Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin‐1β‐mediated signaling pathways

Osteoclasts are bone‐resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic? slides pre‐coated with 0.01–20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate‐resistant acid phosphatase activity and pre‐osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40–60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin‐1β (IL‐1β). IL‐1β production was inhibited using the NO‐synthase inhibitor only on FN indicating a FN‐specific cross‐talk between NO and IL‐1β signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL‐1β signaling. J. Cell. Biochem. 111: 1020–1034, 2010. © 2010 Wiley‐Liss, Inc.     

Bone protects proteins over thousands of years: extraction, analysis, and interpretation of extracellular matrix proteins in archeological skeletal remains

In a good state of preservation, bone conserves the entire protein pattern of extracellular bone matrix proteins over thousands of years. The quality of the profiles of matrix proteins isolated from ancient bones (ranging from the pre-Pottery Neolithic Phase to Early Modern Times from different archaeological sites in different geographical areas), separated by electrophoresis, is as good as those from recent bones. Molecules arising from collagenous proteins (e.g., collagen type I), from the noncollagenous group (e.g., osteonectin), and from the immune system (e.g., immunoglobulin G) were identified in Western blots by specific antibodies. A comparative study of the immunoglobulin G content of the bones of five prehistoric children showed the lowest immunoglobulin G content in a child who suffered from chronic scurvy. Ancient bone proteins were also separated by two-dimensional electrophoresis. This technique makes fractionation of the complex protein mixtures of extracellular bone matrix more reproducible. Bone retains a chemical memory of earlier metabolic stimuli in its configuration of collagenous and noncollagenous proteins. In combination with the results of the microscopic examination of ancient bone, it should be possible to obtain more reliable information on the history and the evolution of diseases, based on analysis of intact proteins.     

Distribution of extracellular matrix proteins in odontogenic tumours and developing teeth

The distribution of two cellular fibronectins (cFn), tenascin, laminin, as well as type VII collagen was studied in 14 benign odontogenic tumours of epithelial (ameloblastoma) and epithelial-ectomesenchymal (ameloblastic fibroma) origins, as well as in developing human teeth by immunocytochemical means using monoclonal antibodies (Mabs). An extradomain sequence-A-containing form of cFn (EDA-cFn) was seen in the extracellular matrix (ECM) of all tumours studied and in the mesenchyme of the developing tooth germs, indicating that cFn in these tissues are predominantly produced locally. A form of cFn containing an oncofetal domain (Onc-cFn), hitherto found only in carcinomas, was detected focally in the stroma of most ameloblastomas but was absent from ameloblastic fibromas and tooth germs. Tenascin was strongly expressed in the basement membrane (BM) zone of all odontogenic tumours and in that of the early tooth germs. Focal absence of laminin and type VII collagen from the BM of some ameloblastomas and the presence of Onc-cFn in the ECM of most ameloblastomas may correlate with their aggressive behaviour. The results also suggest that EDA-cFn and tenascin are involved in epithelial-mesenchymal interactions during tooth development and in odontogenic tumours.     

Changes in the expression of small intestine extracellular matrix proteins in streptozotocin-induced diabetic rats

Diabetes mellitus is characterized by anatomical and functional alterations of the intestinal tract. However, the aetiology of these disturbances remains unclear. The aim of the present work was to investigate the effects of diabetes on the expression of laminin-1 and fibronectin in the small intestine of Streptozotocin (STZ)-induced diabetic rats. The Western immunoblotting of the extracts from the small intestine revealed that experimental diabetes resulted in a marked increase in the intensity of the bands corresponding to laminin-1 and fibronectin. Immunohistochemical studies demonstrated a strong labelling to these two extracellular matrix (ECM) proteins in the small intestine of diabetic rats, mainly localized in the smooth muscle layer. These results occur together with a thickening of the basement membrane (BM) of the smooth muscle cells, demonstrated by transmission electron microscopy (TEM). We propose that the accumulation of ECM proteins in the smooth muscle layer may be an effect mediated by hyperglycaemia, since insulin treatment of diabetic rats reversed this accumulation. These results could provide information on the potential role of the ECM in the intestine, an organ which is known to exhibit important alterations in diabetes.     

Extracellular matrix proteins secreted from both the endometrium and the embryo are required for attachment: A study using a co‐culture model of rat blastocysts and Ishikawa cells

Integrins are expressed in a highly regulated manner at the maternal‐fetal interface during implantation. However, the significance of extracellular matrix (ECM) ligands during the integrin‐mediated embryo attachment to the endometrium is not fully understood. Thus, the distribution of fibronectin in the rat uterus and blastocyst was studied at the time of implantation. Fibronectin was absent in the uterine luminal epithelial cells but was intensely expressed in the trophoblast cells and the inner cell mass suggesting that fibronectin secreted from the blastocyst may be a possible bridging ligand for the integrins expressed at the maternal‐fetal interface. An Arg‐Gly‐Asp (RGD) peptide was used to block the RGD recognition sites on integrins, and the effect on rat blastocyst attachment to Ishikawa cells was examined. There was a significant reduction in blastocyst attachment when either the blastocysts or the Ishikawa cells were pre‐incubated with the RGD‐blocking peptide. Thus, successful attachment of the embryo to the endometrium requires the interaction of integrins on both the endometrium and the blastocyst with the RGD sequence of ECM ligands, such as fibronectin. Pre‐treatment of both blastocysts and Ishikawa cells with the RGD peptide also inhibited blastocyst attachment, but not completely, suggesting that ECM bridging ligands that do not contain the RGD sequence are also involved in embryo attachment. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Streptococcus suis serotype 2 binding to extracellular matrix proteins

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.     

