Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.     

Components, structure, biogenesis and function of the Hydra extracellular matrix in regeneration, pattern formation and cell differentiation

The body wall of Hydra is organized as an epithelial bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM), termed mesoglea by early biologists. Morphological studies have determined that Hydra ECM is composed of two basal lamina layers positioned at the base of each epithelial layer with an intervening interstitial matrix. Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrate species. These components include such macromolecules as laminin, type IV collagen, and various fibrillar collagens. These components are synthesized in a complicated manner involving cross-talk between the epithelial bilayer. Any perturbation to ECM biogenesis leads to a blockage in Hydra morphogenesis. Blockage in ECM/cell interactions in the adult polyp also leads to problems in epithelial transdifferentiation processes. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. This is in contrast to the more rigid matrices often found in vertebrates. The flexible nature of Hydra ECM can in part now be explained by the unique structure of the organism's type IV collagen and fibrillar collagens. This review will focus on Hydra ECM in regard to: 1) its general structure, 2) its molecular composition, 3) the biophysical basis for the flexible nature of Hydra's ECM, 4) the relationship of the biogenesis of Hydra ECM to regeneration of body form, and 5) the functional role of Hydra ECM during pattern formation and cell differentiation.     

Characterization of a biofilm-like extracellular matrix in FLO1-expressing Saccharomyces cerevisiae cells

Like bacteria, fungi growing in biofilms are often embedded in a so-called extracellular matrix (ECM), a complex and species-specific mixture of compounds secreted by cells in the biofilm. The precise physiological role of this ECM and its importance for the stress and drug resistance that is so characteristic of biofilms remain vague. Here, we describe the discovery of an ECM produced by flocculating Saccharomyces cerevisiae cells. Although S. cerevisiae has long been believed not to produce an ECM, our results indicate that flocculating cells secrete a mixture of glucose and mannose polysaccharides that surrounds flocculating cells. This matrix impedes the penetration of large molecules into the floc, but does not seem to play a role in the resistance of flocculating cultures to drugs and ethanol. Together, our results provide a new model system to study the formation and biological role of microbial extracellular matrices.     

Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases

As a member of the phylum Cnidaria, the body wall of hydra is organized as an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM). Previous studies have established the general molecular structure of hydra ECM and indicate that it is organized as two subepithelial zones that contain basement membrane components such as laminin and a central fibrous zone that contains interstitial matrix components such as a unique type I fibrillar collagen. Because of its simple structure and high regenerative capacity, hydra has been used as a developmental model to study cell-ECM interaction during epithelial morphogenesis. The current study extends previous studies by focusing on the relationship of ECM biogenesis to epithelial morphogenesis in hydra, as monitored during head regeneration or after simple incision of the epithelium. Histological studies indicated that decapitation or incision of the body column resulted in an immediate retraction of the ECM at the wound site followed by a re-fusion of the bilayer within 1 hour. After changes in the morphology of epithelial cells at the regenerating pole, initiation of de novo biogenesis of an ECM began within hours while full reformation of the mature matrix required approximately 2 days. These processes were monitored using probes to three matrix or matrix-associated components: basement membrane-associated hydra laminin beta1 chain (HLM-beta1), interstitial matrix-associated hydra fibrillar collagen (Hcol-I) and hydra matrix metalloproteinase (HMMP). While upregulation of mRNA for both HLM-beta1 and Hcol-I occurred by 3 hours, expression of the former was restricted to the endoderm and expression of the latter was restricted to the ectoderm. Upregulation of HMMP mRNA was also associated with the endoderm and its expression paralleled that for HLM-beta1. As monitored by immunofluorescence, HLM-beta1 protein first appeared in each of the two subepithelial zones (basal lamina) at about 7 hours, while Hcol-I protein was first observed in the central fibrous zone (interstitial matrix) between 15 and 24 hours. The same temporal and spatial expression pattern for these matrix and matrix-associated components was observed during incision of the body column, thus indicating that these processes are a common feature of the epithelium in hydra. The correlation of loss of the ECM, cell shape changes and subsequent de novo biogenesis of matrix and matrix-associated components were all functionally coupled by antisense experiments in which translation of HLM-beta1 and HMMP was blocked and head regeneration was reversibly inhibited. In addition, inhibition of translation of HLM-beta1 caused an inhibition in the appearance of Hcol-I into the ECM, thus suggesting that binding of HLM-beta1 to the basal plasma membrane of ectodermal cells signaled the subsequent discharge of Hcol-I from this cell layer into the newly forming matrix. Given the early divergence of hydra, these studies point to the fundamental importance of cell-ECM interactions during epithelial morphogenesis.     

