Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.     

Factor XIIIA transglutaminase expression and secretion by osteoblasts is regulated by extracellular matrix collagen and the MAP kinase signaling pathway

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 β-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.     

Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation,Homo-oligomerization and c-Src Signaling in Osteoblasts

Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9—which can alter TG enzyme conformation—localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts.     

Factor XIIIA transglutaminase crosslinks AT1 receptor dimers of monocytes at the onset of atherosclerosis

Many G protein-coupled receptors form dimers in cells. However, underlying mechanisms are barely understood. We report here that intracellular factor XIIIA transglutaminase crosslinks agonist-induced AT1 receptor homodimers via glutamine315 in the carboxyl-terminal tail of the AT1 receptor. The crosslinked dimers displayed enhanced signaling and desensitization in vitro and in vivo. Inhibition of angiotensin II release or of factor XIIIA activity prevented formation of crosslinked AT1 receptor dimers. In agreement with this finding, factor XIIIA-deficient individuals lacked crosslinked AT1 dimers. Elevated levels of crosslinked AT1 dimers were present on monocytes of patients with the common atherogenic risk factor hypertension and correlated with an enhanced angiotensin II-dependent monocyte adhesion to endothelial cells. Elevated levels of crosslinked AT1 receptor dimers on monocytes could sustain the process of atherogenesis, because inhibition of angiotensin II generation or of intracellular factor XIIIA activity suppressed the appearance of crosslinked AT1 receptors and symptoms of atherosclerosis in ApoE-deficient mice.     

Expression and localization of plasma transglutaminase factor XIIIA in bone.

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.     

Oligomerization and transglutaminase cross-linking of the cystatin CRES in the mouse epididymal lumen: potential mechanism of extracellular quality control

CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDS-resistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway.     

A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate.

Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His(6)-Xpress). His(6)-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247-258, 2001)     

Osteopontin, a substrate for transglutaminase and factor XIII activity.

Osteopontin (OPN), an extracellular matrix cell adhesion protein, was found to serve as a substrate for the incorporation of radiolabelled putrescine mediated by a commercial preparation of guinea pig liver transglutaminase. Preliminary evidence also suggests that OPN serves as a substrate for the plasma transglutaminase, Factor XIIIa. While the protein substrates to which OPN is linked in vivo have not been identified, it is reasonable to speculate that this capacity of OPN may dictate its extracellular location and thereby affect its role in bone homeostasis, tumorigenesis, metastasis, resistance to bacterial infections or, perhaps, wound repair.     

A human IgM signals axon outgrowth: coupling lipid raft to microtubules

Mouse and human IgMs support neurite extension from primary cerebellar granule neurons. In this study using primary hippocampal and cortical neurons, we demonstrate that a recombinant human IgM, rHIgM12, promotes axon outgrowth by coupling membrane domains (lipid rafts) to microtubules. rHIgM12 binds to the surface of neuron and induces clustering of cholesterol and ganglioside GM1. After cell binding and membrane fractionation, rHIgM12 gets segregated into two pools, one associated with lipid raft fractions and the other with the detergent-insoluble cytoskeleton-containing pellet. Membrane-bound rHIgM12 co-localized with microtubules and co-immuno precipitated with β3-tubulin. rHIgM12-membrane interaction also enhanced the tyrosination of α-tubulin indicating a stabilization of new neurites. When presented as a substrate, rHIgM12 induced axon outgrowth from primary neurons. We now demonstrate that a recombinant human mAb can induce signals in neurons that regulate membrane lipids and microtubule dynamics required for axon extension. We propose that the pentameric structure of the IgM is critical to cross-link membrane lipids and proteins resulting in signaling cascades.     

Tubulin Genes in the Algal Protist Euglena gracilis

ABSTRACT Alpha- and beta-tubulin cDNA were selected from a Euglenaλgt11 expression library, recloned and either sequenced (α-tubulin cDNA) or hybridized to Euglena RNA and DNA (α- and β-tubulin cDNA). RNA for hybridization was extracted at 30 minute intervals after flagellar amputation and quantitated for cDNA binding. Unlike previous reports on most other flagellates, no net increase in either α- or β-tubulin RNA could be detected during regeneration—suggesting steady state or constitutive tubulin RNA synthesis. Incubation of the cDNA with genomic DNA after restriction digestion produced patterns of hybridization consistent with the presence of one to two kinds each of the α- and β-tubulin genes. The deduced amino acid sequence of the α-tubulin cDNA was more than 90% identical to the α-tubulins of Trypanosoma, Chlamydomonas, Naegleria, Tetrahymena and higher plants. The carboxy terminus of the α-tubulin cDNA and the previously sequenced β-tubulin of Euglena showed greatest identity to the carboxy terminus of the tubulins from Trypanosoma brucei. The sequence data for α and β-tubulins of Euglena provides direct evidence for the similarity of two gene products from euglenas and trypanosomes and adds support to earlier suggestions that these organisms are phylogenetically related.     

Cytosolic carboxypeptidase 1 is involved in processing α- and β-tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.     

Molecular dynamics modeling of tubulin C-terminal tail interactions with the microtubule surface

Tubulin, an α/β heterodimer, has had most of its 3D structure analyzed; however, the carboxy (C)-termini remain elusive. Importantly, the C-termini play critical roles in regulating microtubule structure and function. They are sites of most of the post-translational modifications of tubulin and interaction sites with molecular motors and microtubule-associated proteins. Simulated annealing was used in our molecular dynamics modeling to predict the interactions of the C-terminal tails with the tubulin dimer. We examined differences in their flexibility, interactions with the body of tubulin, and the existence of structural motifs. We found that the α-tubulin tail interacts with the H11 helix of β-tubulin, and the β-tubulin tail interacts with the H11 helix of α-tubulin. Tail domains and H10/B9 loops interact with each other and compete for interactions with positively-charged residues of the H11 helix on the neighboring monomer. In a simulation in which α-tubulin's H10/B9 loop switches on sub-nanosecond intervals between interactions with the C-terminal tail of α-tubulin and the H11 helix of β-tubulin, the intermediate domain of α-tubulin showed more fluctuations compared to those in the other simulations, indicating that tail domains may cause shifts in the position of this domain. This suggests that C-termini may affect the conformation of the tubulin dimer which may explain their essential function in microtubule formation and effects on ligand binding to microtubules. Our modeling also provides evidence for a disordered-helical/helical double-state system of the T3/H3 region of the microtubule, which could be linked to depolymerization following GTP hydrolysis.     

Immunoelectron Microscopy of Giardia lamblia Cytoskeleton Using Antibody to Acetylated α-Tubulin

Giardia lamblia trophozoites contain acetylated α-tubulin but lack detectable levels of tyrosinolated α-tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, acetylated α-tubulin is localized in axonemes, median bodies and in the adhesive disk. Post-embeddment immunogold labeling of thin sections of cells was used to evaluate acetylation at the level of individual microtubules by electron microscopy. Cells were fixed with glutaraldehyde and embedded in the acrylic resin LR Gold. Results indicate all microtubules in adhesive disk, axonemes, basal bodies, funis and the median bodies contain acetylated α-tubulin. Unlike immunofluorescence labeling, all microtubules of the adhesive disk and the funis could be gold labeled. No nonspecific labeling of the cytoplasm or of structures other than microtubules was observed. Acetylated microtubules in G. lamblia do not appear to be a subset of microtubules and acetylation appears uniform along the entire length of individual microtubules. Acetylation and the tyrosinolation state of microtubules in Giardia are discussed in the context of microtubule stability and crosslinked features of the cytoskeleton.     

Plasma Transglutaminase in Hypertrophic Chondrocytes: Expression and Cell-specific Intracellular Activation Produce Cell Death and Externalization

We previously used subtractive hybridization to isolate cDNAs for genes upregulated in chick hypertrophic chondrocytes (Nurminskaya, M., and T.F. Linsenmayer. 1996. Dev. Dyn. 206:260–271). Certain of these showed homology with the “A” subunit of human plasma transglutaminase (factor XIIIA), a member of a family of enzymes that cross-link a variety of intracellular and matrix molecules. We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA. Northern and enzymatic analyses confirm that the molecule is upregulated in hypertrophic chondrocytes (as much as eightfold). The enzymatic analyses also show that appreciable transglutaminase activity in the hypertrophic zone becomes externalized into the extracellular matrix. This externalization most likely is effected by cell death and subsequent lysis—effected by the transglutaminase itself. When hypertrophic chondrocytes are transfected with a cDNA construct encoding the zymogen of factor XIIIA, the cells convert the translated protein to a lower molecular weight form, and they initiate cell death, become permeable to macromolecules and eventually undergo lysis. Non-hypertrophic cells transfected with the same construct do not show these degenerative changes. These results suggest that hypertrophic chondrocytes have a novel, tissue-specific cascade of mechanisms that upregulate the synthesis of plasma transglutaminase and activate its zymogen. This produces autocatalytic cell death, externalization of the enzyme, and presumably cross-linking of components within the hypertrophic matrix. These changes may in turn regulate the removal and/or calcification of this hypertrophic matrix, which are its ultimate fates.     

