Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the KD was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and >100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pKa values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate- and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.     

STIM1- and Orai1-mediated Ca2+ oscillation orchestrates invadopodium formation and melanoma invasion

Ca²⁺ signaling has been increasingly implicated in cancer invasion and metastasis, and yet, the underlying mechanisms remained largely unknown. In this paper, we report that STIM1- and Orai1-mediated Ca²⁺ oscillations promote melanoma invasion by orchestrating invadopodium assembly and extracellular matrix (ECM) degradation. Ca²⁺ oscillation signals facilitate invadopodial precursor assembly by activating Src. Disruption of Ca²⁺ oscillations inhibited invadopodium assembly. Furthermore, STIM1 and Orai1 regulate the proteolysis activity of individual invadopodia. Mechanistically, Orai1 blockade inhibited the recycling of MT1–matrix metalloproteinase (MMP) to the plasma membrane and entrapped MT1-MMP in the endocytic compartment to inhibit ECM degradation. STIM1 knockdown significantly inhibited melanoma lung metastasis in a xenograft mouse model, implicating the importance of this pathway in metastatic dissemination. Our findings provide a novel mechanism for Ca²⁺-mediated cancer cell invasion and shed new light on the spatiotemporal organization of store-operated Ca²⁺ signals during melanoma invasion and metastasis.     

PpAtg9 trafficking during micropexophagy in Pichia pastoris

PpAtg9 is essential for the selective degradation of peroxisomes (e.g., pexophagy) in Pichia pastoris. This integral membrane protein is synthesized in the endoplasmic reticulum (ER) and transported to a unique peripheral compartment (Atg9-PC). A putative ER exit motif has been identified and when deleted results in the accumulation of PpAtg9 within the ER. Upon the onset of micropexophagy, PpAtg9 transits from the Atg9-PC to perivacuolar structures (PVS) and sequestering membranes (SM) that arise from the vacuole to engulf the peroxisomes. In this article, we will discuss the transport pathways of PpAtg9 and those factors responsible for its trafficking.     

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.     

Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP-Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.     

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix

A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be -190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G. metallireducens cytochrome c did not show Fe(III)-reducing activity.     

Nardilysin, a basic residues specific metallopeptidase that mediates cell migration and proliferation

Nardilysin (NRDc), a metallopeptidase of the M16 family, presents, in vitro, cleavage specificity for basic residues. Depending on the cell type, it is cytoplasmic, exported or cell surface associated. As a new receptor for heparin-binding EGF-like growth factor (HB-EGF), NRDc was recently shown to be involved in cellular migration and proliferation. Since for those processes its enzymatic activity is not required, it is now evident that nardilysin fulfills at least two distinct functions, i.e. an HB-EGF modulator and a peptidase.     

Annexin-1 mediates TNF-alpha-stimulated matrix metalloproteinase secretion from rheumatoid arthritis synovial fibroblasts

Annexins are intracellular molecules implicated in the down-regulation of inflammation. Recently, annexin-1 has also been identified as a secreted molecule, suggesting it may have more complex effects on inflammation than previously appreciated. We studied the role of annexin-1 in mediating MMP-1 secretion from rheumatoid arthritis (RA) synovial fibroblasts (SF) stimulated with TNF-alpha. TNF-alpha induced a biphasic secretion of annexin-1 from RA SF. Early (< or = 60 min), cycloheximide-independent secretion from preformed intracellular pools was followed by late (24 h) cycloheximide-inhibitable secretion requiring new protein synthesis. Exogenous annexin-1 N-terminal peptide Ac2-26 stimulated MMP-1 secretion in a dose- (EC(50) approximately 25 microM) and time- (8-24 h) dependent manner; full-length annexin-1 had a similar effect. Down-regulation of annexin-1 using small interfering RNA resulted in decreased secretion of both annexin-1 and MMP-1, confirming that annexin-1 mediates TNF-alpha-stimulated MMP-1 secretion. Erk, Jnk, and NF-kappaB have been implicated in MMP-1 secretion. Erk, Jnk, and NF-kappaB inhibitors had no effect on annexin-1 secretion stimulated by TNF-alpha but inhibited MMP-1 secretion in response to Ac2-26, indicating that these molecules signal downstream of annexin-1. Annexin-1 stimulation of MMP-1 secretion was inhibited by both a formyl peptide receptor antagonist and pertussis toxin, suggesting that secreted annexin-1 acts via formyl peptide family receptors, most likely FPLR-1. In contrast to its commonly appreciated anti-inflammatory roles, our data indicate that annexin-1 is secreted by RA SF in response to TNF-alpha and acts in an autacoid manner to engage FPRL-1, activate Erk, Jnk, and NF-kappaB, and stimulate MMP-1 secretion.     

Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.     

Intracellular trafficking and secretion of inflammatory cytokines

The secretion of cytokines by immune cells plays a significant role in determining the course of an inflammatory response. The levels and timing of each cytokine released are critical for mounting an effective but confined response, whereas excessive or dysregulated inflammation contributes to many diseases. Cytokines are both culprits and targets for effective treatments in some diseases. The multiple points and mechanisms that have evolved for cellular control of cytokine secretion highlight the potency of these mediators and the fine tuning required to manage inflammation. Cytokine production in cells is regulated by cell signaling, and at mRNA and protein synthesis levels. Thereafter, the intracellular transport pathways and molecular trafficking machinery have intricate and essential roles in dictating the release and activity of cytokines. The trafficking machinery and secretory (exocytic) pathways are complex and highly regulated in many cells, involving specialized membranes, molecules and organelles that enable these cells to deliver cytokines to often-distinct areas of the cell surface, in a timely manner. This review provides an overview of secretory pathways – both conventional and unconventional – and key families of trafficking machinery. The prevailing knowledge about the trafficking and secretion of a number of individual cytokines is also summarized. In conclusion, we present emerging concepts about the functional plasticity of secretory pathways and their modulation for controlling cytokines and inflammation.     

Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation

Phospholamban (PLB) associates with the Ca²⁺-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to -adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca²⁺-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca²⁺-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca²⁺-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca²⁺-ATPase. sarco(endo)plasmic reticulum calcium-adenosine triphosphatase; differentiation; C2C12 myocytes; vesicle trafficking     

Sphingomyelin synthases regulate protein trafficking and secretion

Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.     

Extracellular matrix dynamics during vertebrate axis formation

The first evidence for the dynamics of in vivo extracellular matrix (ECM) pattern formation during embryogenesis is presented below. Fibrillin 2 filaments were tracked for 12 h throughout the avian intraembryonic mesoderm using automated light microscopy and algorithms of our design. The data show that these ECM filaments have a reproducible morphogenic destiny that is characterized by directed transport. Fibrillin 2 particles initially deposited in the segmental plate mesoderm are translocated along an unexpected trajectory where they eventually polymerize into an intricate scaffold of cables parallel to the anterior-posterior axis. The cables coalesce near the midline before the appearance of the next-formed somite. Moreover, the ECM filaments define global tissue movements with high precision because the filaments act as passive motion tracers. Quantification of individual and collective filament "behaviors" establish fate maps, trajectories, and velocities. These data reveal a caudally propagating traveling wave pattern in the morphogenetic movements of early axis formation. We conjecture that within vertebrate embryos, long-range mechanical tension fields are coupled to both large-scale patterning and local organization of the ECM. Thus, physical forces or stress fields are essential requirements for executing an emergent developmental pattern-in this case, paraxial fibrillin cable assembly.     

EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity

EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.     

Morphogen gradient formation and vesicular trafficking

Morphogens are secreted signaling molecules which form spatial concentration gradients while moving away from a restricted source of production. A simple model of gradient formation postulates that the morphogens dilute as they diffuse between cells. In this review we discuss recent data supporting the idea that movement of the morphogen could also occur via vesicular trafficking through the cells. We explore the implications of these results for the control of gradient formation and the determination of the gradient slope which ultimately encodes the coordinates of positional information.     

Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.     

VLA-2 mediates the interaction of collagen with endothelium during in vitro vascular tube formation.

A confluent endothelial monolayer can be induced to form vascular tubes in response to collagen. We investigated possible mechanisms of collagen-induced tube formation by using antibodies to the VLA-2 integrin receptor and protein kinase C inhibitors. Pre-incubation of cells with anti-VLA-2 (which recognises both the α2 and β1 chains) and AK7 (which recognises only the α2 chain) showed a dose-dependent inhibition of tube formation. At 50 μg/ml, anti-VLA-2 completely inhibited collagen-induced tube formation, whereas AK7 caused only partial inhibition. Both chlorpromazine and trifluoperazine, at concentrations of 10μM, prevented tube formation (> 40% inhibition), In summary, the VLA-2 integrin receptor plays a role in the induction of tube formation by type I collagen. Protein kinase C may be activated during this process.     

Expression of matrix metalloproteinases-2 and -9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.