processes, such as tumor progression, fibrosis, chronic inflammation, tissue destruction, and more. A key step in regulating MMP proteolysis is the conversion of the zymogen into an active proteinase. Several proMMPs are activated in the secretion pathway by furin proprotein convertases, but for most the activation mechanisms are largely not known. In this review, we discuss both authentic and potential mechanisms of proMMP activation.     

The role of extracellular matrix metalloproteinase inducer protein in prostate cancer progression

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is a multifunctional membrane glycoprotein overexpressed in many solid tumors, and involved in tumor invasion and angiogenesis. We investigated EMMPRIN expression in human prostate cancer (CaP) tissues and cells, and evaluated whether EMMPRIN expression is related to tumor progression and matrix metalloproteinase (MMPs) expression in human CaP. An immunohistochemical study using tissue microarrays of 120 primary CaPs of different grades and 20 matched lymph node metastases from untreated patients was performed. The association of EMMPRIN expression with clinicopathological parameters was evaluated. Co-immunolocalization for EMMPRIN and MMP-1, MMP-2 or MMP-9 in primary tumors was examined using confocal microscopy. Flow cytometry and immunoblotting were used to examine EMMPRIN expression in 11 metastatic CaP cell lines. Heterogeneous expression of EMMPRIN was found in 78/120 (65%) CaPs, correlated significantly with progression parameters including pre-treatment PSA level (P < 0.05) and increased with progression of CaP (Gleason score, P < 0.05; pathological stage, P < 0.01; nodal involvement, P < 0.05 and surgical margin, P < 0.05). Heterogeneous cytoplasmic MMP-1, MMP-2 and MMP-9 associated with EMMPRIN immunolabeling was observed, particularly in tumors with Gleason scores >3 + 4. Metastatic CaP cell lines, except DuCaP, expressed abundant EMMPRIN protein, indicating highly ( approximately 45 to approximately 65 kDa) and less ( approximately 30 kDa) glycosylated forms, although with no relationship to cells being either androgen responsive or nonresponsive. Our results suggest that EMMPRIN may regulate MMPs and be involved in CaP progression, and as such, could provide a target for treating metastatic CaP disease.     

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.     

Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling

Isoliquiritigenin (ISL, 4,2′,4′-trihydroxychalcone), which is found in licorice, shallot and bean sprouts, is a potent antioxidant with anti-inflammatory and anti-carcinogenic effects. The purpose of this study was to investigate the effects of ISL treatment on the migration, invasion and adhesion characteristics of DU145 human prostate cancer cells. DU145 cells were cultured in the presence of 0–20 μmol/L ISL with or without 10 μg/L epidermal growth factor (EGF). ISL inhibited basal and EGF-induced cell migration, invasion and adhesion dose dependently. ISL decreased EGF-induced secretion of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF), but increased TIMP-2 secretion in a concentration-dependent manner. In addition, ISL decreased the protein levels of integrin-α2, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), and mRNA levels of uPA, MMP-9, VEGF, ICAM and integrin-α2. Furthermore, basal and EGF-induced activator protein (AP)-1 binding activity and phosphorylation of Jun N-terminal kinase (JNK), c-Jun and Akt were decreased after ISL treatment. However, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase was not altered. The JNK inhibitor SP600125 inhibited basal and EGF-induced secretion of uPA, VEGF, MMP-9 and TIMP-1, as well as AP-1 DNA binding activity and cell migration. These results provide evidence for the role of ISL as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of prostate cancer cells. The inhibition of JNK/AP-1 signaling may be one of the mechanisms by which ISL inhibits cancer cell invasion and migration.     

Dissecting the functional domain requirements of cortactin in invadopodia formation

Cells degrade extracellular matrix (ECM) barriers at focal locations by the formation of membrane protrusions called invadopodia. Polymerization of the actin cytoskeleton is critical to the extension of these processes into the ECM. We used a short interference RNA/rescue strategy to investigate the role of cortactin in the formation of Src-induced invadopodia in 3T3 fibroblasts, and subsequent degradation of the ECM. Cortactin-depleted cells did not form invadopodia or degrade the ECM. Functional invadopodia were restored in cortactin-depleted cells by expression of full-length cortactin, and fragments that contained the intact actin-binding repeats. Mutation of the three Src-targeted Tyr sites to Phe caused a loss in its rescuing ability, while mutation of the Erk phosphorylation sites had little effect on invadopodia formation. Interestingly, knock-down of cortactin did not affect the formation of lamellipodia and only slightly attenuated random cell motility. Our data shows that formation of functional invadopodia requires interaction between cortactin and filamentous actin, while interaction with SH3- and NTA-binding partners plays a less significant role. Furthermore, phosphorylation of cortactin by Src, but not by Erk, is essential for functional invadopodia formation. These results also suggest that cortactin plays a different role in invadopodia-dependent ECM degradation and lamellipodia formation in cell movement.     

