Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates neuromuscular junction development via a beta1 integrin-mediated mechanism

Extracellular matrix (ECM) molecules play critical roles in muscle function by participating in neuromuscular junction (NMJ) development and the establishment of stable, cytoskeleton-associated adhesions required for muscle contraction. Matrix metalloproteinases (MMPs) are neutral endopeptidases that degrade all ECM components. While the role of MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), has been investigated in many tissues, little is known about their role in muscle development and mature function. TIMP-2 -/- mice display signs of muscle weakness. Here, we report that TIMP-2 is expressed at the NMJ and its expression is greater in fast-twitch (extensor digitorum longus, EDL) than slow-twitch (soleus) muscle. EDL muscle mass is reduced in TIMP-2-/- mice without a concomitant change in fiber diameter or number. The TIMP-2-/- phenotype is not likely due to increased ECM proteolysis because net MMP activity is actually reduced in TIMP-2-/- muscle. Most strikingly, TIMP-2 colocalizes with beta1 integrin at costameres in the wild-type EDL and beta1 integrin expression is significantly reduced in TIMP-2-/- EDL. We propose that reduced beta1 integrin in fast-twitch muscle may be associated with destabilized ECM-cytoskeletal interactions required for muscle contraction in TIMP-2-/- muscle; thus, explaining the muscle weakness. Given that fast-twitch fibers are lost in muscular dystrophies and age-related sarcopenia, if TIMP-2 regulates mechanotransduction in an MMP-independent manner it opens new potential therapeutic avenues.     

Matrix metalloproteinase-2 is involved in A549 cell migration on laminin-10/11

We have reported that laminin-10/11 strongly promotes migration of A549 human lung carcinoma cells by activating the alpha3beta1 integrin-dependent signaling pathway. To elucidate the mechanism involved, we investigated whether matrix metalloproteinases (MMPs) are involved in cell migration on laminin-10/11. Here, we demonstrate that laminin-10/11, but not fibronectin which does not greatly promote A549 cell movement, stimulated MMP-2 secretion approximately 3-fold. The cell migration-promoting activity of laminin-10/11 was down-regulated by an MMP inhibitor. In addition, cell motility was significantly increased when cells adhered to a mixture of fibronectin and laminin-10/11 with a concomitant decrease of focal contacts, compared with those adhering to fibronectin alone. The enhanced cell migration was partially suppressed by the MMP inhibitor. Furthermore, an anti-alpha3 integrin, but not an anti-alpha5 integrin, antibody induced the activated form of MMP-2. These data suggest that MMP-2 may play an important role in A549 cell migration on laminin-10/11 through an alpha3beta1 integrin-dependent pathway.     

Tissue inhibitor of metalloproteinase-2 (TIMP-2) expression is regulated by multiple neural differentiation signals

Neuronal differentiation requires exquisitely timed cell cycle arrest for progenitors to acquire an appropriate neuronal cell fate and is achieved by communication between soluble signals, such as growth factors and extracellular matrix molecules. Here we report that the expression of TIMP-2, a matrix metalloproteinase inhibitor, is up-regulated by signals that control proliferation (bFGF and EGF) and differentiation (retinoic acid and NGF) in neural progenitor and neuroblastoma cell lines. TIMP-2 expression coincides with the appearance of neurofilament-positive neurons, indicating that TIMP-2 may play a role in neurogenesis. The up-regulation of TIMP-2 expression by proliferate signals suggests a role in the transition from proliferation to neuronal differentiation. Live labeling experiments demonstrate TIMP-2 expression only on alpha(3) integrin-positive cells. Thus, TIMP-2 function may be mediated via interaction with integrin receptor(s). We propose that TIMP-2 represents a component of the neurogenic signaling cascade induced by mitogenic stimuli that may withdraw progenitor cells from the cell cycle permitting their terminal neuronal differentiation.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metallorpoteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.     

Tissue inhibitor of metalloproteinase-2 (TIMP-2) has erythroid-potentiating activity.

