Transglutaminase 2 as a novel activator of LRP6/ß-catenin signaling.

The ß-catenin signaling axis is critical for normal embryonic development and tissue homeostasis in adults. We have previously shown that extracellular enzyme transglutaminase 2 (TG2) activates ß-catenin signaling in vascular smooth muscle cells (VSMCs). In this study, we provide several lines of evidence that TG2 functions as an activating ligand of the LRP5/6 receptors. Specifically, we show that TG2 synergizes with LRP6 in the activation of ß-catenin-dependent gene expression in Cos-7 cells. Interfering with the LRP5/6 receptors attenuates TG2-induced activation of ß-catenin in Cos-7 cells. Further, we show that TG2 binds directly to the extracellular domain of LRP6, which is also able to act as a substrate for TG2-mediated protein cross-linking. Furthermore, inhibitors of TG2 protein cross-linking quench the observed TG2-induced ß-catenin activation, implicating protein cross-linking as a novel regulatory mechanism for this pathway. Together, our findings identify and characterize a new activating ligand of the LRP5/6 receptors and uncover a novel activity of TG2 as an agonist of ß-catenin signaling, contributing to the understanding of diverse developmental events and pathological conditions in which transglutaminase and ß-catenin signaling are implicated.

Immunohistological analysis of transglutaminase factor XIIIA expression in mouse embryonic growth plate.

Previously we demonstrated the expression of Factor XIIIA (FXIIIA), a coagulation transglutaminase, in avian embryonic growth plate. To explore whether FXIIIA is also expressed by chondrocytes of the mammalian cartilage anlagen of bones, we analyzed the mouse embryonic growth plate by immunostaining using anti-FXIIIA antibodies developed against human and chicken proteins. We revealed the expression of FXIIIA in the epiphyseal growth plate, where FXIIIA appears first intracellularly in the zone of proliferation/maturation, and remains intra- and extracellularly throughout the hypertrophic zone. Externalization of FXIIIA occurs before mineralization. Transglutaminase activity was assayed in organ cultures using rhodamine-labeled synthetic substrate Pro-Val-Lys-Gly. Enzymatic activity shows a restricted distribution in cartilage and correlates with FXIIIA expression pattern, suggesting that cartilagenous transglutaminase activity is due, at least partially, to the FXIIIA isoform. We conclude, that coagulation factor FXIIIA is expressed by chondrocytes of embryonic mouse long bone cartilages in a strictly regulated pattern, which correlates with chondrocyte differentiation and matrix mineralization.

Transglutaminase factor XIIIA in the cartilage of developing avian long bones.

Previously, we showed that mRNA for transglutaminase factor XIIIA (FXIIIA) is up-regulated in the hypertrophic zone of the growth plate of the chicken tibiotarsus, a well-characterized model of long bone development. In the present study, we have studied the distribution of the FXIIIA protein and of transglutaminase enzymatic activity in this growth plate, as well as in the cartilage of the epiphysis, which includes that of the articular surface. By immunohistochemical analysis, the protein is detected in the zone of maturation, where it is mostly intracellular, and in the hypertrophic zone, where it is present both intracellularly and in the extracellular matrix. The intracellular enzyme is mostly a zymogen, as determined with an antibody specific for the activation peptide. Externalization of FXIIIA is accompanied by enzyme activation. To study the pattern of transglutaminase activity, a synthetic transglutaminase substrate, rhodamine-conjugated tetrapeptide (Pro-Val-Lys-Gly), was used for pulse labeling in organ cultures. Intensive incorporation of the fluorescent substrate was observed throughout the hypertrophic zone and in the cells surrounding the forming blood vessels. The patterns of FXIIIA immunostaining and substrate incorporation overlap almost completely. The cartilaginous factor XIIIA is different from the plasma form in that, both intracellularly and extracellularly, it exists as a monomer, as determined by Western analysis, whereas the plasma form of FXIII is a tetrameric complex composed of both A and B subunits. We also identified FXIIIA and transglutaminase activity within the articular and condylar regions of the tarsus, suggesting a possible involvement of mechanical pressure and/or stress in the production of the molecule and subsequent cross-linking of the cartilage matrix. Thus, transglutaminases, in particular FXIIIA, are involved in the formation of long bones through its activity both in the hypertrophic region of the growth plate and in the formation of articular/epiphyseal cartilages.