Fluorescent and bioluminescent nanoprobes for in vitro and in vivo detection of matrix metalloproteinase activity

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix (ECM) and regulate the extracellular microenvironment. Despite the significant role that MMP activity plays in cell-cell and cell-ECM interactions, migration, and differentiation, analyses of MMPs in vitro and in vivo have relied upon their abundance using conventional immunoassays, rather than their enzymatic activities. To resolve this issue, diverse nanoprobes have emerged and proven useful as effective activity-based detection tools. Here, we review the recent advances in luminescent nanoprobes and their applications in in vitro diagnosis and in vivo imaging of MMP activity. Nanoprobes with the purpose of sensing MMP activity consist of recognition and detection units, which include MMP-specific substrates and luminescent (fluorescent or bioluminescent) nanoparticles, respectively. With further research into improvement of the optical performance, it is anticipated that luminescent nanoprobes will have great potential for the study of the functional roles of proteases in cancer biology and nanomedicine. [BMB Reports 2015; 48(6): 313-318]     

基质金属蛋白酶与体外循环肺损伤研究进展

肺泡 毛细血管基底膜损伤是体外循环 (CPB)术后肺损伤发生和发展的主要病理过程 ,基质金属蛋白酶 (MMPs)可能通过降解细胞外基质、调节细胞因子而参与CPB所致肺损伤的发生 ,研究MMPs在CPB肺损伤中的作用机制 ,对于防治CPB术后肺损伤的发生和发展具有重要意义     

Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells

Invadopodia are filopodia-like projections possessing protease activity that participate in tumor cell invasion. We demonstrate that co-localization of cortactin and phosphotyrosine identifies a subset of cortactin puncta termed "invadopodial complexes" that we find to be closely associated with the plasma membrane at active sites of focal degradation of the extracellular matrix in MDA-MB-231 breast cancer cells. Manipulation of c-Src activity in cells by transfection with kinase activated c-Src(527) or kinase inactive c-Src(295) results in a dramatic increase or decrease, respectively, in the number of these structures associated with changes in the number of sites of active matrix degradation. Overexpression of kinase-inactive c-Src(295) does not prevent localization of cortactin at the membrane; however, co-localized phosphotyrosine staining is decreased. Thus, elevated phosphotyrosine at invadopodial complexes is specifically associated with the proteolytic activity of invadopodia. Further, invadopodial complexes are spatially, morphologically and compositionally distinct from focal adhesions as determined by localization of focal adhesion kinase (FAK), which is not present in invadopodial complexes. Expression of kinase-inactive c-Src(295) blocks invadopodia activity, but does not block filopodia formation. Thus, invadopodia, but not filopodia, are highly correlated with matrix invasion, and sites of invadopodial activity can be identified by the formation of invadopodial complexes.     

A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.     

Focal adhesion kinase is not required for Src-induced formation of invadopodia in KM12C colon cancer cells and can interfere with their assembly

Overexpression of active Src induces invadopodia formation and associated matrix degradation in KM12C colon cancer cells. FAK is present with active Src at sites of matrix-degrading activity (invadopodia), specifically residing in rings surrounding the cortactin-containing invadopodia cores. Since FAK is a key effector protein in many aspects of Src function, we addressed whether FAK is necessary for Src-induced invadopodia formation and matrix degradation in KM12C colon cancer cells. We found that efficient knockdown of FAK expression by siRNA had no effect on invadopodia formation or matrix degradation. However, overexpression of FAK could actually suppress invadopodia formation and matrix degradation. FAK phosphorylation on the putative auto-phosphorylation tyrosine 397 and the Src-specific sites are all required for overexpressed FAK to inhibit invadopodia formation, while the kinase activity of exogenous FAK is apparently not required. These data imply that kinase activities other than FAK auto-phosphorylation may contribute to the phosphorylation of FAK tyrosine 397 in some contexts to promote an activity of FAK that can counteract invadopodia formation. Further work is required to determine how the strength of signalling through FAK suppresses invadopodia, but we propose that FAK controls the balance of adhesion types in cells, and that this is one of the determinants of whether a cancer cell can make stable matrix-degrading invadopodia.     

Expression and function of matrix metalloproteinase (MMP)-28

Matrix metalloproteinase-28 (MMP-28, epilysin) is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues. In epithelial cells, over-expression of MMP-28 mediates irreversible epithelial to mesenchymal transition concomitant with loss of E-cadherin from the cell surface and an increase in active transforming growth factor beta. We recently reported the expression of MMP-28 in both cartilage and synovium where expression is increased in patients with osteoarthritis.In human chondrosarcoma cells MMP-28 was activated by proprotein convertases and the active form of the enzyme preferentially associated with the extracellular matrix in a C-terminal independent manner. over-expression of MMP-28 in chondrosarcoma cells led to altered cell morphology with increased organisation of actin. Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased. MMP-28 was localised to the cell surface, at least transiently, in a C-terminal dependent manner. Heparin prevented both extracellular matrix association and cell surface binding of MMP-28 suggesting that both are via heparan sulphate proteoglycans. Over-expression of activatable MMP-28, but not catalytically inactive EA mutant increased the expression and activity of MMP-2, and all forms of MMP-28 tested increased expression of MMP19 and TIMP3 mRNA.These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour. Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.