Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma - PubMed (original) (raw)

. 2004 Dec 6;167(5):925-34.

doi: 10.1083/jcb.200409187.

Sandra A Johnson, Natalie A Sims, Melanie K Trivett, John L Slavin, Brian P Rubin, Paul Waring, Grant A McArthur, Carl R Walkley, Andrew J Holloway, Dileepa Diyagama, Jonathon E Grim, Bruce E Clurman, David D L Bowtell, Jong-Seo Lee, Gabriel M Gutierrez, Denise M Piscopo, Shannon A Carty, Philip W Hinds

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Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma

David M Thomas et al. J Cell Biol. 2004.

Abstract

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

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Figures

Figure 1.

Figure 1.

Runx2-dependent osteogenic differentiation is disrupted in osteosarcoma cell lines. (A) Gene expression arrays were performed using RNA extracted from confluent cultures of SAOS2, MG63, HOS, B143, SJSA, and G292 cell lines, normalized to reference RNA as described in Materials and methods section. Data presented are the median log-transformed data for six cell lines. (B) Cells were transfected with 6ose2-luc (or with 6ose2mut-luc) and cytomegalo virus (CMV)–βgal plasmids. After 24 h, luciferase activity was measured and normalized to β-galactosidase. The ratio of the activity of 6ose2-luc to 6ose2mut-luc activity is shown. Ost, osteogenic osteoma cell line CCL-7672. Data shown are means ± SEM. (C) Cells were transfected with 6ose2-luc and CMV-βgal plasmids, with or without a runx2 expression vector. After 24 h, luciferase activity was measured and normalized for transfection efficiency with β-galactosidase. The ratio of luciferase activity in the presence or absence of runx2 is shown. Data shown are means ± SEM. (D) Western blot for runx2 and pRb in nuclear extracts from the indicated cell lines. HOb, human primary osteoblast.

Figure 2.

Figure 2.

Runx2 induces a growth arrest through induction of p27_KIP1_. (A) Effect of ectopic expression of runx2 on colony suppression assay. (B) Retroviral vectors expressing runx2 27ala or runx2 3ala were used to infect 3T3 or RB−/− 3T3 cells, followed by selection for 3 d in 2 μg/ml puromycin. Cell cycle profile was determined by flow cytometry. (C) IMR90, CCL-211, SAOS2, and U2OS cells were infected with adenoviral constructs expressing FLAG-tagged runx2. Western blot for runx2, p27_KIP1_, and p21_CIP1_. The percentage of cells in S-phase, derived from parallel cultures subjected to DNA analysis by flow cytometry, is indicated (bottom). (D) CCL-211 cells were infected with adenoviral constructs expressing runx2. Cyclin A immunoprecipitates were subjected to Western blot for p27_KIP1_, Cdk2, and cyclin A. The bottom panel is directly blotted for p27_KIP1_. Asterisks indicate Ig heavy chain bands. (E) Cyclin A immunoprecipitates assayed for kinase activity in the presence of runx2. CCL-211 cells were treated as in C. Direct Western blot for Rb, cyclin A, cyclin E, Cdk2, p27_KIP1_, p21_CIP1_, runx2, and GFP to demonstrate equal titers of virus in each culture. (F) Runx2 binds the hypophosphorylated form of pRb. COS-7 cells were transfected with vector, pRb, and HA-tagged pRb. A pulldown was performed using GST-runx2. Input is 5% of that used for the pulldown. ppRb, phosphorylated species of pRb. Black lines indicate that intervening lanes have been spliced out.

Figure 3.

Figure 3.

Osteogenic differentiation in vitro is associated with induction of p27_KIP1_ mRNA and protein. (A) MC3T3E1 cells were cultured in differentiation media containing 50 μg/ml ascorbic acid and 2 mM β-glycerophosphate. (top) Western blot of p27_KIP1_ protein after 2–8 d in differentiation media. (bottom) Alizarin red staining of mineralized cultures over 3–12 d under identical conditions. (B) Murine embryonic fibroblasts (MEFs) cultured in the presence of a BMP4/7 fusion protein for 14 d. (top) Induction of AP activity. Data shown are means ± SEM. (bottom left) Alizarin red staining for mineralization. (bottom right) RT-PCR for runx2, p27_KIP1_, p21_CIP1_, and glyceraldehyde phosphate dehydrogenase (GAPDH). Fold changes in gene expression relative to GAPDH are given below each panel.