UDP-glucose pyrophosphorylase: up-regulation in hypertrophic cartilage and role in hyaluronan synthesis.

Previously, we have performed subtractive hybridization to identify genes up-regulated in hypertrophic chondrocytes of the avian epiphyseal growth plate. In the present study, we report the identification of one of the clones as UDP-glucose pyrophosphorylase (UDPG-PPase) and propose a possible function for this enzyme in regulating hyaluronan (HA) synthesis in hypertrophic cartilage. We have cloned the 2.6 kb full-length cDNA for avian UDPG-PPase and confirmed its up-regulation in hypertrophic versus non-hypertrophic cartilage by Northern-blot analysis. The 6-fold increase in mRNA was paralleled by an equivalent increase in enzymic activity. The enzyme catalyses the conversion of glucose 1-phosphate into UDP-glucose, which is used to synthesize a number of cellular components, including HA. Overexpression of enzymically active UDPG-PPase in non-hypertrophic chondrocytes resulted in a 2-3-fold increase in total HA, as determined by a competitive binding assay and immunohistochemistry. In the developing growth plate, HA synthesis was elevated in the hypertrophic zone along with the up-regulation of the HA synthase (HAS)-2 gene. Our data suggest that an increase in both activities, UDPG-PPase and HAS-2, is required for non-hypertrophic chondrocytes to synthesize an amount of HA comparable with that in hypertrophic chondrocytes. Therefore we conclude that HA synthesis during chondrocyte differentiation is regulated at the level of the substrate-provider gene, UDPG-PPase, as well as the HAS genes.

Selective sweep of a newly evolved sperm-specific gene in Drosophila.

The pattern of genetic variation across the genome of Drosophila melanogaster is consistent with the occurrence of frequent 'selective sweeps', in which new favourable mutations become incorporated into the species so quickly that linked alleles can 'hitchhike' and also become fixed. Because of the hitchhiking of linked genes, it is generally difficult to identify the target of any putative selective sweep. Here, however, we identify a new gene in D. melanogaster that codes for a sperm-specific axonemal dynein subunit. The gene has a new testes-specific promoter derived from a protein-coding region in a gene encoding the cell-adhesion protein annexin X (AnnX), and it contains a new protein-coding exon derived from an intron in a gene encoding a cytoplasmic dynein intermediate chain (Cdic). The new transcription unit, designated Sdic (for sperm-specific dynein intermediate chain), has been duplicated about tenfold in a tandem array. Consistent with the selective sweep of this gene, the level of genetic polymorphism near Sdic is unusually low. The discovery of this gene supports other results that point to the rapid molecular evolution of male reproductive functions.

Cytoplasmic dynein intermediate-chain isoforms with different targeting properties created by tissue-specific alternative splicing.

The intermediate chains (ICs) are the subunits of the cytoplasmic dynein that provide binding of the complex to cargo organelles through interaction of their N termini with dynactin. We present evidence that in Drosophila, the IC subunits are represented by at least 10 structural isoforms, created by the alternative splicing of transcripts from a unique Cdic gene. The splicing pattern is tissue specific. A constitutive set of four IC isoforms is expressed in all tissues tested; in addition, tissue-specific isoforms are found in the ovaries and nervous tissue. The structural variations between isoforms are limited to the N terminus of the IC molecule, where the interaction with dynactin takes place. This suggests differences in the dynactin-mediated organelle binding by IC isoforms. Accordingly, when transiently expressed in Drosophila Schneider-3 cells, the IC isoforms differ in their intracellular targeting properties from each other. A mechanism is proposed for the regulation of dynein binding to organelles through the changes in the content of the IC isoform pool.

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.