Tissue inhibitor of metalloproteinase (TIMP) was purified and molecularly cloned on the basis of its erythroid-potentiating activity (EPA). TIMP/EPA appears to be a bifunctional molecule with both growth factor and anti-enzymatic activity. Recently, a second TIMP-related molecule was identified and we have investigated its possible erythroid-potentiating activity. Native, purified human TIMP-2 was assayed for erythroid-potentiating activity using an in vitro erythroid burst formation assay and was compared with that of previously characterized recombinant EPA/TIMP-1. The results demonstrate that both members of the tissue inhibitor of metalloproteinase family, TIMP-1 and TIMP-2, possessed erythroid potentiating activity which was inhibited by antibodies developed to neutralize EPA. These results suggest that TIMP-2 shares a common structural domain with EPA/TIMP-1 that is responsible for the erythroid-potentiating activity of these inhibitors. Therefore, TIMP-1 and TIMP-2, with both anti-protease activity and growth factor activity, join a family of bifunctional molecules such as fibroblast growth factor and thrombin which have both enzymatic and growth factor activity.     

Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin

Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.     

Tissue inhibitor of metalloproteinase-2(TIMP-2)-deficient mice display motor deficits

The degradation of the extracellular matrix is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Matrix components of the basement membrane play critical roles in the development and maintenance of the neuromuscular junction (NMJ), yet almost nothing is known about the regulation of MMP and TIMP expression in either the pre- or postsynaptic compartments. Here, we demonstrate that TIMP-2 is expressed by both spinal motor neurons and skeletal muscle. To determine whether motor function is altered in the absence of TIMP-2, motor behavior was assessed using a battery of tests (e.g., RotaRod, balance beam, hindlimb extension, grip strength, loaded grid, and gait analysis). TIMP-2(-/-) mice fall off the RotaRod significantly faster than wild-type littermates. In addition, hindlimb extension is reduced and gait is both splayed and lengthened in TIMP-2(-/-) mice. Motor dysfunction is more pronounced during early postnatal development. A preliminary analysis revealed NMJ alterations in TIMP-2(-/-) mice. Juvenile TIMP-2(-/-) mice have increased nerve branching and acetylcholine receptor expression. Adult TIMP-2(-/-) endplates are enlarged and more complex. This suggests a role for TIMP-2 in NMJ sculpting during development. In contrast to the increased NMJ nerve branching, cerebellar Purkinje cells have decreased neurite outgrowth. Thus, the TIMP-2(-/-) motor phenotype is likely due to both peripheral and central defects. The tissue specificity of the nerve branching phenotype suggests the involvement of different MMPs and/or extracellular matrix molecules underlying the TIMP-2(-/-) motor phenotype.     

MMP-9 and MMP-2 gelatinases and TIMP-1 and TIMP-2 inhibitors in breast cancer: correlations with prognostic factors

The goal of our study was to analyse the prognostic values for some matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in breast cancer. We evaluated the activity and the expression levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 in malignant versus benign fresh breast tumor extracts. For this purpose, gelatinzymography, immunoblotting and ELISA were used to analyse the activity and expression of MMPs and TIMPs. We found that MMP-9 expression level and activity are increased in malignant tumors. In addition, MMP-9/TIMP-1 and MMP-2/TIMP-2 ratio values obtained by us were significantly different in malignant tumors compared to benign tumors. We suggest that the abnormal MMP-9/TIMP-1 balance plays a role in the configuration of breast invasive carcinoma of no special type and also in tumor growth, while altered MMP-2/TIMP-2 ratio value could be associated with lymph node invasion and used as a prognostic marker in correlation with Nottingham Prognostic Index. Finally, we showed that in malignant tumors high expression of estrogen receptors is associated with enhanced activity of MMP-2 and increased bcl- 2 levels, while high expression of progesterone receptors is correlated with low TIMP-1 protein levels.     

