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1.
Acta Biomater ; 89: 343-358, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30853609

ABSTRACT

Immunomodulation strategies are believed to improve the integration and clinical performance of synthetic bone substitutes. One potential approach is the modification of biomaterial surface chemistry to mimic bone extracellular matrix (ECM). In this sense, we hypothesized that coating synthetic dicalcium phosphate (DCP) bioceramics with bone ECM proteins would modulate the host immune reactions and improve their regenerative performance. To test this, we evaluated the in vitro proteomic surface interactions and the in vivo performance of ECM-coated bioceramic scaffolds. Our results demonstrated that coating DCP scaffolds with bone extracts, specifically those containing calcium-binding proteins, dramatically modulated their interaction with plasma proteins in vitro, especially those relating to the innate immune response. In vivo, we observed an attenuated inflammatory response against the bioceramic scaffolds and enhanced peri-scaffold new bone formation supported by the increased osteoblastogenesis and reduced osteoclastogenesis. Furthermore, the bone extract rich in calcium-binding proteins can be 3D-printed to produce customized hydrogels with improved regeneration capabilities. In summary, bone extracts containing calcium-binding proteins can enhance the integration of synthetic biomaterials and improve their ability to regenerate bone probably by modulating the host immune reaction. This finding helps understand how bone allografts regenerate bone and opens the door for new advances in tissue engineering and bone regeneration. STATEMENT OF SIGNIFICANCE: Foreign-body reaction is an important determinant of in vivo biomaterial integration, as an undesired host immune response can compromise the performance of an implanted biomaterial. For this reason, applying immunomodulation strategies to enhance biomaterial engraftment is of great interest in the field of regenerative medicine. In this article, we illustrated that coating dicalcium phosphate bioceramic scaffolds with bone-ECM extracts, especially those rich in calcium-binding proteins, is a promising approach to improve their surface proteomic interactions and modulate the immune responses towards such biomaterials in a way that improves their bone regeneration performance. Collectively, the results of this study may provide a conceivable explanation for the mechanisms involved in presenting the excellent regenerative efficacy of natural bone grafts.


Subject(s)
Bone Regeneration/drug effects , Bone and Bones , Calcium Phosphates/pharmacology , Ceramics , Complex Mixtures/pharmacology , Hydrogels/pharmacology , Immunologic Factors , Osteogenesis/drug effects , Printing, Three-Dimensional , Tissue Scaffolds/chemistry , Animals , Bone and Bones/chemistry , Bone and Bones/physiology , Ceramics/chemistry , Ceramics/pharmacology , Complex Mixtures/chemistry , Female , Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Rats
2.
Cell Death Differ ; 24(5): 844-854, 2017 05.
Article in English | MEDLINE | ID: mdl-28387755

ABSTRACT

Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation.


Subject(s)
Adipocytes/metabolism , Bone Resorption/genetics , Factor XIIIa/genetics , Fibronectins/genetics , GTP-Binding Proteins/genetics , Osteoblasts/metabolism , Osteoclasts/metabolism , Transglutaminases/genetics , Adipocytes/cytology , Adipogenesis/genetics , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Bone Resorption/metabolism , Bone Resorption/pathology , Bone and Bones/cytology , Bone and Bones/metabolism , Cell Differentiation , Factor XIIIa/metabolism , Fibronectins/blood , GTP-Binding Proteins/deficiency , Gene Expression Regulation , Homeostasis/genetics , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Osteoblasts/cytology , Osteoclasts/cytology , Osteogenesis/genetics , Protein Glutamine gamma Glutamyltransferase 2 , RANK Ligand/genetics , RANK Ligand/metabolism , Signal Transduction , Transglutaminases/deficiency , Transglutaminases/metabolism
3.
Sci Rep ; 6: 35574, 2016 10 19.
Article in English | MEDLINE | ID: mdl-27759118

ABSTRACT

F13A1 gene, which encodes for Factor XIII-A blood clotting factor and a transglutaminase enzyme, was recently identified as a potential causative gene for obesity in humans. In our previous in vitro work, we showed that FXIII-A regulates preadipocyte differentiation and modulates insulin signaling via promoting plasma fibronectin assembly into the extracellular matrix. To understand the role of FXIII-A in whole body energy metabolism, here we have characterized the metabolic phenotype of F13a1-/- mice. F13a1-/- and F13a1+/+ type mice were fed chow or obesogenic, high fat diet for 20 weeks. Weight gain, total fat mass and fat pad mass, glucose handling, insulin sensitivity, energy expenditure and, morphological and biochemical analysis of adipose tissue was performed. We show that mice lacking FXIII-A gain weight on obesogenic diet, similarly as wild type mice, but exhibit a number of features of metabolically healthy obesity such as protection from developing diet-induced insulin resistance and hyperinsulinemia. Mice also show normal fasting glucose levels, larger adipocytes, decreased extracellular matrix accumulation and inflammation of adipose tissue, as well as decreased circulating triglycerides. This study reveals that FXIII-A transglutaminase can regulate whole body insulin sensitivity and may have a role in the development of diet-induced metabolic disturbances.