Vitronectin is a substrate for transglutaminases

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.     

Distribution of microtubules during the growth of tobacco pollen tubes

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α-, β- and tyrosinated α-tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer-assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.     

The Characterization of Tubulin in CNS Membrane Fractions

Abstract— Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and a plasma membrane (PM) fraction enriched in synaptic membranes were isolated from rat forebrain. The proteins in these membrane fractions were analyzed by two-dimensional gel electrophoresis (2DGE) in the isoelectric range of 5.1 to 6.0 by a modification of the O'Farrell procedure. Proteins were detected by Coomassie Brilliant Blue staining of the electrophoretograms. The results of these analyses were compared with 2DGE analysis of cytosol proteins, with particular attention given to tubulin subunits and actin. The RER contained one major protein (53K 5.4) in the β-tubulin region with a molecular weight of 53,000 and an isoelectric point of 5.4. The SER contained at least two major proteins in the β-tubulin region; one with a migration identical to 53K 5.4 and other proteins with slightly higher apparent molecular weights and more acidic isoelectric points (54K, 5.4 to 5.3), identical to cytoplasmic β-tubulin. The PM fraction also contained multiple overlapping proteins (54K, 5.4 to 5.3) in the β-tubulin area and a trace amount of the 53K 5.4 protein. The proteins in the β-tubulin region were removed from the 2DGE electrophoretogram and digested by Staphylococcus aureus V8 protease, and the peptides separated on one-dimensional polyacrylamide gels. The peptide patterns of 53K 5.4 protein from RER and SER were almost identical and differed significantly from the cytoplasmic β-tubulin pattern; however, the peptide maps of the PM and SER β-tubulin region were identical to the cytoplasmic β-tubulin. The 2DGE analysis of RER did not contain proteins in the region of cytoplasmic α-tubulin. SER and PM contained proteins in the α-tubulin region with a similar, but not identical, peptide analysis to cytoplasmic α-tubulin. Significant amounts of actin were detected in 2DGE analysis of SER and PM, and the peptide analysis of the actin was identical to the cytoplasmic actin analysis. The RER fraction contained only trace amounts of actin. The cytosol and all membrane fractions contained a protein (68K 5.6) found among microtubule-associated proteins, as judged by molecular weight and isoelectric point. Several proteins present in all membrane fractions (61K 5.1 and 58K 5.1) bound to concanavalin A agarose.     

Isolation of separate mRNAs for α- and β-Tubulin and characterization of the corresponding in vitro translation products

The messenger RNAs coding for α- and β-tubulin have been isolated from embryonic chick brain. Although the mRNAs for the two tubulin subunits have been resolved on native gels, they are very similar in molecular weight (650,000 daltons) as judged by mobility on denaturing gels containing methyl mercury. The mRNAs for β- and γ-actin have also been resolved on native gels, but migrate as an unresolved peak (molecular weight 650,000–700,000 daltons) under denaturing conditions. Since the nonmuscle actins are substantially smaller proteins than α- and β-tubulin, the large size of chick nonmuscle actin mRNAs suggests an unusually long untranslated region.Since tubulin and actin polypeptides are internal structural proteins, one would expect them to be synthesized only on free polysomes. Translation of mRNA derived directly from a purified membrane fraction or by puromycin release from that fraction, however, showed the synthesis of a small proportion of these proteins on polysomes that are membrane-associated. Peptide mapping has in all cases confirmed the identity of the products of cell-free synthesis with authentic α-tubulin, β-tubulin and actin. Approximately 67% of the α- and 13% of the β-tubulin chains produced by in vitro translation are competent for co-assembly into microtubules with added carrier microtubule protein.