Effects of E-cadherin on mouse embryo implantation and expression of matrix metalloproteinase-2 and -9

E-cadherin is a cell surface glycoprotein, which is responsible for adhesion between epithelial cells. Whether it is involved in embryo implantation is still unknown. In a mouse intrauterine horn injection model, one uterine horn in each mouse was injected with different doses of E-cadherin antibody on day 3 of pregnancy. The results showed that embryo implantation was significantly inhibited in the mice injected with 3 microg E-cadherin antibody. The mouse uteri in this group were collected on days 5, 6, and 7 of pregnancy and expressions of MMP-2 and -9 were studied. In situ hybridization and RT-PCR results showed that the expression of MMP-2 and -9 mRNAs in uteri of E-cadherin antibody treated group was increased on days 5-7. The results of gelatin zymography of MMPs showed that the activities of pro-MMP-2, MMP-2, and pro-MMP-9 were increased significantly on days 5 and 6, and pro-MMP-9 activity was increased on day 7. The present study suggested that E-cadherin was involved in embryo implantation through decreasing the expressions and activities of MMP-2 and -9.     

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.     

Vascular endothelial growth factor A (VEGF-A) induces endothelial and cancer cell migration through direct binding to integrin {alpha}9{beta}1: identification of a specific {alpha}9{beta}1 binding site

Integrin α9β1 mediates accelerated cell adhesion and migration through interactions with a number of diverse extracellular ligands. We have shown previously that it directly binds the vascular endothelial growth factors (VEGF) A, C, and D and contributes to VEGF-induced angiogenesis and lymphangiogenesis. Until now, the α9β1 binding site in VEGF has not been identified. Here, we report that the three-amino acid sequence, EYP, encoded by exon 3 of VEGF-A is essential for binding of VEGF to integrin α9β1 and induces adhesion and migration of endothelial and cancer cells. EYP is specific for α9β1 binding and neither requires nor activates VEGFR-2, the cognate receptor for VEGF-A. Following binding to EYP, integrin α9β1 transduces cell migration through direct activation of the integrin signaling intermediates Src and focal adhesion kinase. This interaction is biologically important because it mediates in vitro endothelial cell tube formation, wound healing, and cancer cell invasion. These novel findings identify EYP as a potential site for directed pharmacotherapy.     

Interleukin-4 improves the migration of human myogenic precursor cells in vitro and in vivo

Different molecules are available to recruit new neighboring myogenic cells to the site of regeneration. Formerly called B cell stimulatory factor-1, IL-4 can now be included in the list of motogenic factors. The present report demonstrates that human IL-4 is not required for fusion between mononucleated myoblasts but is required for myotube maturation. In identifying IL-4 as a pro-migratory agent for myogenic cells, these results provide a mechanism which partly explains IL-4 demonstrated activity during differentiation. Among the different mechanisms by which IL-4 might enhance myoblast migration processes, our results indicate that there are implications of some integrins and of three major components of the fibrinolytic system. Indeed, increases in the amount of active urokinase plasminogen activator and its receptor were observed following an IL-4 treatment, while the plasminogen activator inhibitor-1 decreased. Finally, IL-4 did not modify the amount of cell surface alpha5 integrin but increased the presence of beta3 and beta1 integrins. This integrin modulation might favor myogenic cell migration and its interaction with newly formed myotubes. Therefore, IL-4 co-injection with transplanted myoblasts might be an approach to enhance the migration of transplanted cells for the treatment of a damaged myocardium or of a Duchenne Muscular Dystrophy patient.     

Differential regulation of Na(v)beta subunits during myogenesis

Voltage-gated sodium channels (Na_v) consist of a pore-forming α subunit (Na_vα) associated with β regulatory subunits (Na_vβ). Adult skeletal myocytes primarily express Na_v1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na_v1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na_v1.5 currents than those recorded from myoblasts. Unlike myoblasts, I_Na recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na_vβ subunits modulate channel gating and pharmacology, the goal of the present work was to study Na_vβ subunits during myogenesis. All four Na_vβ (Na_vβ1-4) isoforms were present in L6E9 myocytes. While Na_vβ1-3 subunits were up-regulated by myogenesis, Na_vβ4 subunits were not. These results show that Na_vβ genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na⁺ channels during development and myotube formation.     

