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First published online 2 October 2008
doi: 10.1242/dev.028118

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PTEN deficiency causes dyschondroplasia in mice by enhanced hypoxia-inducible factor 1 signaling and endoplasmic reticulum stress

State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, 20 Dongdajie, Beijing 100071, China.

View larger version (50K): [in this window] [in a new window]   Fig. 1. Targeted disruption of the Pten gene in mouse chondrocytes results in skeleton overgrowth. (A) Phenotype of Pten+/+;Col2a1-Cre (left), PtenCo/+;Col2a1-Cre (middle) and PtenCo/Co;Col2a1-Cre (right) littermates at P40. (B) Tail length measurements of female (left) and male (right) mice at various time points. Each point represents the mean ±s.d. from six mice. (C) Soft X-ray images of femur and tibia from P50 mice showing increased width (red arrowheads) and bone density (white arrowheads) of long bones in mutant mice. (D) Tibia and femur length were not significantly changed in mutant mice. Mean ±s.d. of eight samples from four mice of each genotype. P>0.05. (E-G) Efficient deletion of Pten in mutant cartilage as revealed by immunohistochemistry (E), Southern blot (F) and western blot (G). Loss of Pten also caused phosphorylation of AKT (p-AKT) within chondrocytes (E,G). Scale bar: 50 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 2. Dyschondroplasia in adult PtenCo/Co;Col2a1-Cre mice. (A-F) Safranine O staining performed on sections of femur (A-D) and tibia (E,F) from control (A,C,E) or mutant (B,D,F) littermates. Cartilaginous nodules were present in the epiphysis (B, black arrowhead), metaphysis (D, black arrowhead) or diaphysis (F, black arrowhead) of long bones in mutant mice. Cartilaginous nodules resulted in ectopic ossification within cortical bone (D, red arrowhead) or within the bone marrow cavity (F, red arrowhead). Age of bones: P32 in A,B; P58 in C,D; P48 in E,F. Scale bar: 500 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 3. Impaired, but resumable, proliferation of the center-located chondrocytes within PtenCo/Co;Col2a1-Cre growth plates. (A,B,D-L) BrdU labeling was performed on sections of control (A,D,G,J) or mutant (B,E,H,K) growth plates from P3 (A,B), P5 (D,E), P7 (G,H) and P38 (J,K) mice. (C) The percentage of BrdU-positive chondrocytes in the proliferating zone of P3 control (A) or mutant (B) growth plates was determined in consecutive columns, as delineated by the grids, and plotted. Mean ±s.d. of six sections from three mice of each genotype. *P<0.01. (E,H) The central region of mutant growth plate cartilage lost the ability to proliferate and gradually formed a neoplastic core. (F,I,L) Higher magnification of the boxed regions in E,H,K. Note the small number of BrdU-positive chondrocytes within the lower portion of the core (F,I, yellow arrowheads) and within the cartilaginous nodule (L, black arrowhead). Some balloon-like chondrocytes within the lower portion of the core also started to undergo hypertrophy (I, red arrowhead). Hours of BrdU labeling: A,B, 2; D,E, 5; G,H, 6; J,K, 2.5. Scale bar: 275 µm in A,B,J,K; 340 µm in D,E; 400 µm in G,H.

View larger version (113K): [in this window] [in a new window]   Fig. 4. Delayed and asynchronous chondrocyte differentiation within PtenCo/Co;Col2a1-Cre growth plates. (A,B) Long-term BrdU labeling-chasing assay. BrdU was retained in the nuclei of resting chondrocytes and of the cells within the neoplastic core after a 7-day chase in both control (A) and mutant (B) growth plates. (C,D) Short-term BrdU labeling-chasing assay. Hypertrophic chondrocytes that transited from proliferating chondrocytes were labeled after a 1.5-day chase. (E) Statistical analysis of the quantities of BrdU-positive hypertrophic chondrocytes in the femur and tibia. Mean ±s.d. of six sections from two mice of each genotype. **P<0.01. (F-M) Sections from P5 control (F,H,J,L) or mutant (G,I,K,M) femoral growth plate were hybridized in situ with RNA probes for Ppr (F,G), Ihh (H,I) or Col10a1 (J,K), or subjected to von-Kossa staining (L,M). Red bars indicate layers of terminal hypertrophic chondrocytes. Scale bar: 400 µm in A,B; 275 µm in C,D,J,K; 544 µm in F-I; 222 µm in L,M.