Subject(s)
Adipose Tissue/pathology , Factor XIIIa/genetics , Insulin Resistance/physiology , Obesity, Metabolically Benign/metabolism , Transglutaminases/genetics , Adipose Tissue/metabolism , Animals , Diet, High-Fat , Disease Models, Animal , Energy Metabolism , Humans , Insulin/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity, Metabolically Benign/genetics , Triglycerides/blood , Weight Gain
4.
J Cell Physiol ; 227(7): 2936-46, 2012 Jul.
Article in English | MEDLINE | ID: mdl-21959563

ABSTRACT

Osteoblast differentiation is regulated by the presence of collagen type I (COL I) extracellular matrix (ECM). We have recently demonstrated that Factor XIIIA (FXIIIA) transglutaminase (TG) is required by osteoblasts for COL I secretion and extracellular deposition, and thus also for osteoblast differentiation. In this study we have further investigated the link between COL I and FXIIIA, and demonstrate that COL I matrix increases FXIIIA levels in osteoblast cultures and that FXIIIA is found as cellular (cFXIIIA) and extacellular matrix (ecmFXIIIA) forms. FXIIIA mRNA, protein expression, cellular localization and secretion were enhanced by ascorbic acid (AA) treatment and blocked by dihydroxyproline (DHP) which inhibits COL I externalization. FXIIIA mRNA was regulated by the MAP kinase pathway. Secretion of ecmFXIIIA, and its enzymatic activity in conditioned medium, were also decreased in osteoblasts treated with the lysyl oxidase inhibitor ß-aminopropionitrile, which resulted in a loosely packed COL I matrix. Osteoblasts secrete a latent, inactive dimeric ecmFXIIIA form which is activated upon binding to the matrix. Monodansyl cadaverine labeling of TG substrates in the cultures revealed that incorporation of the label occurred at sites where fibronectin co-localized with COL I, indicating that ecmFXIIIA secretion could function to stabilize newly deposited matrix. Our results suggest that FXIIIA is an integral part of the COL I deposition machinery, and also that it is part of the ECM-feedback loop, both of which regulate matrix deposition and osteoblast differentiation.


Subject(s)
Collagen Type I/metabolism , Factor XIIIa/metabolism , MAP Kinase Signaling System , Osteoblasts/metabolism , Transglutaminases/biosynthesis , Aminopropionitrile/pharmacology , Animals , Ascorbic Acid/metabolism , Cell Differentiation/physiology , Cells, Cultured , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Factor XIIIa/genetics , Fibronectins/metabolism , Mice , Osteoblasts/enzymology , Protein-Lysine 6-Oxidase/antagonists & inhibitors , RNA, Messenger/genetics , Transglutaminases/genetics , Transglutaminases/metabolism
5.
J Histochem Cytochem ; 55(7): 675-85, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17341477

ABSTRACT

Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immunohistochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immunoreactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.


Subject(s)
Bone and Bones/enzymology , Factor XIIIa/metabolism , Animals , Cell Differentiation , Cell Line , Cyclic AMP-Dependent Protein Kinases/physiology , Factor XIIIa/biosynthesis , Factor XIIIa/genetics , Femur/enzymology , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred BALB C , Mice, Knockout , Osteoblasts/cytology , Osteoblasts/enzymology , Osteocytes/enzymology , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Tibia/enzymology , Tissue Extracts/metabolism
6.
Matrix Biol ; 25(7): 419-29, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16919430

ABSTRACT

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.


Subject(s)
Aggrecans/metabolism , Cartilage, Articular/metabolism , Collagen Type II/metabolism , Peptides/pharmacology , Aggrecans/drug effects , Amino Acid Sequence , Animals , Cartilage, Articular/drug effects , Cattle , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/metabolism , Collagen Type II/drug effects , Collagenases/drug effects , Collagenases/metabolism , Gene Expression Regulation/drug effects , Immune Sera/immunology , In Vitro Techniques , Matrix Metalloproteinase 1/drug effects , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 13/drug effects , Matrix Metalloproteinase 13/genetics , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptides/immunology
7.
Arthritis Rheum ; 52(1): 128-35, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15641080

