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Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse.

Wilson DG, Phamluong K, Li L, Sun M, Cao TC, Liu PS, Modrusan Z, Sandoval WN, Rangell L, Carano RA, Peterson AS, Solloway MJ - J. Cell Biol. (2011)

Bottom Line: These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton.Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality.Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

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Early chondrocyte proliferation, differentiation, and critical patterning pathways are preserved in 14.5-dpc humeri despite abnormal chondrocyte morphology in Mia3−/− embryos. (A–D) Ki67 labeling of 14.5-dpc (A and B) and 16.5-dpc (C and D) humeri demonstrate normal proliferation of mutant chondrocytes. RZ, PZ, PHZ, and HZ denote resting, proliferative, prehypertrophic, and hypertrophic zones, respectively. (E and F) The majority of terminally differentiated hypertrophic chondrocytes are TUNEL positive in wt 14.5-dpc humeri (E), whereas Mia3- hypertrophic chondrocytes fail to die (F). (G and H) Scattered dying cells are restricted to the hypertrophic zone in 16.5-dpc wt and Mia3- humeri (red brackets). (I) Ki67-positive (+ve) cells within the proliferative zone. Mean values are presented, and SEM is shown from two independent wt samples and three Mia3−/− humeri with 4–10 sections/animal. (J–U) Section in situ analysis comparing expression of Col9a1, Col2a1, Col10a1, Ihh, Ptc, and Pth1r between wt and Mia3- 14.5-dpc humeri demonstrates an intact Ihh and Pth1r signaling axis despite dysmorphism of the cartilage. (V and W) Comparison of 14.5-dpc wt and Mia3−/− forelimb RNA on Mouse Genome 430 v2.0 arrays (Affymetrix) reveals an overall reduction in genes associated with chondrogenic progression (V) and osteoblast expansion (W; n = 5 per group, P < 0.05). Expression values are represented as colors, in which the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest). Bars, 100 µm.
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fig4: Early chondrocyte proliferation, differentiation, and critical patterning pathways are preserved in 14.5-dpc humeri despite abnormal chondrocyte morphology in Mia3−/− embryos. (A–D) Ki67 labeling of 14.5-dpc (A and B) and 16.5-dpc (C and D) humeri demonstrate normal proliferation of mutant chondrocytes. RZ, PZ, PHZ, and HZ denote resting, proliferative, prehypertrophic, and hypertrophic zones, respectively. (E and F) The majority of terminally differentiated hypertrophic chondrocytes are TUNEL positive in wt 14.5-dpc humeri (E), whereas Mia3- hypertrophic chondrocytes fail to die (F). (G and H) Scattered dying cells are restricted to the hypertrophic zone in 16.5-dpc wt and Mia3- humeri (red brackets). (I) Ki67-positive (+ve) cells within the proliferative zone. Mean values are presented, and SEM is shown from two independent wt samples and three Mia3−/− humeri with 4–10 sections/animal. (J–U) Section in situ analysis comparing expression of Col9a1, Col2a1, Col10a1, Ihh, Ptc, and Pth1r between wt and Mia3- 14.5-dpc humeri demonstrates an intact Ihh and Pth1r signaling axis despite dysmorphism of the cartilage. (V and W) Comparison of 14.5-dpc wt and Mia3−/− forelimb RNA on Mouse Genome 430 v2.0 arrays (Affymetrix) reveals an overall reduction in genes associated with chondrogenic progression (V) and osteoblast expansion (W; n = 5 per group, P < 0.05). Expression values are represented as colors, in which the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest). Bars, 100 µm.

Mentions: Histological analysis confirmed the chondrogenic delay and highlighted defects in chondrocytic ECM deposition. Cartilaginous templates of the bone first become morphologically distinct in the Mia3 mutants at 13.5 dpc (Fig. 3, I and J). In controls, Masson’s trichrome stain reveals collagen deposition typical of maturing chondrocytes that begin to secrete increasing amounts of collagen and other structural proteins in the ECM (Fig. 3 I; Kronenberg, 2003). However, very little stain is seen in the mutants (Fig. 3 J). This is not simply because of a delay in cellular differentiation, as the ECM stain surrounding the hypertrophic cells is still significantly reduced in the mutant embryos at 15.5 dpc (Fig. 3, K and L). Mineralized bone collar, which is stained darkly blue, is embedded in the perichondrium of controls at this stage but missing from the mutants. Hematoxylin and eosin–stained sections of Mia3−/− humeri show a complete absence of well-defined trabeculae seen at the diaphysis of controls (Fig. 3, M and N). Ki67 labeling of 14.5-dpc humeri indicates that chondrocytes populating the resting and proliferative zones maintain their proliferative capacity in the knockout mice (Fig. 4, A, B, and I). However, consistent with the arrest in chondrogenic progression, dying TUNEL-positive cells are completely absent from the center of the mutant bone (Fig. 4, E and F). By 16.5 dpc, the majority of cells residing internal to the bone collar are Ki67 positive, whereas in the mutant, the terminally differentiated chondrocytes remain unlabeled (Fig. 4, C and D), and a small number of these are now TUNEL positive (Fig. 4, G and H). Dying cells are not apparent elsewhere in the mutant bone. Thus, the generalized dwarfing of the knockout skeleton is driven primarily by the delay and arrest of chondrogenic maturation, lack of vascular recruitment, and the failure to elaborate a primary ossification center in Mia3 mutants.


Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse.