View larger version (99K): [in this window] [in a new window]   Fig. 5. Abnormal cellularity and Col II properties in PtenCo/Co;Col2a1-Cre growth plates. (A,B) Immunohistochemistry (IHC) for type II collagen (Col II) proteins performed on mouse P5 proximal humeral growth plate sections. The neoplastic core was clearly distinguishable by its abnormal cellularity and Col II properties (B). (C-F) Electron microscopy images of chondrocytes and surrounding matrix from resting zone (C) and proliferating zone (E) control growth plate, or from the periphery (D) and central region (F) of the core. The insets show higher magnification views from positions indicated in A or B. The ER of the abnormal cells was extremely distended and fragmented (D,F, asterisks) compared with the normal ER (C,E). Note the premature `string-bead' fibrils implanted inside the deformed ER (F, red arrowhead), which were similar to those surrounding the resting chondrocytes (C, red arrowhead). (G-J) IHC for Col II proteins of wild-type (G,I) and mutant (H,J) chondrocytes cultured under normoxic (G,H) or hypoxic (I,J) conditions. Note that Pten mutant chondrocytes were detached from each other and were swollen by pools of Col II fibrils within the cytoplasm under hypoxic conditions (J, red arrowheads). (K) Col2a1 mRNA expression in control and mutant primary chondrocytes cultured under hypoxic conditions as evaluated by northern blot. Scale bar: 500 µm in A,B; 2 µm in C; 2.5 µm in D; 2.9 µm in E; 3.1 µm in F; 90 µm in insets, C-F; 120 µm in G-J.

View larger version (125K): [in this window] [in a new window]   Fig. 6. Activated HIF1 pathway in PtenCo/Co;Col2a1-Cre growth plates. (A-D,F-M,O-V) Sections of proximal tibial growth plates from E16 mouse embryos (A,B,F,G,J,K,O,P,S,T) and P5 femoral growth plates (C,D,H,I,L,M,Q,R,U,V) were subjected to IHC for HIF1 protein expression (A-D) or in situ hybridization with RNA probes for Vegf (F-I), Pgk (J-M), p21Cip1 (O-R) or p57Kip2 (S-V). Note the ectopic activation of HIF1 protein within mutant growth plates (B,D, red arrowheads). (E) Western blot analysis of HIF1 protein accumulation in the nuclei of control or mutant chondrocytes isolated from knee joints. (N) mRNA expression of total Vegf, Vegf isoforms (Vegf120, Vegf164) and Pgk within control or mutant cartilage from knee joints as revealed by real-time RT-PCR. Error bars represent means ± s.d. from six replicate PCR reactions of a single experiment.*P<0.05, **P<0.01. Scale bar: 200 µm in A,B,F,G,J,K,O,P,S,T; 470 µm in C,D,H,I,L,M,Q,R,U,V.

View larger version (61K): [in this window] [in a new window]   Fig. 7. Activated HIF1 pathway causes ER stress in chondrocytes. In situ hybridization for BiP mRNA in mouse E16 proximal tibial (A,B) and P5 proximal humeral (C,D) growth plates. Note the ectopic activation of BiP mRNA around the neoplastic core of mutant growth plates (D, arrowhead). (E) BiP protein expression in control and mutant primary chondrocytes cultured under normoxic or hypoxic conditions for 1 or 5 days as revealed by western blot analysis. (F) Control and mutant chondrocytes were treated with increasing concentrations of DMOG for 2 days and then analyzed by western blot with antibodies against HIF1 and BiP. (G) Control and mutant chondrocytes were treated with 1 mM DMOG for increasing times and then analyzed by western blot with antibodies against HIF1 and BiP. Scale bar: 200 µm in A,B; 470 µm in C,D.

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