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First published online 7 April 2009
doi: 10.1242/jcs.040709

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PTHrP prevents chondrocyte premature hypertrophy by inducing cyclin-D1-dependent Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation

Ming Zhang^1,*, Rong Xie^2,*, Wei Hou^2,3, Baoli Wang², Run Shen², Xiumei Wang², Qing Wang¹, Tianhui Zhu¹, Jennifer H. Jonason² and Di Chen^2,

¹ Medical College of Nankai University, Tianjin 300071, PR China
² Department of Orthopaedics, Center for Musculoskeletal Research, University of Rochester School of Medicine, Rochester, NY 14642, USA
³ Spine Research Institute, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, PR China

View larger version (55K): [in this window] [in a new window]   Fig. 1. Cyclin D1 induces Runx3 degradation. (A) A Runx3 expression plasmid was cotransfected with different amounts of cyclin A and cyclin D1 expression plasmids (0.2, 0.6 and 1.8 µg/6-cm culture dish) into COS cells. Western blotting was performed 48 hours after transfection. Cyclin D1 induced Runx3 degradation. By contrast, cyclin A had no effect on Runx3 degradation. (B) Myc-Runx3 and cyclin D1 expression plasmids were cotransfected into COS cells. Immunoprecipitation (IP) was performed using the anti-CDK4 antibody followed by western blotting (WB) with an anti-Myc or anti-cyclin D1 antibody. CDK4 interacts with Runx3 in COS cells. The interaction between cyclin D1 and CDK4 was also detected as a positive control. (C,D) Cell lysates were collected from chondrogenic RCJ3.1C5.18 cells and immunoprecipitation was performed using either an anti-CDK4 antibody or an anti-Runx3 antibody followed by western blotting using the anti-Runx3 or anti-CDK4 antibody. Interaction was detected between endogenous Runx3 and CDK4.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Ser356 of Runx3 is critical for Runx3 stability. (A) Protein decay assay. Equal amounts of Myc-Runx3, FLAG-S356A-Runx3 or FLAG-S356E-Runx3 expression plasmid were transfected into COS cells. Cell lysates were extracted after treatment with 80 µg/ml cycloheximide for 0, 20, 60, 120 or 300 minutes and western blotting was performed. WT Runx3 protein levels were considerably reduced at 120 minutes after protein synthesis was inhibited by cycloheximide. FLAG-S356A-Runx3 remained stable during the entire 300 minute period after cycloheximide treatment. By contrast, FLAG-S356E-Runx3 expression began to be considerably reduced at 60 minutes after cells were treated with cycloheximide. (B) Myc-Runx3 and FLAG-S356A-Runx3 expression plasmids were cotransfected with different amounts of cyclin D1 plasmid (0.2, 0.6, and 1.8 µg/dish) into COS cells. Cell lysates were extracted 24 hours after transfection and western blotting was performed. WT Runx3 protein levels were considerably reduced by cyclin D1 in a dose-dependent manner. By contrast, S356A-Runx3 levels were not affected by the overexpression of cyclin D1.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Cyclin D1 induces Runx3 phosphorylation. (A) COS cells were transfected with equal amounts of Myc-Runx3 and FLAG-S356A-Runx3 plasmids and treated with PS1 (10 µM, 4 hours incubation). Cell lysates were extracted 24 hours after transfection. Phosphorylation of Runx3 was detected by western blotting (WB) using the anti-phosphoserine antibody after WT and mutant Runx3 protein was immunoprecipitated (IP) using either the anti-Myc or anti-FLAG antibody. Strong phosphorylation was detected in WT Runx3-transfected cells. However, only trace amount of phosphorylated mutant Runx3 (P-Runx3) was detected. (B) In vitro phosphorylation assay. CDK4 was immunoprecipitated from 300 µg C2C12 cell lysate using 1.5 µg anti-CDK4 antibody and was used in the Runx3 kinase assay (1 µM substrate). GST-Rb (379-928) was used as a positive control and GST-Smad4 was used as a negative control. GST-Runx3 exhibits a strong phosphorylation, whereas GST-S356A-Runx3 exhibits a weak phosphorylation. Left: Runx3 phosphorylation by CDK4. Right: protein amounts, detected by Coomassie blue staining.