Wilson DG, Phamluong K, Li L, Sun M, Cao TC, Liu PS, Modrusan Z, Sandoval WN, Rangell L, Carano RA, Peterson AS, Solloway MJ - J. Cell Biol. (2011)

Early chondrocyte proliferation, differentiation, and critical patterning pathways are preserved in 14.5-dpc humeri despite abnormal chondrocyte morphology in Mia3−/− embryos. (A–D) Ki67 labeling of 14.5-dpc (A and B) and 16.5-dpc (C and D) humeri demonstrate normal proliferation of mutant chondrocytes. RZ, PZ, PHZ, and HZ denote resting, proliferative, prehypertrophic, and hypertrophic zones, respectively. (E and F) The majority of terminally differentiated hypertrophic chondrocytes are TUNEL positive in wt 14.5-dpc humeri (E), whereas Mia3- hypertrophic chondrocytes fail to die (F). (G and H) Scattered dying cells are restricted to the hypertrophic zone in 16.5-dpc wt and Mia3- humeri (red brackets). (I) Ki67-positive (+ve) cells within the proliferative zone. Mean values are presented, and SEM is shown from two independent wt samples and three Mia3−/− humeri with 4–10 sections/animal. (J–U) Section in situ analysis comparing expression of Col9a1, Col2a1, Col10a1, Ihh, Ptc, and Pth1r between wt and Mia3- 14.5-dpc humeri demonstrates an intact Ihh and Pth1r signaling axis despite dysmorphism of the cartilage. (V and W) Comparison of 14.5-dpc wt and Mia3−/− forelimb RNA on Mouse Genome 430 v2.0 arrays (Affymetrix) reveals an overall reduction in genes associated with chondrogenic progression (V) and osteoblast expansion (W; n = 5 per group, P < 0.05). Expression values are represented as colors, in which the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest). Bars, 100 µm.
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fig4: Early chondrocyte proliferation, differentiation, and critical patterning pathways are preserved in 14.5-dpc humeri despite abnormal chondrocyte morphology in Mia3−/− embryos. (A–D) Ki67 labeling of 14.5-dpc (A and B) and 16.5-dpc (C and D) humeri demonstrate normal proliferation of mutant chondrocytes. RZ, PZ, PHZ, and HZ denote resting, proliferative, prehypertrophic, and hypertrophic zones, respectively. (E and F) The majority of terminally differentiated hypertrophic chondrocytes are TUNEL positive in wt 14.5-dpc humeri (E), whereas Mia3- hypertrophic chondrocytes fail to die (F). (G and H) Scattered dying cells are restricted to the hypertrophic zone in 16.5-dpc wt and Mia3- humeri (red brackets). (I) Ki67-positive (+ve) cells within the proliferative zone. Mean values are presented, and SEM is shown from two independent wt samples and three Mia3−/− humeri with 4–10 sections/animal. (J–U) Section in situ analysis comparing expression of Col9a1, Col2a1, Col10a1, Ihh, Ptc, and Pth1r between wt and Mia3- 14.5-dpc humeri demonstrates an intact Ihh and Pth1r signaling axis despite dysmorphism of the cartilage. (V and W) Comparison of 14.5-dpc wt and Mia3−/− forelimb RNA on Mouse Genome 430 v2.0 arrays (Affymetrix) reveals an overall reduction in genes associated with chondrogenic progression (V) and osteoblast expansion (W; n = 5 per group, P < 0.05). Expression values are represented as colors, in which the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest). Bars, 100 µm.
Mentions: Histological analysis confirmed the chondrogenic delay and highlighted defects in chondrocytic ECM deposition. Cartilaginous templates of the bone first become morphologically distinct in the Mia3 mutants at 13.5 dpc (Fig. 3, I and J). In controls, Masson’s trichrome stain reveals collagen deposition typical of maturing chondrocytes that begin to secrete increasing amounts of collagen and other structural proteins in the ECM (Fig. 3 I; Kronenberg, 2003). However, very little stain is seen in the mutants (Fig. 3 J). This is not simply because of a delay in cellular differentiation, as the ECM stain surrounding the hypertrophic cells is still significantly reduced in the mutant embryos at 15.5 dpc (Fig. 3, K and L). Mineralized bone collar, which is stained darkly blue, is embedded in the perichondrium of controls at this stage but missing from the mutants. Hematoxylin and eosin–stained sections of Mia3−/− humeri show a complete absence of well-defined trabeculae seen at the diaphysis of controls (Fig. 3, M and N). Ki67 labeling of 14.5-dpc humeri indicates that chondrocytes populating the resting and proliferative zones maintain their proliferative capacity in the knockout mice (Fig. 4, A, B, and I). However, consistent with the arrest in chondrogenic progression, dying TUNEL-positive cells are completely absent from the center of the mutant bone (Fig. 4, E and F). By 16.5 dpc, the majority of cells residing internal to the bone collar are Ki67 positive, whereas in the mutant, the terminally differentiated chondrocytes remain unlabeled (Fig. 4, C and D), and a small number of these are now TUNEL positive (Fig. 4, G and H). Dying cells are not apparent elsewhere in the mutant bone. Thus, the generalized dwarfing of the knockout skeleton is driven primarily by the delay and arrest of chondrogenic maturation, lack of vascular recruitment, and the failure to elaborate a primary ossification center in Mia3 mutants.

Bottom Line: These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton.Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality.Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

Show MeSH
Related in: MedlinePlus