Endoplasmic Reticulum Stress-Unfolding Protein Response-Apoptosis Cascade Causes Chondrodysplasia in a <i>col2a1</i> p.Gly1170Ser Mutated Mouse Model

<div><p>The collagen type II alpha 1 (<i>COL2A1</i>) mutation causes severe skeletal malformations, but the pathogenic mechanisms of how this occurs are unclear. To understand how this may happen, a <i>col2a1</i> p.Gly1170Ser mutated mouse model was constructed and in homozygotes, the chondrodysplasia phenotype was observed. Misfolded procollagen was largely synthesized and retained in dilated endoplasmic reticulum and the endoplasmic reticulum stress (ERS)-unfolded protein response (UPR)-apoptosis cascade was activated. Apoptosis occurred prior to hypertrophy, prevented the formation of a hypertrophic zone, disrupted normal chondrogenic signaling pathways, and eventually caused chondrodysplasia. Heterozygotes had normal phenotypes and endoplasmic reticulum stress intensity was limited with no abnormal apoptosis detected. Our results suggest that earlier chondrocyte death was related to the ERS-UPR-apoptosis cascade and that this was the chief cause of chondrodysplaia. The <i>col2a1</i> p.Gly1170Ser mutated mouse model offered a novel connection between misfolded collagen and skeletal malformation. Further investigation of this mouse mutant model can help us understand mechanisms of type II collagenopathies.</p></div

Experimental evidence for apoptosis.

<p>(A) Immunostaining of cleaved caspase-3 in the growth plates. (B) TUNEL assay results showed apoptosis chondrocytes (green fluorescence) with DAPI labeled nucleuses. (C) Statistical analysis of the positive rates within littermates (≥10 sections for each genotype) showed increased apoptosis in homozygotes (*<i>P</i><0.01). Scale bar = 100 µm.</p

Relative ERS-related genes in rib cartilages of littermates.

<p>Each genotype contained more than 3 littermates. Gene expression was measured by real-time quantitative RT-PCR and normalized to GAPDH expression. Relative expression was calculated using the 2-ΔΔCt method. *<i>P</i><0.05 was considered statistically significant.</p

Skeletal analysis of mutant fetuses.

<p>(A) Alizarin Red and Alcian blue staining results of E16.5, E18.5 embryos and newborns. (B) Details of the skeletal structures between 3 genotypes: 1. forelimbs 2. hindlimbs 3. front paws 4. hind paws 5. ribs 6. lumbar spines. Note that in homozygotes, the middle phalanges were non-ossified, the intercostal spaces were decreased, and the vertebrae were shortened and widened. Heterozygotes were normal.</p

EdU study in growth plates.

<p>Short term labeled (3 h) EdU assay results in growth plates of E18.5 embryos. (A) Green fluorescent signals indicated proliferated cells and DAPI staining for nucleuses. (B) Statistical analysis of the positive rates within littermates (nWT = 2/nHetero = 5/nHomo = 3, each limbs had ≥3 sections for analysis) showed that proliferating chondrocytes were significantly decreased in homozygotes (*<i>P</i><0.01).</p

Construction of mutated mouse model.

<p>(A) Schematic description of transgene construction. A c.3508G>A mutation in Ex50 was generated by PCR-based site-directed mutagenesis and introduced into the targeting vector containing exons form Ex40 to Ex53 of the <i>col2a1</i> gene. A PGK-Neo gene and a PGK-TK gene were also introduced for the positive-negative selection. After being transfected into ES cells, the mutation was transferred into the genome by homologous recombination. The double resistant colonies which were verified with 5′arm+3′arm PCR and sequencing were considered correctly generated and further used for microinjection. (B) Sequencing results of mice of the 3 genotypes. Arrow denotes the c.3508G>A mutation in Ex50. (C) Phenotypes of the neonates of 3 genotypes. Homozygotes were cyanotic immediately after birth and died rapidly; heterozygotes had normal phenotypes.</p

Confocal microscope analysis in chondrocytes.

<p>Confocal microscope analysis results of chondrocytes that were isolated from articular cartilages of E19.5 embryos, cultured for 5 days, and processed with immunofluorescence with antibodies for type II collagen (left) and Grp78 (middle, an ER marker). Merged photos (right) showed abnormal assembly and intracellular retention of mutated type II collagen in homozygous cells. Scale bar = 10 µm.</p

Quantitative analysis of growth plate and immunostaining.

<p>(A) Total growth plate and chondrocyte zone heights of wild types and heterozygotes. (B) Integrated optical density (IOD) values of collagen type II for all 3 groups. (C) IOD values of Sox9 for all 3 groups. (D) IOD values of collagen type X for all 3 groups. Sample size ≥3 littermates/genotype. IOD value of each sample was obtained from the average of 3 different sections under the same power lens. *<i>P</i><0.05 was considered statistically significant.</p

Morphological measurements of mutant fetuses and mice.

<p>(A) Heights of E16.5, E18.5 embryos and newborns. (B) Weights of E16.5, E18.5 embryos and newborns. (C) Humeri lengths of E16.5, E18.5 embryos and newborns. (D) Femur lengths of E16.5, E18.5 embryos and newborns. Sample size ≥3 littermates/genotype/time point. *<i>P</i><0.05 was considered statistically significant.</p

Transmission electron microscope analysis.

<p>Transmission electron microscope analysis of the extracellular matrix (A, B, C) and chondrocytes (D, E, F) in the proliferating zone of the growth plates from E19.5 embryos. Dilated vesicles, such as the ER (1), and Golgi body (2), were commonly seen in transgenic chondrocytes (E, F). Scale bar for a–c corresponded to 1 nm, and bar for D–F corresponded to 200 nm.</p