MMP Secretion Rate and Inter-invadopodia Spacing Collectively Govern Cancer Invasiveness

Invadopodia are micron-sized invasive structures that mediate extracellular matrix (ECM) degradation through a combination of membrane-bound and soluble matrix metalloproteinases (MMPs). However, how such localized degradation is converted into pores big enough for cancer cells to invade, and the relative contributions of membrane-bound versus soluble MMPs to this process remain unclear. In this article, we address these questions by combining experiments and simulations. We show that in MDA-MB-231 cells, an increase in ECM density enhances invadopodia-mediated ECM degradation and decreases inter-invadopodia spacing. ECM degradation is mostly mediated by soluble MMPs, which are activated by membrane-bound MT1-MMP. We present a computational model of invadopodia-mediated ECM degradation, which recapitulates the above observations and identifies MMP secretion rate as an important regulator of invadopodia stability. Simulations with multiple invadopodia suggest that inter-invadopodia spacing and MMP secretion rate collectively dictate the size of the degraded zones. Taken together, our results suggest that for creating pores conducive for cancer invasion, cells must tune inter-invadopodia spacing and MMP secretion rate in an ECM density-dependent manner, thereby striking a balance between invadopodia penetration and ECM degradation.     

Fenofibrate Attenuated Glucose-Induced Mesangial Cells Proliferation and Extracellular Matrix Synthesis via PI3K/AKT and ERK1/2

Excess mesangial extracellular matrix (ECM) and mesangial cell proliferation is the major pathologic feature of diabetic nephropathy (DN). Fenofibrate, a PPARα agonist, has been shown to attenuate extracellular matrix formation in diabetic nephropathy. However, the mechanisms underlying this effect remain to be elucidated. In this study, the effect of fenofibrate on high-glucose induced cell proliferation and extracellular matrix exertion and its mechanisms were investigated in cultured rat mesangial cells by the methylthiazoletetrazolium (MTT) assay, flow cytometry and western blot. The results showed that treatment of mesangial cells (MCs) with fenofibrate repressed high-glucose induced up-regulation of extracellular matrix Collagen-IV, and inhibited entry of cell cycle into the S phase. This G1 arrest and ECM inhibition was caused by the reduction of phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT. On the contrary, PPARα siRNA accelerated high glucose-induced cell cycle progression by ERK1/2 and AKT activation. Taken together, fenofibrate ameliorated glucose-induced mesangial cell proliferation and matrix production via its inhibition of PI3K/AKT and ERK1/2 signaling pathways. Such mechanisms may contribute to the favorable effects of treatment using fenofibrate in diabetic nephropathy.     

An antibody that inhibits fibronectin-independent adhesion of fibroblasts to extracellular matrix material

Chinese hamster ovary (CHO) fibroblasts adhere to the extracellular matrix by both fibronectin-dependent and -independent mechanisms (Harper and Juliano, 1981a,b). Previous studies have suggested that a trypsin-sensitive, 265,000-dalton membrane glycoprotein (gp265) is involved in the fibronectin-independent adhesion process. Using a polyclonal antibody against soluble products obtained from trypsin-treated CHO cells, we have been able to further analyze this involvement. This antibody immunoprecipitates a trypsin-sensitive 265,000-dalton protein from detergent-solubilized cells. Incubation of AdvF11, a variant cell line that does not utilize fibronectin for adhesion, with this antibody blocks their adhesion to extracellular matrix material (ECM). The immunoglobulin fraction will also partially block adhesion of the parental cell line to ECM particularly when the ECM is first treated with an antifibronectin antibody. Taken together these results add support for the involvement of gp265 in fibronectin-independent adhesion and provide a methodology for further characterization.     