Pitx2c modulates Pax3+/Pax7+ cell populations and regulates Pax3 expression by repressing miR27 expression during myogenesis

Pitx2 is a paired-related homeobox gene that is expressed in muscle progenitors during myogenesis. We have previously demonstrated that overexpression of Pitx2c isoform in myoblasts maintained these cells with a high proliferative capacity and completely blocked terminal differentiation by inducing high Pax3 expression levels (Martinez et al., 2006). We now report that Pitx2c-mediated proliferation vs. differentiation effect is maintained during in vivo myogenesis. In vivo Pitx2c loss of function leads to a decrease in Pax3+/Pax7− cell population in the embryo accompanied by an increase of Pax3+/Pax7+ cells. Pitx2c transient-transfection experiments further supported the notion that Pitx2c can modulate Pax3/Pax7 expression. Pitx2c but not Pitx3 controls Pax3/Pax7 expression, although redundant roles are elicited at the terminal myoblast differentiation. Contrary to Pitx2c, Pitx3 does not regulate cell proliferation or Pax3 expression, demonstrating the specificity of Pitx2c mediating these actions in myoblasts. Furthermore we demonstrated that Pitx2c modulates Pax3 by repressing miR27 expression and that Pax3-miR-27 modulation mediated by Pitx2c is independent of Pitx2c effects on cell proliferation. Therefore, this study sheds light on previously unknown function of Pitx2c balancing the different myogenic progenitor populations during myogenesis.     

Mitofusin 2 (Mfn2): a key player in insulin-dependent myogenesis in vitro

We have previously shown that mitochondrial activity increases in response to insulin in differentiating muscle cells. Moreover, the protein kinase kinase/extracellular-signal-regulated kinase (MAPKK/ERK-MEK) inhibitor PD98059 accelerates insulin-mediated myogenesis, whereas the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or blockade of mitochondrial respiration abrogates insulin-mediated myogenesis. Our present study focuses on the mitochondrial transmembrane protein, hyperplasia suppressor gene/mitofusin2 (HSG/Mfn2), which regulates both mitochondrial fusion (as demonstrated by perinuclear mitochondria clustering) and insulin-dependent myogenesis in vitro. Increased mitochondrial length and interconnectivity are not observed after the inhibition of PI3-K activity with LY294002. Insulin induces Mfn2 and subunits I and IV of cytochrome-c oxidase (MTCOI and NCOIV) in L6 myoblasts. Inhibition of the MEK-dependent signalling pathway elevates the Mfn-2 protein level. The molecular mechanism of this phenomenon is unknown, although immunoprecipitation studies indicate that, during insulin-mediated myogenesis, Ras protein (an upstream activator of the MAPK/ERK1/2 cascade) interacts with HSG/Mfn2 in muscle cells. Interaction of Ras with Mfn2 continues unless insulin is present and is reduced after PD98059 co-treatment indicating that insulin-mediated myogenesis is increased by the inhibition of MEK, most probably by the lack of mitogenic signals opposing muscle differentiation. We conclude that insulin-mediated myogenesis depends on PI3-K activity, which stimulates mitochondrial activity and the extensive fusion of mitochondria. We further suggest that insulin stimulates the expression of Mfn2 protein, which in turn binds to Ras and inhibits the MEK-dependent signalling pathway. At the same time, the PI3-K-dependent signalling pathway is boosted, mitochondrial respiration increases and the rate of myogenesis is accelerated. This work was supported by the State Committee for Scientific Research in Poland (grant no. 2 P06D 015 29) and by grant no. 117/E-385/SPB/COST/P-06/DWM within the framework of COST 925 Action on “The importance of prenatal events for postnatal muscle growth in relation to the quality of muscle based foods”.     

Identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human Fas ligand

Soluble Fas ligand (sFasL) is released from the cell surface by matrix metalloproteinases (MMPs), one of which is MMP-7. We have reported that MMP-7-generated sFasL is pro-apoptotic in both in vitro and in vivo systems. However, there are contradictory reports that the soluble form of FasL is inactive or anti-apoptotic, resulting in significant controversy in the literature. One potential explanation for these discrepancies is that forms of sFasL with different amino-terminal sequences have been demonstrated to have varying activities. Here we report that MMP-7 cleaves murine and human FasL at sites that are distinct from previously reported cleavage sites resulting in production of novel forms of sFasL. Cleavage of FasL by MMP-7 occurs at the leucine residues in the sequence "ELAELR" within the region between the transmembrane and trimerization domains. When this site is unavailable, a more c-terminal site, "SL," is cleaved. MMP-7 differentially processes murine and human FasL since it cleaves human FasL not only at the "ELAELR" site but also at a previously identified site. Additionally, MMP-3, but not MMP-2, was found to have the same cleavage specificity for murine FasL as MMP-7. We conclude that the controversy regarding the biological activity of sFasL may be explained, in part, by the generation of distinct forms of sFasL as a result of cleavage at specific sequences.     

Type XVII collagen regulates lamellipod stability, cell motility, and signaling to Rac1 by targeting bullous pemphigoid antigen 1e to alpha6beta4 integrin

Rac1 activity, polarity, lamellipodial dynamics, and directed motility are defective in keratinocytes exhibiting deficiency in β4 integrin or knockdown of the plakin protein Bullous Pemphigoid Antigen 1e (BPAG1e). The activity of Rac, formation of stable lamellipodia, and directed migration are restored in β4 integrin-deficient cells by inducing expression of a truncated form of β4 integrin, which lacks binding sites for BPAG1e and plectin. In these same cells, BPAG1e, the truncated β4 integrin, and type XVII collagen (Col XVII), a transmembrane BPAG1e-binding protein, but not plectin, colocalize along the substratum-attached surface. This finding suggested to us that Col XVII mediates the association of BPAG1e and α6β4 integrin containing the truncated β4 subunit and supports directed migration. To test these possibilities, we knocked down Col XVII expression in keratinocytes expressing both full-length and truncated β4 integrin proteins. Col XVII-knockdown keratinocytes exhibit a loss in BPAG1e-α6β4 integrin interaction, a reduction in lamellipodial stability, an impairment in directional motility, and a decrease in Rac1 activity. These defects are rescued by a mutant Col XVII protein truncated at its carboxyl terminus. In summary, our results suggest that in motile cells Col XVII recruits BPAG1e to α6β4 integrin and is necessary for activation of signaling pathways, motile behavior, and lamellipodial stability.     

基质金属蛋白酶及其抑制剂在肝纤维化中的表达变化

目的 观察肝纤维化形成过程中基质金属蛋白酶MMP-1及其抑制剂TIMP-1的表达变化,从细胞外基质降解代谢的角度研究四氯化碳(CCl4)中毒性肝纤维化发生的机制.方法 雄性Wistar大鼠20只,分为正常组和肝纤维化模型组.肝纤维化组采用CCl4、饮酒、高脂低蛋白饮食等复合病因刺激制备肝纤维化动物模型,造模时间为8周.实验结束后测定肝脏指数、血清透明质酸(HA)、谷丙转氨酶(ALT)及尿羟脯氨酸(HYP)排出量,光镜下观察肝组织纤维化程度,并用免疫组化SABC法检测肝组织中Ⅰ、Ⅲ型胶原蛋白及MMP-1、TIMP-1的表达,同时用荧光实时定量PCR(RT-PCR)的方法检测肝组织中MMP-1、TIMP-1 mRNA的表达.结果 与正常对照组比较,肝纤维化模型组大鼠肝脏指数、血清HA及ALT显著增高,尿羟脯氨酸的排出量明显增加,病理组织学检查发现肝组织内纤维结缔组织增生明显,有假小叶形成;免疫组化的结果显示肝组织内Ⅰ、Ⅲ型胶原蛋白、MMP-1及TIMP-1的表达较正常组显著增加.结论 肝组织中MMP-1及TIMP-1的表达变化可能是导致肝纤维化的重要机制之一.