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p38 α MAPK regulates proliferation and differentiation of osteoclast progenitors and bone remodeling in an aging-dependent manner

View Article: PubMed Central - PubMed

ABSTRACT

Bone mass is determined by the balance between bone formation, carried out by mesenchymal stem cell-derived osteoblasts, and bone resorption, carried out by monocyte-derived osteoclasts. Here we investigated the potential roles of p38 MAPKs, which are activated by growth factors and cytokines including RANKL and BMPs, in osteoclastogenesis and bone resorption by ablating p38α MAPK in LysM+monocytes. p38α deficiency promoted monocyte proliferation but regulated monocyte osteoclastic differentiation in a cell-density dependent manner, with proliferating p38α−/− cultures showing increased differentiation. While young mutant mice showed minor increase in bone mass, 6-month-old mutant mice developed osteoporosis, associated with an increase in osteoclastogenesis and bone resorption and an increase in the pool of monocytes. Moreover, monocyte-specific p38α ablation resulted in a decrease in bone formation and the number of bone marrow mesenchymal stem/stromal cells, likely due to decreased expression of PDGF-AA and BMP2. The expression of PDGF-AA and BMP2 was positively regulated by the p38 MAPK-Creb axis in osteoclasts, with the promoters of PDGF-AA and BMP2 having Creb binding sites. These findings uncovered the molecular mechanisms by which p38α MAPK regulates osteoclastogenesis and coordinates osteoclastogenesis and osteoblastogenesis.

No MeSH data available.


Related in: MedlinePlus

p38α regulated osteoclast differentiation in a cell density-dependent manner.(A) TRAP staining showed that p38α deficiency promoted osteoclast differentiation in low cell density culture but slightly inhibited osteoclast differentiation in high cell density cultures. Monocytes were isolated from LysM-Cre; p38αf/f and control mice, plated at different densities, and cultured in the presence of RANKL and M-CSF. After 7 days, the cultures were stained for TRAP (left panels). Right panels: quantitation data. Scale bar, 200 μm. N = 3. (B) Quantitative PCR results showed that p38α deficiency promoted the expression of genes required for osteoclast differentiation at low cell densities. Monocytes described in Fig. 2A (low cell density) were collected and used to isolate total RNA, which was used to run quantitative PCR assays. N = 3. (C) Quantitative PCR results showed that p38α deficiency slightly inhibited the expression of genes required for osteoclast differentiation at high cell densities. (D) Western blot results showed that p38α deficient monocytes cultures at low densities exhibited an increase in activation of Tak1 and the protein levels of NF-κB isoforms p50/52 and p65. Scale bar, 100 μm. (E) Inhibition of Tak1 down-regulated NF-κB levels and suppressed osteoclast differentiation. Left panel: western blot showed that Tak1 inhibitor down-regulated NF-κB levels. Middle panel: Tak1 inhibitor suppressed osteoclast differentiation. Right panel: Quantitation data. N = 3. (F) Western blot results revealed that p38α deficient monocyte cultures at high densities showed a decrease in c-Fos protein levels. (G) p38α deficiency did not affect the resorbing activity of osteoclasts on dentine slices. WT and p38 deficient monocytes were induced to differentiate into osteoclasts by M-CSF and RANKL for 2 days and then counted and the same numbers of cells were plated onto dentine slices. After 7 days, the dentine slices were sonicated and stained with Gill’s hematoxylin. Right panel: quantitation data. Scale bar, 200 μm. N = 3. For all results in Fig. 2, P-values are based on Student’s t-test. *p < 0.05, **p < 0.01 when the value of mutant mice or cells was compared to that of control mice or cells, or the drugs-treated group compared to control group.
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f2: p38α regulated osteoclast differentiation in a cell density-dependent manner.(A) TRAP staining showed that p38α deficiency promoted osteoclast differentiation in low cell density culture but slightly inhibited osteoclast differentiation in high cell density cultures. Monocytes were isolated from LysM-Cre; p38αf/f and control mice, plated at different densities, and cultured in the presence of RANKL and M-CSF. After 7 days, the cultures were stained for TRAP (left panels). Right panels: quantitation data. Scale bar, 200 μm. N = 3. (B) Quantitative PCR results showed that p38α deficiency promoted the expression of genes required for osteoclast differentiation at low cell densities. Monocytes described in Fig. 2A (low cell density) were collected and used to isolate total RNA, which was used to run quantitative PCR assays. N = 3. (C) Quantitative PCR results showed that p38α deficiency slightly inhibited the expression of genes required for osteoclast differentiation at high cell densities. (D) Western blot results showed that p38α deficient monocytes cultures at low densities exhibited an increase in activation of Tak1 and the protein levels of NF-κB isoforms p50/52 and p65. Scale bar, 100 μm. (E) Inhibition of Tak1 down-regulated NF-κB levels and suppressed osteoclast differentiation. Left panel: western blot showed that Tak1 inhibitor down-regulated NF-κB levels. Middle panel: Tak1 inhibitor suppressed osteoclast differentiation. Right panel: Quantitation data. N = 3. (F) Western blot results revealed that p38α deficient monocyte cultures at high densities showed a decrease in c-Fos protein levels. (G) p38α deficiency did not affect the resorbing activity of osteoclasts on dentine slices. WT and p38 deficient monocytes were induced to differentiate into osteoclasts by M-CSF and RANKL for 2 days and then counted and the same numbers of cells were plated onto dentine slices. After 7 days, the dentine slices were sonicated and stained with Gill’s hematoxylin. Right panel: quantitation data. Scale bar, 200 μm. N = 3. For all results in Fig. 2, P-values are based on Student’s t-test. *p < 0.05, **p < 0.01 when the value of mutant mice or cells was compared to that of control mice or cells, or the drugs-treated group compared to control group.