Fibronectin-based masking molecule blocks platelet adhesion

Vessel wall extracellular matrix, which underlies the endothelium, is a potent stimulator of platelet adhesion and activation. Exposure of this matrix can result from damage incurred by vascular interventions, such as saphenous vein bypass grafting and angioplasty. Fibrillar collagens are an important component of the thrombogenic extracellular matrix. Herein we describe a means of targeting poly(ethylene glycol) (PEG)-mediated blockade directly to platelet-binding ECM molecules, such as type I collagen, thereby selectively blocking platelet adhesion to vascular matrix. Purified fibronectin (FN), a matrix protein that interacts with fibrillar collagens and platelets, was selectively pegylated to generate a targeted molecular shielding reagent that masked ECM ligands from platelet recognition and adhesion. This approach protects the functions of other vascular proteins, including surface proteins on intact endothelium. To mask the platelet-binding site of FN, PEG-propyl moieties (5000 Da) were covalently appended to lysine residues on the surface of FN, generating FNPEG-5K. To preserve the collagen-binding function of FN, it was pegylated while bound to a gelatin agarose matrix. We demonstrate that FNPEG-5K blocks platelet adhesion to purified type I collagen. Moreover, the same preparation blocks platelet adhesion to vascular wall components, including collagens.     

Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I

Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF- I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors.     

Selective integrin subunit reduction disrupts fibronectin extracellular matrix deposition and fibrillin 1 gene expression

Integrins are transmembrane receptors that can specifically bind extracellular matrix (ECM) proteins. Assembly of the ECM protein fibronectin into fibrils has been shown to be a cell-mediated process that requires integrins. Like fibronectin, fibrillin 1 is an ECM glycoprotein that can assemble into fibrils, but the role of integrins in fibril formation is not understood. To investigate the role of integrins in fibrillin 1 ECM deposition, cells that normally produce and assemble fibrillin 1 fibers in vitro were stably transfected with plasmid constructs encoding short interfering RNAs that target specific integrin subunits. Cells that were deficient in α2- and β3-integrin subunits produced and deposited fibronectin normally, but cells that were deficient for α5 and αV were unable to elaborate a fibronectin matrix, although they continued to produce and secrete the protein. Surprisingly, the cells that were unable to elaborate a fibronectin matrix also lost fibrillin 1 gene expression.     

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.     

Velocity of Ellman's reaction and its implication for kinetic studies in the millisecond time range

A detailed study of the velocity of the reaction between Ellman's reagent and thiocholine was undertaken, in order to test the possibilities of this reaction as a detection method for the earlier stages of cholinesterases reactions. Experiments were carried out on a stopped-flow apparatus with a built-in spectrophotometer. The obtained experimental data were analyzed by fitting the data to theoretical kinetic equations derived for the reaction. In this way, a complete kinetic characterization of the reaction was obtained. An important practical result derived from our investigations is the finding that, under most experimental conditions, the Ellman's reactions is more than sufficiently rapid as a detection method. However, in the case of reactions in the time scale of 200 milliseconds or less, this being 5 times the half life of Ellman's reaction at standard conditions, one has to consider the interference of this reaction with the enzyme reaction itself.     

Growth factor-extracellular matrix synergy in the control of trophoblast invasion

At the periphery of the human placenta, trophoblast attaches to the uterine wall. The tissue interface contains many anchoring sites, with cytotrophoblast columns that form bridges between the overlying extraembryonic (villous) mesenchyme and the maternal decidual stroma beneath. From the periphery of these columns, large numbers of trophoblast cells detach, migrate through the decidua and eventually colonize and transform maternal arteries. In this way the placenta increases and gives priority to the maternal blood supply to the conceptus. We have shown that when early villous tissue is explanted on a collagen gel in serum-free medium, anchoring-site morphogenesis occurs. Thus, in the presence of placental mesenchyme but in the absence of maternal cells, contact with a permissive extracellular matrix (ECM) is necessary and sufficient for cytotrophoblast column development. Proliferation of trophoblast occurs, followed by differentiation into a columnar cell phenotype in which cells remain attached to one another and to the ECM. At this stage, interaction between fibronectin and integrin alpha5beta1 at the cell surface stabilizes the column and the cells remain as a contiguous multilayered sheet. However, the addition of serum-free conditioned medium from first-trimester placental fibroblasts stimulates cytotrophoblast to detach from the distal column and migrate in streams across the ECM. The removal of insulin-like growth factor I (IGF-I) from the fibroblast medium decreases streaming activity, whereas the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produces a streaming phenotype. In contrast, transforming growth factor beta1 (10 ng/ml) maintains the cells in a tight sheet. These results suggest the possibility of a paracrine interaction between villous mesenchyme and cytotrophoblast in anchoring sites to stimulate the infiltration of the maternal ECM by trophoblast. Such a mechanism would be self-limiting because the signal diminishes with distance from the placenta.     

The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico "matrisome" defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.     

Autogenic feeder free system from differentiated mesenchymal progenitor cells,maintains pluripotency of the MEL-1 human embryonic stem cells

Human embryonic stem cells (hESc) are known for its pluripotency and self renewal capability, thus possess great potential in regenerative medicine. However, the lack of suitable xenofree extracellular matrix substrate inhibits further applications or the use of hESc in cell-based therapy. In this study, we described a new differentiation method, which generates a homogeneous population of mesenchymal progenitor cells (hESc–MPC) from hESc via epithelial–mesenchymal transition. The extracellular matrix (ECM) proteins from hESc–MPC had in turn supported the undifferentiated expansion of hESc. Immunocytochemistry and flow cytometry characterization of hESc–MPC revealed the presence of early mesenchymal markers. Tandem mass spectometry analysis of ECM produced by hESc–MPC revealed the presence of a mixture of extracellular proteins which includes tenascin C, fibronectin, and vitronectin. The pluripotency of hESc (MEL-1) cultured on the ECM was maintained as shown by the expression of pluripotent genes (FoxD3, Oct-4, Tdgf1, Sox-2, Nanog, hTERT, Rex1), protein markers (SSEA-3, SSEA-4, TRA-1-81, TRA-1-60, Oct-4) and the ability to differentiate into cells representative of ectoderm, endoderm and mesoderm. In summary, we have established a xeno-free autogenic feeder free system to support undifferentiated expansion of hESc, which could be of clinical relevance.     

Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells

Background  

Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.     

Trichostatin A inhibits beta-casein expression in mammary epithelial cells.

Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous beta-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of beta-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types.     

Exosome-mediated transduction of mechanical force regulates prostate cancer migration via microRNA

Physical cues in the extracellular microenvironment regulate cancer cell metastasis. Functional microRNA (miRNA) carried by cancer derived exosomes play a critical role in extracellular communication between cells and the extracellular microenvironment. However, little is known about the role of exosomes loaded miRNAs in the mechanical force transmission between cancer cells and extracellular microenvironment. Herein, our results suggest that stiff extracellular matrix (ECM) induced exosomes promote cancer cell migration. The ECM mechanical force regulated the exosome miRNA cargo of prostate cancer cells. Exosome miRNAs regulated by the ECM mechanical force modulated cancer cell metastasis by regulating cell motility, ECM remodeling and the interaction between cancer cells and nerves. Focal adhesion kinase mediated-ECM mechanical force regulated the intracellular miRNA expression, and F-actin mediate-ECM mechanical force regulated miRNA packaging into exosomes. The above results demonstrated that the exosome miRNA cargo promoted cancer metastasis by transmitting the ECM mechanical force. The ECM mechanical force may play multiple roles in maintaining the microenvironment of cancer metastasis through the exosome miRNA cargo.