Type X collagen and other up-regulated components of the avian hypertrophic cartilage program.

Elucidating the cellular and molecular processes involved in growth and remodeling of skeletal elements is important for our understanding of congenital limb deformities. These processes can be advantageously studied in the epiphyseal growth zone, the region in which all of the increase in length of a developing long bone is achieved. Here, young chondrocytes divide, mature, become hypertrophic, and ultimately are removed. During cartilage hypertrophy, a number of changes occur, including the acquisition of synthesis of new components, the most studied being type X collagen. In this review, which is based largely on our own work, we will first examine the structure and properties of the type X collagen molecule. We then will describe the supramolecular forms into which the molecule becomes assembled within tissues, and how this changes its physical properties, such as thermal stability. Certain of these studies involve a novel, immunohistochemical approach that utilizes an antitype X collagen monoclonal antibody that detects the native conformation of the molecule. We describe the developmental acquisition of the molecule, and its transcriptional regulation as deduced by in vivo footprinting, transient transfection, and gel-shift assays. We provide evidence that the molecule has unique diffusion and regulatory properties that combine to alter the hypertrophic cartilage matrix. These conclusions are derived from an in vitro system in which exogenously added type X collagen moves rapidly through the cartilage matrix and subsequently produces certain changes mimicking ones that have been shown normally to occur in vivo. These include altering the cartilage collagen fibrils and effecting changes in proteoglycans. Last, we describe the subtractive hybridization, isolation, and characterization of other genes up-regulated during cartilage hypertrophy, with specific emphasis on one of these--transglutaminase.

Differential expression of genes associated with collagen fibril growth in the chicken tendon: identification of structural and regulatory genes by subtractive hybridization.

Collagen fibril growth is a very rapid and abrupt process, resulting in a 4- to 5-fold increase in fibril length between 16 and 18 days of chicken metatarsal tendon development. This fibril growth is due to a postdepositional fusion/association of preformed intermediates, termed fibril segments. We propose that the regulated assembly of collagen fibrils from the segment intermediates is mediated by interactions of structural macromolecules. The cells could modulate this process by responding to cytokines and altering cell-matrix signaling, transcription, and translation. To identify the genes involved in this process a subtractive hybridization procedure was utilized. Genes of cell proliferation were excluded as major contributors to differential gene expression in avian tendon on days 14 and 19 of development after analysis of BrdUr incorporation. The BrdUr incorporation studies revealed little, if any, tendon fibroblast proliferation at both stages. This suggested that observed alterations in gene expression would be related to the pre- and postfibril growth phases in developing tendons. A total of 80 unique up- and down-regulated cDNA fragments were isolated and 26 of these were identified. There was an up-regulation of structural proteins (for example, collagen types I, VI, and XI and fibromodulin), a number of regulatory proteins (including TGF-beta 2 and IGF-1), as well as other enzymes/proteins. Northern analysis confirmed the up-regulation of mRNAs for all the structural proteins. The observed 20-fold increase of mRNA for the isolated clone corresponding to the 3' UTR of alpha 1(VI) collagen makes it a possible marker for the postfibril growth stage of collagen fibrillogenesis. The large number of isolated genes differentially expressed during the rapid phase of fibril growth reveals a fine and possibly tissue-specific control of fibrillogenesis.

Differential expression of fibromodulin mRNA associated with tendon fibril growth: isolation and characterization of a chicken fibromodulin cDNA.

