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1.
Amino Acids ; 46(7): 1751-61, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24710705

ABSTRACT

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and ß1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of ß1 and ß3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.


Subject(s)
Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Collagen Type I/chemistry , GTP-Binding Proteins/chemistry , Transglutaminases/chemistry , Alkaline Phosphatase/metabolism , Antibodies/pharmacology , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Gene Expression Regulation/drug effects , Humans , Integrins/immunology , Materials Testing , Oligopeptides/pharmacology , Osteoblasts/drug effects , Peptides/chemistry , Peptides/pharmacology , Protein Glutamine gamma Glutamyltransferase 2 , Signal Transduction/drug effects
2.
Amino Acids ; 41(4): 909-21, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21046178

ABSTRACT

The multifunctional enzyme tissue transglutaminase (TG2) is reported to both mediate and inhibit tumour progression. To elucidate these different roles of TG2, we established a series of stable-transfected mouse colon carcinoma CT26 cells expressing a catalytically active (wild type) and a transamidating-inactive TG2 (Cys277Ser) mutant. Comparison of the TG2-transfected cells with the empty vector control indicated no differences in cell proliferation, apoptosis and susceptibility to doxorubicin, which correlated with no detectable changes in the activation of the transcription factor NF-κB. TG2-transfected cells showed increased expression of integrin ß3, and were more adherent and less migratory on fibronectin than control cells. Direct interaction of TG2 with ß3 integrins was demonstrated by immunoprecipitation, suggesting that TG2 acts as a coreceptor for fibronectin with ß3 integrins. All cells expressed the same level of TGFß receptors I and II, but only cells transfected with active TG2 had increased levels of TGFß1 and matrix-deposited fibronectin, which could be inhibited by TG2 site-directed inhibitors. Moreover, only cells transfected with active TG2 were capable of inhibiting tumour growth when compared to the empty vector controls. We conclude that in this colon carcinoma model increased levels of active TG2 are unfavourable to tumour growth due to their role in activation of TGFß1 and increased matrix deposition, which in turn favours increased cell adhesion and a lowered migratory and invasive behaviour.


Subject(s)
Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , GTP-Binding Proteins/metabolism , Transglutaminases/metabolism , Animals , Apoptosis/genetics , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Colonic Neoplasms/drug therapy , Doxorubicin/pharmacology , Extracellular Matrix/metabolism , Female , Fibronectins , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , Integrin beta Chains , Integrin beta1 , Integrin beta3 , Mice , Mice, Inbred BALB C , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , Syndecan-4 , Transforming Growth Factor beta1/metabolism , Transglutaminases/genetics , Transglutaminases/immunology
3.
J Biol Chem ; 284(43): 29547-58, 2009 Oct 23.
Article in English | MEDLINE | ID: mdl-19657147

ABSTRACT

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-kappaB activation, resulting in the increased expression of transforming growth factor (TGF) beta(1). In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFbeta(1) in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-kappaB and TGFbeta1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-kappaB and TGFbeta(1) production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.


Subject(s)
Collagen/biosynthesis , Fibrin/biosynthesis , GTP-Binding Proteins/biosynthesis , Nitric Oxide/metabolism , Transforming Growth Factor beta1/biosynthesis , Transglutaminases/biosynthesis , 3T3 Cells , Animals , Collagen/genetics , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Fibrin/genetics , GTP-Binding Proteins/genetics , Humans , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , Nitric Oxide/genetics , Nitric Oxide Donors/pharmacology , Protein Glutamine gamma Glutamyltransferase 2 , S-Nitroso-N-Acetylpenicillamine/pharmacology , Transforming Growth Factor beta1/genetics , Transglutaminases/genetics
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