Limits...
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.

Show MeSH

Related in: MedlinePlus

Osteogenic differentiation in vitro is associated with induction of p27KIP1 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 p27KIP1 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, p27KIP1, p21CIP1, and glyceraldehyde phosphate dehydrogenase (GAPDH). Fold changes in gene expression relative to GAPDH are given below each panel.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172443&req=5

fig3: Osteogenic differentiation in vitro is associated with induction of p27KIP1 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 p27KIP1 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, p27KIP1, p21CIP1, and glyceraldehyde phosphate dehydrogenase (GAPDH). Fold changes in gene expression relative to GAPDH are given below each panel.

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)

Osteogenic differentiation in vitro is associated with induction of p27KIP1 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 p27KIP1 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, p27KIP1, p21CIP1, and glyceraldehyde phosphate dehydrogenase (GAPDH). Fold changes in gene expression relative to GAPDH are given below each panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172443&req=5

fig3: Osteogenic differentiation in vitro is associated with induction of p27KIP1 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 p27KIP1 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, p27KIP1, p21CIP1, and glyceraldehyde phosphate dehydrogenase (GAPDH). Fold changes in gene expression relative to GAPDH are given below each panel.
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