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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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Fluorescence micrographs of hypertrophic (A and B)  and non-hypertrophic chondrocytes (C and D) transfected with  the full-length (A–C) and truncated (constitutively active) (D)  pFXIIIA-GFP constructs. Transfected cells (arrows) were identified by the intrinsic fluorescence of the GFP fusion protein (left  column); nuclear morphology was visualized by Hoechst staining  (middle column), and the integrity of cell membranes was determined by intracellular reactivity of non-permeabilized cells with  rhodamine-conjugated anti-GFP antibody (right column). 2 d  after transfection of hypertrophic cells with the full-length  FXIIIA-GFP construct (A) no changes are seen in the nuclear  morphology compared with non-transfected cells. Also, the fusion FXIIIA-GFP protein in these non-permeabilized cells is not  reactive with the anti-GFP antibody. By 4 d (B), the Hoechst  staining shows shrunken and fragmented nuclei in those cells  transfected with pFXIIIA-GFP, and now the anti-GFP antibody  can penetrate the non-permeabilized cells and shows reactivity.  Non-hypertrophic chondrocytes, transfected with the pFXIIIA-GFP construct, still maintain a normal nuclear morphology 6 d  after transfection (C). However, the nuclei of non-hypertrophic  chondrocytes transfected with the truncated (constitutively active) pFXIIIA-GFP (D) become shrunken and fragmented by  day 4.
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Figure 5: Fluorescence micrographs of hypertrophic (A and B) and non-hypertrophic chondrocytes (C and D) transfected with the full-length (A–C) and truncated (constitutively active) (D) pFXIIIA-GFP constructs. Transfected cells (arrows) were identified by the intrinsic fluorescence of the GFP fusion protein (left column); nuclear morphology was visualized by Hoechst staining (middle column), and the integrity of cell membranes was determined by intracellular reactivity of non-permeabilized cells with rhodamine-conjugated anti-GFP antibody (right column). 2 d after transfection of hypertrophic cells with the full-length FXIIIA-GFP construct (A) no changes are seen in the nuclear morphology compared with non-transfected cells. Also, the fusion FXIIIA-GFP protein in these non-permeabilized cells is not reactive with the anti-GFP antibody. By 4 d (B), the Hoechst staining shows shrunken and fragmented nuclei in those cells transfected with pFXIIIA-GFP, and now the anti-GFP antibody can penetrate the non-permeabilized cells and shows reactivity. Non-hypertrophic chondrocytes, transfected with the pFXIIIA-GFP construct, still maintain a normal nuclear morphology 6 d after transfection (C). However, the nuclei of non-hypertrophic chondrocytes transfected with the truncated (constitutively active) pFXIIIA-GFP (D) become shrunken and fragmented by day 4.

Mentions: We transfected hypertrophic chondrocytes with this construct and then examined the effect on the cells by fluorescence microscopy. After several days, the transfected cells showed a spectrum of changes indicative of cellular death, including shrunken and fragmented nuclei (Fig. 5 B, middle column). Eventually, all that remained of the transfected cells were patches of GFP-containing cytoplasm (Fig. 5 B, right column). Control hypertrophic cells transfected with a plasmid expressing GFP only showed no cellular changes, and several days after transfection were found frequently in nests of two or four cells, suggesting that they had continued to undergo division (data not shown).


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)

Fluorescence micrographs of hypertrophic (A and B)  and non-hypertrophic chondrocytes (C and D) transfected with  the full-length (A–C) and truncated (constitutively active) (D)  pFXIIIA-GFP constructs. Transfected cells (arrows) were identified by the intrinsic fluorescence of the GFP fusion protein (left  column); nuclear morphology was visualized by Hoechst staining  (middle column), and the integrity of cell membranes was determined by intracellular reactivity of non-permeabilized cells with  rhodamine-conjugated anti-GFP antibody (right column). 2 d  after transfection of hypertrophic cells with the full-length  FXIIIA-GFP construct (A) no changes are seen in the nuclear  morphology compared with non-transfected cells. Also, the fusion FXIIIA-GFP protein in these non-permeabilized cells is not  reactive with the anti-GFP antibody. By 4 d (B), the Hoechst  staining shows shrunken and fragmented nuclei in those cells  transfected with pFXIIIA-GFP, and now the anti-GFP antibody  can penetrate the non-permeabilized cells and shows reactivity.  Non-hypertrophic chondrocytes, transfected with the pFXIIIA-GFP construct, still maintain a normal nuclear morphology 6 d  after transfection (C). However, the nuclei of non-hypertrophic  chondrocytes transfected with the truncated (constitutively active) pFXIIIA-GFP (D) become shrunken and fragmented by  day 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132883&req=5

Figure 5: Fluorescence micrographs of hypertrophic (A and B) and non-hypertrophic chondrocytes (C and D) transfected with the full-length (A–C) and truncated (constitutively active) (D) pFXIIIA-GFP constructs. Transfected cells (arrows) were identified by the intrinsic fluorescence of the GFP fusion protein (left column); nuclear morphology was visualized by Hoechst staining (middle column), and the integrity of cell membranes was determined by intracellular reactivity of non-permeabilized cells with rhodamine-conjugated anti-GFP antibody (right column). 2 d after transfection of hypertrophic cells with the full-length FXIIIA-GFP construct (A) no changes are seen in the nuclear morphology compared with non-transfected cells. Also, the fusion FXIIIA-GFP protein in these non-permeabilized cells is not reactive with the anti-GFP antibody. By 4 d (B), the Hoechst staining shows shrunken and fragmented nuclei in those cells transfected with pFXIIIA-GFP, and now the anti-GFP antibody can penetrate the non-permeabilized cells and shows reactivity. Non-hypertrophic chondrocytes, transfected with the pFXIIIA-GFP construct, still maintain a normal nuclear morphology 6 d after transfection (C). However, the nuclei of non-hypertrophic chondrocytes transfected with the truncated (constitutively active) pFXIIIA-GFP (D) become shrunken and fragmented by day 4.
Mentions: We transfected hypertrophic chondrocytes with this construct and then examined the effect on the cells by fluorescence microscopy. After several days, the transfected cells showed a spectrum of changes indicative of cellular death, including shrunken and fragmented nuclei (Fig. 5 B, middle column). Eventually, all that remained of the transfected cells were patches of GFP-containing cytoplasm (Fig. 5 B, right column). Control hypertrophic cells transfected with a plasmid expressing GFP only showed no cellular changes, and several days after transfection were found frequently in nests of two or four cells, suggesting that they had continued to undergo division (data not shown).

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