A 450 bp cDNA fragment similar to that encoding bovine fibromodulin was isolated using a screening procedure to isolate genes differentially expressed between the pre- and post-growth phases of fibril growth in the developing chicken embryo metatarsal tendon. Using this fragment, a 2.4 kb cDNA clone for chicken fibromodulin was isolated from a lambda ZAP library, and the 5' rapid amplification of cDNA ends technique was employed to clone the 5'end of the fibromodulin cDNA. The full-length cDNA contained an open reading frame coding for a 380-amino-acid protein. There was approximately 80% similarity with human, rat and bovine fibromodulins, which confirmed its identity as fibromodulin. Structural features of the deduced sequence include an 18-amino-acid signal peptide, cysteine residues in conserved positions in the N- and C-terminal regions, and a central leucine-rich domain containing eleven repeats of the sequence LXXLXLXXNXL/I. Features unique to chicken fibromodulin include an additional glycosylation site as well as a decreased number of tyrosine residues that could be sulphated, and therefore potential changes in the charge of the molecule. In addition, there was little similarity among the untranslated regions. When compared with chicken decorin and lumican, fibromodulin showed greater similarity to the other keratan sulphate-containing proteoglycan, lumican. Northern blot analysis revealed a 6-8-fold increase in the fibromodulin mRNA level from day 14 to day 19 of development. In the chicken tendon, collagen fibril growth is a process characterized by a precipitous increase in length during a short developmental period. The necessary changes would require the expression of different genes regulating fibril formation and growth, and interactions between fibromodulin and collagen fibrils may participate in the regulation of collagen fibril growth and matrix assembly.

Identification and characterization of up-regulated genes during chondrocyte hypertrophy.

Chondrocyte hypertrophy involves de novo acquisition and/or increased expression of certain gene products including, among others, type X collagen, alkaline phosphatase, and matrix metalloproteinases. To analyze further the genetic program associated with chondrocyte hypertrophy, we have employed a modification of the polymerase chain reaction-mediated subtractive hybridization method of Wang and Brown (Wang and Brown [1991] Proc. Natl. Acad. Sci 88:11505). Cultures of hypertrophic tibial chondrocytes and nonhypertrophic sternal cells were used for poly A+ RNA isolation. Among 50 individual cDNA fragments isolated for up-regulated hypertrophic genes, 18 were tentatively identified by their similarities to entries in the GenBank database, whereas the other 32 showed no significant similarity. The identified genes included translational and transcriptional regulatory factors, ribosomal proteins, the enzymes transglutaminase and glycogen phosphorylase, type X collagen (highly specific for hypertrophic cartilage matrix), gelsolin, and the carbohydrate-binding protein galectin. Two of these, transglutaminase and galectin, were cloned and were further characterized. The chondrocyte transglutaminase revealed previously in hypertrophic cartilage by immunochemical methods appears to be the chicken equivalent of mammalian factor XIIIa (showing 75% overall protein similarity). The chicken chondrocyte galectin is a variant of mammalian galectin-3. Galectins are known to bind to components found in hypertrophic cartilage, and factor XIIIa is known to crosslink some of the same components, possibly modifying them for calcification and/or removal.

Collagen fibrillogenesis in situ: fibril segments undergo post-depositional modifications resulting in linear and lateral growth during matrix development.

Elucidating how collagen fibril growth is regulated is important in determining how tissues are assembled. Fibrils are deposited as segments. The growth of these segments is an important determinant of tissue architecture, stability, and mechanical attributes. Fibril segments were isolated from developing tendons and their structure characterized. The post-depositional changes leading to linear and lateral growth of fibrils also were examined. Segments extracted from 14-day chicken embryo tendons had a mean length of 29 microns. The segments were asymmetric, having a short and a long tapered end. Most of the segments were centrosymmetric with respect to molecular packing. Segments extracted from 12- to 16-day tendons had the same structure, but mean segment length increased incrementally due to the addition of an increasingly large population of longer segments. At 17 days of development there was a precipitous increase in segment length. The morphological data indicate that the increase in length was the result of lateral associations among adjacent segments. Analysis demonstrated that this fibril growth was associated with a significant decrease in fibril associated decorin. Using immunoelectron microscopy, decorin was seen to decrease significantly at 18 days of development. When decorin content was biochemically determined, a decrease also was observed. Decorin mRNA also decreased relative to fibrillar collagen mRNA during the same period. These data support the hypothesis that a decrease in fibril-associated decorin is necessary for fibril growth associated with tissue maturation. Growth through post-depositional fusion allows for appositional and intercalary growth and would be essential for normal development, growth, and repair.