Figure 4.

Figure 4.

Growth arrest due to expression of runx2 or treatment with BMP2 is reduced by knockdown of p27 KIP1 . (A) Primary MEFs of the indicated genotypes were treated with 100 ng/ml BMP2 for 48 h followed by flow cytometric analysis of DNA content. Data shown are the change in G1 fraction due to treatment. This experiment was repeated twice with similar results. (B) Western blot for p27_KIP1_ in MC3T3E1 cells infected with a retrovirus expressing either control siRNA or siRNA for p27_KIP1_. (C) DNA analysis using flow cytometry of cultures of cells derived as described in B. (D) Colony suppression assay of cells as described transfected with empty vector or runx2. Each experiment was performed in triplicate.

Figure 5.

Figure 5.

Role of p27_KIP1_ in osteoblast function. (A) MEFs of the indicated genotypes were differentiated in the presence of 100 ng/ml BMP2, ascorbic acid, and β-glycerophosphate for 7 d and analyzed for AP activity. Data shown are means ± SEM of fold change relative to untreated wild-type controls (n = 4 experiments using independently derived littermate-matched cultures, each in triplicate). Genotype effect significant by analysis of variance (ANOVA; P < 0.01). (B) RT-qPCR analysis of gene expression in MEFs of the indicated genotypes, normalized to ARPPo. Data shown are means ± SEM. Genotype effect significant for BMP2 induction of osteocalcin (P < 0.01) and type I collagen (P < 0.05, ANOVA), but not osteopontin. (C) Histomorphometric analysis of long bones in mice between 8 and 12 wk old of the indicated genotypes. Data shown are means ± SEM. Bold-faced data are significantly different from wild-type littermates (P < 0.05).

Figure 6.

Figure 6.

p27_KIP1_ is required for terminal growth arrest in vitro. MEFs were cultured in the presence or absence of 100 ng/ml BMP2, ascorbic acid, and β-glycerophosphate for 14 d after confluence. Cells were then passaged. (A) 5 × 104 cells were grown under standard culture conditions for 3 d, and then counted. Data shown are means ± SEM. (B) RT-qPCR analysis of osteocalcin gene expression in MEFs of the indicated genotypes 2 d after passage. Data shown are means ± SEM of cycle number (ΔΔCT) normalized to ARPPo and expression before passage. The interaction between BMP2 and genotype was significant (P < 0.01, ANOVA). (C) Photomicrograph of senescence-associated β-galactosidase–stained cultures. (i) Wild-type untreated cultures; (ii) _p27KIP1−/−_ untreated cultures; (iii) wild-type differentiated cultures; and (iv) _p27KIP1−/−_ differentiated cultures. (D) Cells were stained for senescence-associated (SA) β-galactosidase activity after passage and counted (>200 cells in triplicate cultures). Data shown are means ± SEM. The effect of BMP2 and genotype was significant (P < 0.01), although there was no interaction by ANOVA.

Figure 7.

Figure 7.

Expression of p27 KIP1 , osteocalcin, and proliferating cell nuclear antigen (PCNA) in human osteosarcoma samples. (A–I) High-power photomicrographs of parallel sections from two high-grade (A–C and G–I) and one low-grade (G–I) human osteosarcomas were stained for p27_KIP1_ (A, D, and G), osteocalcin (B, E, and H), and PCNA (C, F, and I). Arrows in D–F indicate multinucleated osteoclast; arrows in G–I indicate osteocytes. Bar, 50 μm. (J) Blinded quantitation of staining for p27_KIP1_ and PCNA in tumors with evidence of osteoblast differentiation (osteoid production) compared with dedifferentiated tumors. Error bars represent SEM. *, P < 0.05.

Figure 8.

Figure 8.

A model for interactions between cell cycle proteins and runx2 in osteoblasts. The interaction of hypophosphorylated pRB with runx2 completes a positive feedback loop, promoting cell cycle withdrawal and expression of the osteoblast phenotype. See Discussion section.

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