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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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Growth arrest due to expression of runx2 or treatment with BMP2 is reduced by knockdown of p27KIP1. (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 p27KIP1 in MC3T3E1 cells infected with a retrovirus expressing either control siRNA or siRNA for p27KIP1. (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.
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fig4: Growth arrest due to expression of runx2 or treatment with BMP2 is reduced by knockdown of p27KIP1. (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 p27KIP1 in MC3T3E1 cells infected with a retrovirus expressing either control siRNA or siRNA for p27KIP1. (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.

Mentions: The data described above suggest a role for p27KIP1 and pRb in mediating runx2-dependent proliferation arrest. We have previously reported a role for pRb in runx2-dependent expression of differentiation-related genes, and so wished to determine the physiologic significance of p27KIP1 in osteoblast differentiation. As reported previously (Drissi et al., 1999), osteoblast differentiation in vitro is associated with increased expression of p27KIP1 (Fig. 3 A). Bone morphogenetic proteins (BMPs) are powerful osteoinductive agents whose effects are mediated by runx2 (Tsuji et al., 1998; Gori et al., 1999). Treatment of murine embryonic fibroblasts (MEFs) with a synthetic BMP4/7 fusion protein induced osteoblast differentiation (and cell cycle arrest), concomitant with expression of runx2 and p27KIP1 mRNA (Fig. 3 B). Similarly, BMP2 induced a G1 cell cycle arrest in MEFs that was dependent in part on the presence of both pRb and p27KIP1 (Fig. 4 A). This is consistent with a role for runx2 in inhibition of cell proliferation and induction of differentiation by BMPs. To confirm these observations in a preosteoblast cell model, we used siRNA to knockdown p27KIP1 in MC3T3E1 cells (Fig. 4 B). The level of knockdown achieved was >75%, which we consider significant because p27KIP1 is believed to act as a haploinsufficient tumor suppressor gene (Fero et al., 1998). Consistent with observations that runx2- osteoblasts have increased rates of proliferation (Pratap et al., 2003), we observed an increased rate of proliferation in MC3T3E1 cells in which p27KIP1 was reduced. BMP2 treatment of MC3T3E1 cells expressing a control siRNA vector resulted in a 42% reduction in S-phase cells, compared with a 17% reduction in cells expressing the p27KIP1 siRNA (Fig. 4 C). Furthermore, as observed in G292 cells and 3T3 fibroblasts, ectopic expression of runx2 suppressed growth of MC3T3E1 cells, and this effect was abolished in cells expressing siRNA for p27KIP1 (Fig. 4 D; Chi square P < 0.001). These data suggest that p27KIP1, like pRb, plays a role in regulating basal rates of proliferation in preosteoblasts and contributes to the growth arrest associated with osteoblastic differentiation.


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)

Growth arrest due to expression of runx2 or treatment with BMP2 is reduced by knockdown of p27KIP1. (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 p27KIP1 in MC3T3E1 cells infected with a retrovirus expressing either control siRNA or siRNA for p27KIP1. (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.
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Related In: Results  -  Collection

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fig4: Growth arrest due to expression of runx2 or treatment with BMP2 is reduced by knockdown of p27KIP1. (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 p27KIP1 in MC3T3E1 cells infected with a retrovirus expressing either control siRNA or siRNA for p27KIP1. (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.
Mentions: The data described above suggest a role for p27KIP1 and pRb in mediating runx2-dependent proliferation arrest. We have previously reported a role for pRb in runx2-dependent expression of differentiation-related genes, and so wished to determine the physiologic significance of p27KIP1 in osteoblast differentiation. As reported previously (Drissi et al., 1999), osteoblast differentiation in vitro is associated with increased expression of p27KIP1 (Fig. 3 A). Bone morphogenetic proteins (BMPs) are powerful osteoinductive agents whose effects are mediated by runx2 (Tsuji et al., 1998; Gori et al., 1999). Treatment of murine embryonic fibroblasts (MEFs) with a synthetic BMP4/7 fusion protein induced osteoblast differentiation (and cell cycle arrest), concomitant with expression of runx2 and p27KIP1 mRNA (Fig. 3 B). Similarly, BMP2 induced a G1 cell cycle arrest in MEFs that was dependent in part on the presence of both pRb and p27KIP1 (Fig. 4 A). This is consistent with a role for runx2 in inhibition of cell proliferation and induction of differentiation by BMPs. To confirm these observations in a preosteoblast cell model, we used siRNA to knockdown p27KIP1 in MC3T3E1 cells (Fig. 4 B). The level of knockdown achieved was >75%, which we consider significant because p27KIP1 is believed to act as a haploinsufficient tumor suppressor gene (Fero et al., 1998). Consistent with observations that runx2- osteoblasts have increased rates of proliferation (Pratap et al., 2003), we observed an increased rate of proliferation in MC3T3E1 cells in which p27KIP1 was reduced. BMP2 treatment of MC3T3E1 cells expressing a control siRNA vector resulted in a 42% reduction in S-phase cells, compared with a 17% reduction in cells expressing the p27KIP1 siRNA (Fig. 4 C). Furthermore, as observed in G292 cells and 3T3 fibroblasts, ectopic expression of runx2 suppressed growth of MC3T3E1 cells, and this effect was abolished in cells expressing siRNA for p27KIP1 (Fig. 4 D; Chi square P < 0.001). These data suggest that p27KIP1, like pRb, plays a role in regulating basal rates of proliferation in preosteoblasts and contributes to the growth arrest associated with osteoblastic differentiation.

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