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. 2014 Feb 18;9(2):e85145.
doi: 10.1371/journal.pone.0085145. eCollection 2014.

Abnormal Chondrocyte Apoptosis in the Cartilage Growth Plate Is Influenced by Genetic Background and Deletion of CHOP in a Targeted Mouse Model of Pseudoachondroplasia

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Abnormal Chondrocyte Apoptosis in the Cartilage Growth Plate Is Influenced by Genetic Background and Deletion of CHOP in a Targeted Mouse Model of Pseudoachondroplasia

Katarzyna A Piróg et al. PLoS One. .
Free PMC article

Abstract

Pseudoachondroplasia (PSACH) is an autosomal dominant skeletal dysplasia caused by mutations in cartilage oligomeric matrix protein (COMP) and characterised by short limbed dwarfism and early onset osteoarthritis. Mouse models of PSACH show variable retention of mutant COMP in the ER of chondrocytes, however, in each case a different stress pathway is activated and the underlying disease mechanisms remain largely unknown. T585M COMP mutant mice are a model of moderate PSACH and demonstrate a mild ER stress response. Although mutant COMP is not retained in significant quantities within the ER of chondrocytes, both BiP and the pro-apoptotic ER stress-related transcription factor CHOP are mildly elevated, whilst bcl-2 levels are decreased, resulting in increased and spatially dysregulated chondrocyte apoptosis. To determine whether the abnormal chondrocyte apoptosis observed in the growth plate of mutant mice is CHOP-mediated, we bred T585M COMP mutant mice with CHOP-null mice to homozygosity, and analysed the resulting phenotype. Although abnormal apoptosis was alleviated in the resting zone following CHOP deletion, the mutant growth plates were generally more disorganised. Furthermore, the bone lengths of COMP mutant CHOP null mice were significantly shorter at 9 weeks of age when compared to the COMP mutant mice, including a significant difference in the skull length. Overall, these data demonstrate that CHOP-mediated apoptosis is an early event in the pathobiology of PSACH and suggest that the lack of CHOP, in conjunction with a COMP mutation, may lead to aggravation of the skeletal phenotype via a potentially synergistic effect on endochondral ossification.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Bone measurements in mice lacking CHOP.
A) Long bone measurements (cm) of wild type and CHOP null mice at 3, 6 and 9 weeks of age confirming that deletion of CHOP had no effect on the long bone formation (n = 10). B) Head measurements (cm) in wild type and CHOP null mice demonstrating shorter skulls in CHOP null mice at 9 weeks of age (n = 10; One Way ANOVA). Key: ICD = inner canthal distance; CHOP+/+ = wild type, CHOP−/− = knock-out. Error bars show standard error of the mean (SEM); * P<0.05.
Figure 2
Figure 2. Histochemical analysis of growth plate cartilage.
A) Haematoxylin and eosin staining of wild type and CHOP null growth plates at 3 weeks of age showing normal columnar organisation of chondrocytes in the growth plates of both genotypes and delineating the resting (RZ), proliferating (PZ) and hypertrophic (HZ) zones of the growth plate. B) Immunohistochemistry for COMP showing the normal distribution of COMP in wild type and CHOP null growth plate at 3 weeks of age. The negative control was generated using the secondary antibody only. Key: RZ = resting zone; PZ = proliferative zone; HZ = hypertrophic zone; CHOP+/+ = wild type, CHOP−/− = knock-out. Scale bar for all images is 100 µm.
Figure 3
Figure 3. Histochemical and immunohistochemical analysis of growth plate cartilage.
A) Haematoxylin and eosin staining of C57BL6/J enriched wild type (+/+ C57+) and T585M COMP mutant (m/m C57+) cartilage growth plates at 3 weeks of age showing further disorganisation and more hypocellular areas (red circles) in the m/m C57+ growth plates. B) Immunohistochemistry for COMP at 3 weeks of age showing abnormal columnar organisation of the growth plates in the mutant mice with areas of hypocellularity (red circles) and the retention of COMP in the T585M COMP mutant (COMPm/mC57+) hypertrophic zone (red arrowheads) on the C57BL6/J enriched genetic background. Positive COMP staining is brown with green nuclear counterstain. The negative control was generated using the secondary antibody only. Key: +/+ = wild type mice (mixed background); +/+ C57+ = wild type mice (enriched C57 background); m/m = T585M COMP mutant mice (mixed background); m/m C57+ = T585M COMP mutant mice (enriched C57 background). Scale bar in all images is 100 µm.
Figure 4
Figure 4. BrdU and TUNEL labelling of growth plate cartilage.
