Extracellular matrix of the charophycean green algae

A comprehensive knowledge of cell wallstructure and function throughout the plant kingdom is essential to understanding cell wall evolution. The fundamental understanding of the charophycean green algal cell wall is broadening. The similarities and differences that exist between land plant and algal cell walls provide opportunities to understand plant evolution. A variety of polymers previously associated with higher plants were discovered in the charophycean green algae (CGA), including homogalacturonans, cross-linking glycans, arabinogalactan protein, β-glucans, and cellulose. The cellulose content of CGA cell walls ranged from 6% to 43%, with the higher valuescomparable to that found in the primary cell wall of land plants (20-30%). (1,3)β-glucans were found in the unicellular Chlorokybus atmophyticus, Penium margaritaceum, and Cosmarium turpini, the unbranched filamentous Klebsormidium flaccidum, and the multicellular Chara corallina. The discovery of homogalacturonan in Penium margaritaceum representsthe first confirmation of land plant-type pectinsin desmids and the second rigorous characterization of a pectin polymer from the charophycean algae. Homogalacturonan was also indicated from the basal species Chlorokybus atmophyticus and Klebsormidium flaccidum. There is evidence of branched pectins in Cosmarium turpini and linkage analysis suggests the presence of type I rhamnogalacturonan (RGI). Cross-linking β-glucans are associated with cellulose microfibrils during land plant cell growth, and were found in the cell wall of CGA. The evidence of mixed-linkage glucan (MLG) in the 11 charophytesis both suprising and significant given that MLG was once thought to be specific to some grasses. The organization and structure of Cosmarium turpini and Chara corallina MLG was found to be similar to that of Equisetumspp., whereas the basal species of the CGA, Chlorokybus atmophyticus and Klebsormidium flaccidum, have unique organization of alternating of 3- and 4-linkages. The significance of this result on the evolution of the MLG synthetic pathway has yet to be determined. The extracellular matrix (ECM) of Chlorokybus atmophyticus, Klebsormidium flaccidum, and Spirogyra spp. exhibits significant biochemical diversity, ranging from distinct “land plant” polymers to polysaccharides unique to these algae. The neutral sugar composition of Chlorokybus atmophyticus hot water extract and Spirogyra extracellular polymeric substance (EPS), combined with antibody labeling results, revealed the distinct possibility of an arabinogalactan protein in these organisms. Polysaccharide analysis of Zygnematales (desmid) EPS, indicated a probable range of different EPS backbones and substitution patterns upon the core portions of the molecules. Desmid EPS is predominately composed of a complex matrix of branched, uronic acid containing polysaccharides with ester sulfate substitutions and, as such, has an almost infinite capacity for various hydrogen bonding, hydrophobic interaction and ionic cross-bridging motifs, which characterize their unique function in biofilms. My observations support the hypothesis that members of the CGA represent the phylogenetic line that gave rise to vascular plants and that the primary cell wall of vascular plants many have evolved directly from structures typical of the cell wall of filamentous green algae found in the charophycean green algae.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein.

Dominant-negative mutations in the homopentameric extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) result in inappropriate intracellular retention of misfolded COMP in the rough endoplasmic reticulum of chondrocytes, causing chondrocyte cell death, which leads to two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). COMP null mice show no adverse effects on normal bone development and growth, suggesting a possible therapy involving removal of COMP mRNA. The goal of this study was to assess the ability of a hammerhead ribozyme (Ribo56, designed against the D469del mutation) to reduce COMP mRNA expression. In COS7 cells transfected with plasmids that overexpress wild-type or mutant COMP mRNA and Ribo56, the ribozyme reduced overexpressed normal COMP mRNA by 46% and mutant COMP mRNA by 56% in a dose-dependent manner. Surprisingly, the use of recombinant adenoviruses to deliver wild-type or mutant COMP mRNA and Ribo56 simultaneously into COS7 cells proved problematic for the activity of the ribozyme to reduce COMP expression. However, in normal human costochondral cells (hCCCs) infected only with adenoviruses expressing Ribo56, expression of endogenous wild-type COMP mRNA was reduced in a dose-dependent manner by 50%. In chondrocytes that contain heterozygous COMP mutations (D469del, G427E and D511Y) that cause PSACH, Ribo56 was more effective at reducing COMP mRNA (up to 70%). These results indicate that Ribo56 is effective at reducing mutant and wild-type COMP levels in cells and suggests a possible mode of therapy to reduce the mutant protein load.

Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

Protein cross-linking mediated by tissue transglutaminase correlates with the maturation of extracellular matrices during lung development

At birth, the mammalian lung is still immature. The alveoli are not yet formed and the interairspace walls contain two capillary layers which are separated by an interstitial core. After alveolarization (first 2 postnatal weeks in rats) the alveolar septa mature: their capillary layers merge, the amount of connective tissue decreases, and the mature lung parenchyma is formed (second and third week). During the first 3 wk of life the role of tissue transglutaminase (tTG) was studied in rat lung by immunostaining of cryostat and paraffin sections, by Northern and Western blotting, and by a quantitative determination of gamma-glutamyl-epsilon-lysine. While enzyme activity and intracellular tTG were already present before term, the enzyme product (gamma-glutamyl-epsilon-lysine-crosslink) and extracellular tTG appeared between postnatal days 10 and 19 in the lung parenchyma. In large blood vessels and large airways, which mature earlier than the parenchyma, both the enzyme product and extracellular tTG had already appeared at the end of the first postnatal week. We conclude that tTG is expressed and externalized into the extracellular matrix of lung shortly before maturation of an organ area. Because tTG covalently and irreversibly crosslinks extracellular matrix proteins, we hypothesize that it may prevent or delay further remodeling of basement membranes and may stabilize other extracellular components, such as microfibrils.

Effects of enamel matrix proteins in combination with a bovine-derived natural bone mineral for the repair of bone defects.

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

BACKGROUND The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth. METHODOLOGY/PRINCIPAL FINDINGS We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth. CONCLUSIONS/SIGNIFICANCE We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

Fibulin-2 stabilizes tumor extracellular matrix and drives malignant progression of lung adenocarcinoma

The ECM of epithelial carcinomas undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How tumors maintain ECM integrity in the face of dynamic biophysical forces is still largely unclear. This study addresses these deficiencies using mouse models of human lung adenocarcinoma. Spontaneous lung tumors were marked by disorganized basement membranes, dense collagen networks, and increased tissue stiffness. Metastasis-prone lung adenocarcinoma cells secreted fibulin-2 (Fbln2), a matrix glycoprotein involved in ECM supra-molecular assembly. Fibulin-2 depletion in tumor cells decreased the intra-tumoral abundance of matrix metalloproteinases and reduced collagen cross-linking and tumor compressive properties resulting in inhibited tumor growth and metastasis. Fbln2 deposition within intra-tumoral fibrotic bands was a predictor of poor clinical outcome in patients. Collectively, these findings support a feed-forward model in which tumor cells secrete matrix-stabilizing factors required for the assembly of ECM that preferentially favors malignant progression. To our knowledge, this is the first evidence that tumor cells directly regulate the integrity of their surrounding matrix through the secretion of matrix-stabilizing factors such as fibulin-2. These findings open a new avenue of research into matrix assembly molecules as potential therapeutic targets in cancer patients.

Interactions and structural analysis of the zinc -binding motif in decorin, a small leucine rich proteoglycan in extracellular matrix

Decorin, a dermatan/chondroitin sulfate proteoglycan, is ubiquitously distributed in the extracellular matrix (ECM) of mammals. Decorin belongs to the small leucine rich proteoglycan (SLRP) family, a proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. The decorin core protein appears to mediate the binding of decorin to ECM molecules, such as collagens and fibronectin. It is believed that the interactions of decorin with these ECM molecules contribute to the regulation of ECM assembly, cell adhesions, and cell proliferation. These basic biological processes play critical roles during embryonic development and wound healing and are altered in pathological conditions such as fibrosis and tumorgenesis. ^ In this dissertation, we discover that decorin core protein can bind to Zn2+ ions with high affinity. Zinc is an essential trace element in mammals. Zn2+ ions play a catalytic role in the activation of many enzymes and a structural role in the stabilization of protein conformation. By examining purified recombinant decorin and its core protein fragments for Zn2+ binding activity using Zn2+-chelating column chromatography and Zn2+-equilibrium dialysis approaches, we have located the Zn2+ binding domain to the N-terminal sequence of the decorin core protein. The decorin N-terminal domain appears to contain two Zn2+ binding sites with similar high binding affinity. The sequence of the decorin N-terminal domain does not resemble any other reported zinc-binding motifs and, therefore, represents a novel Zn 2+ binding motif. By investigating the influence of Zn2+ ions on decorin binding interactions, we found a novel Zn2+ dependent interaction with fibrinogen, the major plasma protein in blood clots. Furthermore, a recombinant peptide (MD4) consisting of a 41 amino acid sequence of mouse decorin N-terminal domain can prolong thrombin induced fibrinogen/fibrin clot formation. This suggests that in the presence of Zn2+ the decorin N-terminal domain has an anticoagulation activity. The changed Zn2+-binding activities of the truncated MD4 peptides and site-directed mutagenesis generated mutant peptides revealed that the functional MD4 peptide might contain both a structural zinc-binding site in the cysteine cluster region and a catalytic zinc site that could be created by the flanking sequences of the cysteine cluster region. A model of a loop-like structure for MD4 peptide is proposed. ^

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.