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Plasma transglutaminase in hypertrophic chondrocytes: expression and cell-specific intracellular activation produce cell death and externalization.

Nurminskaya M, Magee C, Nurminsky D, Linsenmayer TF - J. Cell Biol. (1998)

Bottom Line: We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA.This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself.Non-hypertrophic cells transfected with the same construct do not show these degenerative changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
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.

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Enzymatic assays for plasma transglutaminase in cultures of hypertrophic (Hyp) and non-hypertrophic (NH) chondrocytes. Activities were measured without thrombin activation  (solid bar) and with activation (cross-hatched bar). Cell cultures  were initiated with chondrocytes from 14- and 20-d-old embryos.
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Figure 3: Enzymatic assays for plasma transglutaminase in cultures of hypertrophic (Hyp) and non-hypertrophic (NH) chondrocytes. Activities were measured without thrombin activation (solid bar) and with activation (cross-hatched bar). Cell cultures were initiated with chondrocytes from 14- and 20-d-old embryos.

Mentions: To determine whether transglutaminase enzymatic activity also becomes elevated during hypertrophy, we used the standard assay measuring [14C]putrescine incorporation into casein (Demignot et al., 1995). This was examined both in cell cultures (Fig. 3) and in isolated tissues (Fig. 4; Table I). Each sample was analyzed before and after thrombin activation. The activity requiring thrombin should reflect only the factor XIIIA, as this is the member of the transglutaminase family that is synthesized as a zymogen. The activity already present before thrombin treatment (constitutive activity), however, is probably more complex. In the non-hypertrophic cells, the constitutive activity should reflect largely the tissue form, as these cells do not seem able to activate the factor XIIIA zymogen (see below). In the hypertrophic cells, however, the constitutive activity most likely includes both the tissue form and that portion of the factor XIIIA that has already undergone activation—since hypertrophic chondrocytes have the ability to activate the zymogen intracellularly (shown later).


Plasma transglutaminase in hypertrophic chondrocytes: expression and cell-specific intracellular activation produce cell death and externalization.

Nurminskaya M, Magee C, Nurminsky D, Linsenmayer TF - J. Cell Biol. (1998)

Enzymatic assays for plasma transglutaminase in cultures of hypertrophic (Hyp) and non-hypertrophic (NH) chondrocytes. Activities were measured without thrombin activation  (solid bar) and with activation (cross-hatched bar). Cell cultures  were initiated with chondrocytes from 14- and 20-d-old embryos.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132883&req=5

Figure 3: Enzymatic assays for plasma transglutaminase in cultures of hypertrophic (Hyp) and non-hypertrophic (NH) chondrocytes. Activities were measured without thrombin activation (solid bar) and with activation (cross-hatched bar). Cell cultures were initiated with chondrocytes from 14- and 20-d-old embryos.
Mentions: To determine whether transglutaminase enzymatic activity also becomes elevated during hypertrophy, we used the standard assay measuring [14C]putrescine incorporation into casein (Demignot et al., 1995). This was examined both in cell cultures (Fig. 3) and in isolated tissues (Fig. 4; Table I). Each sample was analyzed before and after thrombin activation. The activity requiring thrombin should reflect only the factor XIIIA, as this is the member of the transglutaminase family that is synthesized as a zymogen. The activity already present before thrombin treatment (constitutive activity), however, is probably more complex. In the non-hypertrophic cells, the constitutive activity should reflect largely the tissue form, as these cells do not seem able to activate the factor XIIIA zymogen (see below). In the hypertrophic cells, however, the constitutive activity most likely includes both the tissue form and that portion of the factor XIIIA that has already undergone activation—since hypertrophic chondrocytes have the ability to activate the zymogen intracellularly (shown later).

Bottom Line: We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA.This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself.Non-hypertrophic cells transfected with the same construct do not show these degenerative changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus