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

Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P, McArthur GA, Walkley CR, Holloway AJ, Diyagama D, Grim JE, Clurman BE, Bowtell DD, Lee JS, Gutierrez GM, Piscopo DM, Carty SA, Hinds PW - J. Cell Biol. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ian Potter Foundation Centre for Cancer Genomics and Predictive Medicine, and Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Center, Victoria, Melbourne, Australia. david.thomas@petermac.org

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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Role of p27KIP1 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).
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fig5: Role of p27KIP1 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).

Mentions: Next, we wished to determine whether p27KIP1 is required for the osteoblast phenotype. Loss of p27KIP1 partially attenuated BMP2-induced AP activity (Fig. 5 A), and both basal and BMP2-induced expression of osteocalcin, osteopontin, and type I collagen mRNA were reduced (but not abolished by) the absence of p27KIP1 (Fig. 5 B). To determine the net effect of loss of p27KIP1 in vivo, the long bones of murine wild-type and p27KIP1−/− littermates were analyzed by histomorphometry. There was a minor effect of loss of p27KIP1 on the formation of unmineralized bone (osteoid), both as a function of total bone volume and as measured by osteoid thickness (Fig. 5 C). This effect was not a result of accelerated mineralization caused by loss of p27KIP1, because there was no evidence of altered mineral apposition rate as measured directly by dual calcein labeling. Osteoblast and osteoclast numbers were unchanged (unpublished data). Because osteoclasts do not resorb unmineralized osteoid (Chambers et al., 1985), any defect in osteoclast function is unlikely to account for the reduction in osteoid. These data are consistent with a very minor effect of loss of p27KIP1 on osteoblast differentiation and function.


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

Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P, McArthur GA, Walkley CR, Holloway AJ, Diyagama D, Grim JE, Clurman BE, Bowtell DD, Lee JS, Gutierrez GM, Piscopo DM, Carty SA, Hinds PW - J. Cell Biol. (2004)

Role of p27KIP1 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172443&req=5

fig5: Role of p27KIP1 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).
Mentions: Next, we wished to determine whether p27KIP1 is required for the osteoblast phenotype. Loss of p27KIP1 partially attenuated BMP2-induced AP activity (Fig. 5 A), and both basal and BMP2-induced expression of osteocalcin, osteopontin, and type I collagen mRNA were reduced (but not abolished by) the absence of p27KIP1 (Fig. 5 B). To determine the net effect of loss of p27KIP1 in vivo, the long bones of murine wild-type and p27KIP1−/− littermates were analyzed by histomorphometry. There was a minor effect of loss of p27KIP1 on the formation of unmineralized bone (osteoid), both as a function of total bone volume and as measured by osteoid thickness (Fig. 5 C). This effect was not a result of accelerated mineralization caused by loss of p27KIP1, because there was no evidence of altered mineral apposition rate as measured directly by dual calcein labeling. Osteoblast and osteoclast numbers were unchanged (unpublished data). Because osteoclasts do not resorb unmineralized osteoid (Chambers et al., 1985), any defect in osteoclast function is unlikely to account for the reduction in osteoid. These data are consistent with a very minor effect of loss of p27KIP1 on osteoblast differentiation and function.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ian Potter Foundation Centre for Cancer Genomics and Predictive Medicine, and Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Center, Victoria, Melbourne, Australia. david.thomas@petermac.org

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.

Show MeSH
Related in: MedlinePlus