View larger version (69K): [in this window] [in a new window]   Fig. 4. Cyclin D1 induces ubiquitylation and proteasome degradation of Runx3. (A) Myc-Runx3 and HA-ubiquitin expression plasmids were cotransfected with different amounts of cyclin D1 expression plasmid (0.2 and 0.6 µg/6-cm culture dish) into COS cells in the presence or absence of PS1 (10 µM for 4 hour incubation). Cyclin D1 induced Runx3 ubiquitylation in a dose-dependent manner and this effect was further enhanced by the addition of PS1. (B) Myc-Runx3, FLAG-S356A-Runx3 or FLAG-S356E-Runx3 was cotransfected with HA-ubiquitin plasmid into COS cells in the presence of PS1 (10 µM, 4 hours incubation). Runx3 ubiquitylation was detected in the WT Runx3 and S356E-Runx3 groups but not in the S356A-Runx3 group. (C) To further determine the ubiquitylation of endogenous Runx3, chondrogenic RCJ3.1C5.18 cells were treated without or with MG132 (10 µM, 4 hour incubation) before cell lysates were collected. Immunoprecipitation was performed using the anti-Runx3 antibody followed by western blotting using the anti-ubiquitin antibody. Ubiquitylation of endogenous Runx3 was detected in the presence of MG132 in RCJ3.1C5.18 cells. (D) Myc-Runx3 expression plasmid was cotransfected with different amounts of cyclin D1 expression plasmid (0.2, 0.6 and 1.8 µg/dish) into COS cells. Cells were treated with PS1 (10 µM) for 4 hours after transfection. Cyclin D1 induced a dose-dependent degradation of Runx3 and treatment with PS1 completely reversed cyclin-D1-induced Runx3 degradation. (E) WT and mutant Runx3 (S356A) expression plasmids were transfected into COS cells. Cells were treated with PS1 (10 µM, 4 hour incubation) after transfection. The addition of PS1 increased the level of WT Runx3 protein but had not effect on mutant Runx3 (S356A) protein.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Ser356 is critical for Runx3 function. (A) Wild-type (WT) and mutant Runx3 (S356E and S356A) expression plasmids were cotransfected with 6xOSE2-Luc reporter into C3H10T1/2 cells. Cell lysates were collected 24 hours after transfection and a luciferase assay was performed. The S356A-Runx3 increased activity and S356E-Runx3 decreased activity of the 6xOSE2-Luc reporter compared with the WT Runx3. *P<0.05, compared with the WT Runx3; unpaired Student's t-test. (B) WT and mutant Runx3 (S356E and S356A) expression plasmids were transfected into C3H10T1/2 cells. ALP activity was measured using an ALP assay kit. S356A-Runx3 increased and S356E-Runx3 decreased ALP activity compared with the WT Runx3. *P<0.05, compared with the WT Runx3; unpaired Student's t-test.

View larger version (75K): [in this window] [in a new window]   Fig. 6. Chondrocyte proliferation and differentiation are altered in Ccdn1-knockout mice. (A-F) Histological analyses, including Safranin O/Fast green (A,C) and Alcian blue/Hemotoxylin and Orange G (B) staining, showed that growth plate length (GPL; D) and the lengths of the proliferating zone (PZ; C,E) and the hypertrophic zone (HZ; C,F) were reduced (n=6) and the formation of the secondary ossification center was delayed (black arrows in A and B) in Ccdn1-knockout mice compared to their WT littermates. (G-M) Primary chondrocytes were isolated from 3-day-old Ccdn1-/- mice and WT littermates and expression of chondrocyte marker genes and alkaline phosphatase (ALP) staining were examined. The results demonstrated that the expression of chondrocyte marker genes, such as Alp (G), collagen type X (ColX; H), Mmp9 (I), Mmp13 (J), Vegf (K) and osteocalcin (Oc; L), and ALP activity (M) were significantly increased in Ccdn1- knockout chondrocytes (n=3). *P<0.05, compared with the WT littermate controls; unpaired Student's t-test.

View larger version (33K): [in this window] [in a new window]   Fig. 7. PTHrP downregulates steady-state protein levels of Runx2 and Runx3 in a cyclin-D1-dependent manner. (A,B) The time-course effects of PTHrP on the expression of cyclin D1 mRNA and CDK protein were examined by real-time PCR and western blotting in chondrogenic RCJ3.1C5.18 cells. PTHrP (10-8 M) significantly increased the expression of cyclin D1 mRNA and CDK4 protein (4-12 hour treatment) in RCJ3.1C5.18 cells. (C,D) Effects of PTHrP on Runx2 and Runx3 protein levels were examined by western blotting in primary chondrocytes. PTHrP significantly downregulated Runx2 (C) and Runx3 (D) protein levels in WT chondrocytes. However, these effects were completely inhibited in Ccdn1-knockout chondrocytes. *P<0.05, one-way analysis of variance followed by Dunnett's test, compared with the control group without PTHrP treatment.

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