A) The relative proportion of BrdU positive cells in wild type and T585M COMP mutant growth plates compared to the corresponding C57BL6/J enriched mice (n = 3; One Way ANOVA). This analysis demonstrated a further decrease in chondrocyte proliferation at 3 weeks of age due to the change in genetic background. B) Representative images of TUNEL staining in C57BL6/J enriched wild type and COMP mutant cartilage growth plates at 3 weeks of age. Green staining (yellow arrowheads) identifies TUNEL positive (apoptotic) cells against a DAPI (blue) nuclear counter stain. C) Quantification of the TUNEL staining in wild type and mutant growth plates at 3 weeks of age (n = 3; One Way ANOVA) showing further dysregulation of apoptosis. Key: COMP+/+ = wild type mice (mixed background); COMP+/+C57+ = wild type mice (enriched C57 background); COMPm/m = T585M COMP mutant mice (mixed background); COMPm/m C57+ = T585M COMP mutant mice (enriched C57 background). Error bars show standard error of the mean (SEM); * P<0.05, **P<0.01, *** P<0.001.
Figure 5
Figure 5. Histochemical and immunohistochemical analysis of growth plate cartilage.
A) Haematoxylin and eosin staining of T585M COMP mutant and either CHOP wild type [CHOP+/+COMPm/m] or CHOP null [CHOP−/−COMPm/m] growth plates at 3 weeks of age. Areas of hypocellularity localised to the lower hypertrophic zone (red circles) are seen in [CHOP−/−COMPm/m] cartilage compared to [CHOP+/+COMPm/m] controls. B) Immunohistochemistry for COMP showing abnormal columnar organisation of the growth plate in the COMP mutant CHOP null mice [CHOP−/−COMPm/m] with areas of hypocellularity (yellow circles) in the hypertrophic zone at 3 weeks of age. Mutant COMP was also retained in chondrocytes of the hypertrophic zone of both [CHOP+/+COMPm/m] and [CHOP−/−COMPm/m] due to the enriched C57BL6/J background in this model (see yellow arrows). Positive staining for COMP is brown with a green nuclear counterstain. The negative control was generated using a secondary antibody only. Key: CHOP+/+COMPm/m = CHOP wild type and T585M COMP mutation (homozygous); CHOP−/−COMPm/m = CHOP null and T585M COMP mutation (homozygous). Scale bar in all images is 100 µm.
Figure 6
Figure 6. TUNEL assay and cell quantification in growth plate at 3 weeks of age.
A) Quantification of the TUNEL staining in T585M COMP mutant and either CHOP wild type [CHOP+/+COMPm/m] or CHOP null [CHOP−/−COMPm/m] growth plates. This analysis shows a significant decrease in apoptosis in the resting zone following deletion of CHOP (n = 3; One Way ANOVA). B) The average number of cells per growth plate zone showing a significant increase in the RZ and HZ and a trend towards an increase in the PZ following deletion of CHOP (n = 3; One Way ANOVA). C) The average number of TUNEL positive cells per growth plate zone showing a significant decrease in the RZ and a trend towards an increase in the HZ of the [CHOP−/−COMPm/m] growth plates (n = 3; One Way ANOVA). Key: CHOP+/+COMPm/m = CHOP wild type and T585M COMP mutation (homozygous); CHOP−/−COMPm/m = CHOP null and T585M COMP mutation (homozygous); RZ = resting zone; PZ = proliferative zone; HZ = hypertrophic zone. Error bars show standard error of the mean (SEM); # P = 0.06, * P<0.05, ** P<0.01, *** P<0.001.
Figure 7
Figure 7. Western blot and BrdU analysis of mouse cartilage at 3 weeks of age.
A) A representative image of a bcl-2 Western blot on mouse xiphoid homogenates at 3 weeks. 4 mice per genotype (1–4); GAPDH was used as a loading control. B) Densitometry of Western blots of cartilage homogenates from xiphoid cartilage showing an significant increase in bcl-2 and BiP protein after CHOP removal (n = 3; t-test) B) Quantification of the relative number of BrdU positive cells in T585M COMP mutant and either CHOP wild type [CHOP+/+COMPm/m] or knock-out [CHOP−/−COMPm/m] growth plate showing that CHOP deletion had no effect on chondrocyte proliferation (n = 3; t-test). Key: CHOP+/+COMPm/m = CHOP wild type and T585M COMP mutation (homozygous); CHOP−/−COMPm/m = CHOP null and T585M COMP mutation (homozygous). Error bars show standard error of the mean (SEM); * P<0.05.
Figure 8
Figure 8. Head and bone measurements in COMP mutant and CHOP null mice.
A) Head measurements in T585M COMP mutant and either CHOP wild type [CHOP+/+COMPm/m] or CHOP null [CHOP−/−COMPm/m] showing shorter skulls in [CHOP−/−COMPm/m] at 9 weeks of age (n = 10; One Way ANOVA). B) Long bone measurements in [CHOP−/−COMPm/m] mice showing a further exacerbated short limbed dwarfism following CHOP deletion (n = 10; One Way ANOVA). Error bars represent standard error of the mean (SEM) Key: Key: CHOP+/+COMPm/m = CHOP wild type and T585M COMP mutation (homozygous); CHOP−/−COMPm/m = CHOP null and T585M COMP mutation (homozygous); ICD = inner canthal distance. Error bars represent standard error of the mean (SEM); * P<0.05, ** P<0.01, *** P<0.001.

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