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

A novel human glioma-associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athymic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]-leucine- and [3H]-glucosamine-labeled ECM from the human glioma cell line U-251MG has shown that GMEM is a high-molecular-weight macromolecule (Mr approximately 1,000,000) composed of Mr approximately 230,000 disulfide-bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one-dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U-251MG.     

A strategy to quantitate global phosphorylation of bone matrix proteins

Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured “in bulk” are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.     

Expression of Concern: MiRNA‐218 regulates osteoclast differentiation and inflammation response in periodontitis rats through Mmp9

Periodontitis is a multiple infection and inflammatory disease featured by connective tissue homeostasis loss, periodontal inflammation, and alveolar bone resorption. MicroRNAs (miRNAs) are involved in the mediation of a large scale of pathological processes. Here, we show that miRNA‐218 provides protective effect on periodontitis via regulation of matrix metalloproteinase‐9 (Mmp9). This pathway is aberrant in periodontium from rats with periodontitis and human periodontal ligament progenitor cells stimulated by lipopolysaccharide, with downregulation of miR‐218 and higher levels of Mmp9 compared with periodontium from healthy rats and cells without stimulation. Overexpression of miR‐218 can suppress the degradation of Collagen Types I and IV and dentin sialoprotein (DSP), attenuate osteoclast formation, and inhibit the secretion of proinflammatory cytokines. On the other hand, overexpression of Mmp9 promotes the degradation of Collagen Types I and IV and DSP as well as RANKL‐induced osteoclast formation and elevates inflammatory factors levels. Furthermore, the inhibitory effect of miR‐218 was prevented by rescuing the Mmp9 expression. In addition, we also have showed that miR‐218 was able to attenuate bone resorption and inflammation in a periodontitis rat model. Collectively, our findings therefore suggest that miR‐218 acts as a protective role in periodontitis through the regulation of Mmp9.     

Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

Cytoskeleton organization of normal,scar, and embryonic human fibroblasts spread on extracellular matrix proteins

We assayed the cytoskeleton organization of normal, scar, and embryonic human fibroblasts spread on major proteins of the extracellular matrix (ECM), type-I and-IV collagens, laminin 2/4, and fibronectin. Confocal fluorescent microscopy showed that fibroblasts of different origins were distinguished by their organization of actin structures and focal contacts visualized with antibodies to vinculin. It was found that different fibroblasts spread on identical ECM proteins had a common spatial organization of their cytoskeletons and some modifications of their actin structures and focal contacts. Variations in the organization of actin microfilaments indicate differences in cell interactions with various ECM proteins. The difference may be dependent on the integrin combination exposed on the cell membrane. It is suggested that fibroblasts of different origins differ in their morphogenetic functions.     

Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly

Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in α₅β₁-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes α₅β₁-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605–709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.     

The expression of mRNA of cytokines and of extracellular matrix proteins in triiodothyronine-treated rat hearts

In various models of cardiac hypertrophy, e.g. treatment of rats with norepinephrine infusion or pressure overload, increased expression of cytokines together with increase in extracellular matrix proteins (ECMP) was reported. In this study the effect of triiodothyronine (T3) on the expression of mRNA for cytokines and ECMP was investigated. Female Sprague-Dawley rats were treated daily with T3 in a dose of 0.2 mg.kg^–1 of body weight s.c. Changes in the left (LV) and right (RV) ventricular function were measured 6, 24, 48, 72 h and 7 and 14 days after the first T3-injection using Millar ultraminiature pressure catheter transducers. RNA was isolated from LV and RV tissue, and the expression of cytokines and ECMP was measured using the ribonuclease protection assay. T3-treatment induced a significant increase in LV dP/dt_max and RV dP/dt_max, (p < 0.05) 24 h after the first injection of T3 together with an increase in heart rate (p < 0.01). The RV systolic pressure increased 48 h after the first T3 injection, whereas the LV systolic pressure remained unchanged. After 48 h the heart weight to body weight ratio was increased (p< 0.01). Hypertrophy of the RV was more prominent than that of the LV (155.9 vs. 137.7%).In all groups the expression of mRNA for interleukins (IL) IL-6, IL-1, IL-1 and tumour necrosis factor (TNF)- in both ventricles did not change (p > 0.05). There was a significant increase in the mRNA for colligin 24 h after the T3 injection in both LV (p < 0.01) and RV (p< 0.05). This was followed by an increase in the mRNA for collagen I and III 72 h after the first T3-dose (p < 0.05 in RV; p < 0.01 in LV). At this point, the mRNA for tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was increased (p < 0.01) in the LV only. Moreover, after 7 days also the mRNA for matrix metalloproteinase (MMP)-2 increased (p < 0.01) in the LV. Both, TIMP-2 and MMP-2 were increased in the RV only after 14 days (p < 0.05). The gelatinase activity of MMP-2, however, was unchanged in both ventricles. The T3-induced cardiac hypertrophy was not accompanied by fibrosis as measured by the Sirius red staining after 14-days of T3-treatment. The moderate increase in mRNA for ECMP and MMP may be attributed more to the increasing mass of the ventricles with the accompanying remodelling of the ECM than to increased fibrosis.     

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.     

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low‐dose ultraviolet light irradiation

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild‐type and GzmB‐knockout mice were repeatedly exposed to minimal erythemal doses of solar‐simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild‐type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB‐mediated fibronectin fragments increased the expression of collagen‐degrading matrix metalloproteinase‐1 (MMP‐1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age‐related chronic inflammatory diseases.