ABSTRACT

OBJECTIVE: To determine whether interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha), or both, plays a role in the excessive degradation that is observed in cultured osteoarthritic (OA) articular cartilage. METHODS: Antagonists of IL-1 and TNFalpha, namely, IL-1 receptor antagonist and the PEGylated soluble TNFalpha receptor I, respectively, were added at different concentrations to explant cultures of nonarthritic (5 obtained at autopsy) and OA (15 obtained at arthroplasty) articular cartilage. The cleavage of type II collagen (CII) by collagenase was measured by an immunoassay in cartilage and culture media. Proteoglycan (mainly aggrecan) content and degradation were measured by a colorimetric assay for glycosaminoglycan (GAG) content in cartilage and culture media. Reverse transcriptase-polymerase chain reaction was used to analyze gene expression of matrix metalloproteases (MMPs) 1, 3, and 13, CII, aggrecan, IL-1, and TNFalpha. RESULTS: Antagonists of IL-1 and TNFalpha inhibited the increase in CII cleavage by collagenase as well as the increase in GAG release observed in OA cartilage compared with normal cartilage. Inhibition was significant in tissue from some patients but not from others, although significant inhibition was observed when all the results were analyzed together. An increase in the GAG content in cartilage was seen in 4 of 15 cases. However, this increase was not significant when all the data were combined. Preliminary results indicated no effect of these antagonists on nonarthritic cartilage from 3 different donors. Independent analyses of gene expression in cultured cartilage from 9 other OA patients revealed that IL-1 or TNFalpha blockade, either alone and/or in combination, frequently down-regulated MMP-1, MMP-3, and MMP-13 expression. Expression of IL-1 and TNFalpha was inhibited by either antagonist or by the combination in essentially half the cases. The combined blockade up-regulated aggrecan and CII gene expression in approximately half the cases. CONCLUSION: These results suggest that the autocrine/paracrine activities of TNFalpha and IL-1 in articular cartilage may play important roles in cartilage matrix degradation in OA patients but not in all patients. Inhibition of either or both of these cytokines may offer a useful therapeutic approach to the management of OA by reducing gene expression of MMPs involved in cartilage matrix degradation and favoring its repair.


Subject(s)
Cartilage, Articular/metabolism , Extracellular Matrix/metabolism , Interleukin-1/metabolism , Osteoarthritis, Knee/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adult , Aged , Antirheumatic Agents/pharmacology , Case-Control Studies , Collagen Type II/chemistry , Collagen Type II/metabolism , Collagenases/metabolism , Female , Gene Expression/drug effects , Humans , Interleukin 1 Receptor Antagonist Protein , Male , Middle Aged , Polyethylene Glycols/pharmacology , Proteoglycans/metabolism , Receptors, Tumor Necrosis Factor, Type I , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/pharmacology
8.
Parasitol Res ; 88(6): 583-6, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12107483

ABSTRACT

Differences in codon usage can seriously hamper the expression of cloned cDNAs in heterologous systems. In this study, we show that the expression of a cloned Schistosoma mansoni cDNA in cultured HEK293 cells was dramatically increased by rewriting a portion of the cDNA according to human preferred codon usage, suggesting that codon optimization is a valuable strategy for improving the heterologous expression of helminth sequences. We further describe a simple modification of a recursive PCR-based method, which allows the rewriting of long stretches of DNA sequence in a single PCR reaction. This method can be used to optimize the codon usage of virtually any DNA from helminths and other parasites.


Subject(s)
Codon/genetics , DNA, Complementary/genetics , DNA, Helminth/genetics , Schistosoma mansoni/genetics , Animals , Cell Line , Cells, Cultured , DNA, Complementary/metabolism , Genes, Helminth , Humans , Models, Genetic , Schistosoma mansoni/metabolism , Selection, Genetic
9.
Mol Biochem Parasitol ; 119(1): 75-86, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11755188

ABSTRACT

A new cDNA was cloned from the bloodfluke, Schistosoma mansoni and shown to encode a protein with structural characteristics of a biogenic amine G protein-coupled receptor (GPCR). At the amino acid level, the parasite receptor (SmGPCR) shared about the same level of sequence homology (approximately 30%) with all major types of amine GPCRs and could not be identified on the basis of sequence. SmGPCR exhibited several nonconservative substitutions at key GPCR positions, including an unusual asparagine substitution (Asn(111)) for the highly conserved aspartate of transmembrane (TM) 3. The full-length SmGPCR cDNA was double-tagged with N-terminal FLAG and C-terminal hexahistidine epitopes, and was codon-optimized for expression in cultured HEK293 and COS7 cells. In situ immunofluorescence analyses targeting the two N- and C-terminal epitopes demonstrated that the modified SmGPCR was expressed at high level in mammalian cells and assumed a typical GPCR topology, the N-terminus being extracellular and the C-terminus intracellular. Functional activity assays revealed that SmGPCR was responsive to histamine, which caused a dose-dependent elevation in intracellular Ca2+ (EC50=0.54+/-0.05 microM). An Asn(111)-->Asp mutation had no effect on the responsiveness to histamine, suggesting that SmGPCR does not require the TM3 aspartate for agonist activation, in contrast to most amine GPCRs. None of the other monoamines tested had any significant effect on receptor activity, using assays that measured both Ca2+- and cAMP-mediated signaling. The results suggest that SmGPCR is a novel structural class of histamine receptor that may be unique to flatworms.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Histamine/pharmacology , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Schistosoma mansoni/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Evolution, Molecular , Helminth Proteins/chemistry , Helminth Proteins/genetics , Helminth Proteins/metabolism , Humans , Luminescent Measurements , Microscopy, Fluorescence , Molecular Sequence Data , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Schistosoma mansoni/drug effects , Schistosoma mansoni/genetics , Sequence Homology, Amino Acid
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