Mentions: We then compared the differentiation potential of p38α deficient monocytes isolated from 2.5-month-old mice. We found that in response to M-CSF and RANKL, p38α deficiency either stimulated or impeded osteoclast differentiation depending on the cell densities. When plated at a low density, p38α deficient monocyte cultures showed an increase in the number of TRAP positive osteoclasts (Fig. 2A), and an increase in the mRNA levels of PU.1, c-Fos, and NFATc1, transcription factors required for osteoclastogenesis (Fig. 2B). However, when plated at a high density, p38α deficient monocyte cultures showed a modestly decreased number of TRAP positive osteoclasts and a decrease in the mRNA levels of c-Fos and NFATc1 (Fig. 2A and C). Note that at high cell densities, osteoclasts appeared to be smaller than the ones in low-density cultures, suggesting that the limit of space may have interfered with osteoclast growth and/or differentiation, which warrants further investigation. Moreover, since cells at high densities do not usually have space to proliferate, these results also suggest that p38α’s roles in regulating early osteoclast differentiation may be linked to its effects on cell proliferation.


p38 α MAPK regulates proliferation and differentiation of osteoclast progenitors and bone remodeling in an aging-dependent manner
p38α regulated osteoclast differentiation in a cell density-dependent manner.(A) TRAP staining showed that p38α deficiency promoted osteoclast differentiation in low cell density culture but slightly inhibited osteoclast differentiation in high cell density cultures. Monocytes were isolated from LysM-Cre; p38αf/f and control mice, plated at different densities, and cultured in the presence of RANKL and M-CSF. After 7 days, the cultures were stained for TRAP (left panels). Right panels: quantitation data. Scale bar, 200 μm. N = 3. (B) Quantitative PCR results showed that p38α deficiency promoted the expression of genes required for osteoclast differentiation at low cell densities. Monocytes described in Fig. 2A (low cell density) were collected and used to isolate total RNA, which was used to run quantitative PCR assays. N = 3. (C) Quantitative PCR results showed that p38α deficiency slightly inhibited the expression of genes required for osteoclast differentiation at high cell densities. (D) Western blot results showed that p38α deficient monocytes cultures at low densities exhibited an increase in activation of Tak1 and the protein levels of NF-κB isoforms p50/52 and p65. Scale bar, 100 μm. (E) Inhibition of Tak1 down-regulated NF-κB levels and suppressed osteoclast differentiation. Left panel: western blot showed that Tak1 inhibitor down-regulated NF-κB levels. Middle panel: Tak1 inhibitor suppressed osteoclast differentiation. Right panel: Quantitation data. N = 3. (F) Western blot results revealed that p38α deficient monocyte cultures at high densities showed a decrease in c-Fos protein levels. (G) p38α deficiency did not affect the resorbing activity of osteoclasts on dentine slices. WT and p38 deficient monocytes were induced to differentiate into osteoclasts by M-CSF and RANKL for 2 days and then counted and the same numbers of cells were plated onto dentine slices. After 7 days, the dentine slices were sonicated and stained with Gill’s hematoxylin. Right panel: quantitation data. Scale bar, 200 μm. N = 3. For all results in Fig. 2, P-values are based on Student’s t-test. *p < 0.05, **p < 0.01 when the value of mutant mice or cells was compared to that of control mice or cells, or the drugs-treated group compared to control group.
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f2: p38α regulated osteoclast differentiation in a cell density-dependent manner.(A) TRAP staining showed that p38α deficiency promoted osteoclast differentiation in low cell density culture but slightly inhibited osteoclast differentiation in high cell density cultures. Monocytes were isolated from LysM-Cre; p38αf/f and control mice, plated at different densities, and cultured in the presence of RANKL and M-CSF. After 7 days, the cultures were stained for TRAP (left panels). Right panels: quantitation data. Scale bar, 200 μm. N = 3. (B) Quantitative PCR results showed that p38α deficiency promoted the expression of genes required for osteoclast differentiation at low cell densities. Monocytes described in Fig. 2A (low cell density) were collected and used to isolate total RNA, which was used to run quantitative PCR assays. N = 3. (C) Quantitative PCR results showed that p38α deficiency slightly inhibited the expression of genes required for osteoclast differentiation at high cell densities. (D) Western blot results showed that p38α deficient monocytes cultures at low densities exhibited an increase in activation of Tak1 and the protein levels of NF-κB isoforms p50/52 and p65. Scale bar, 100 μm. (E) Inhibition of Tak1 down-regulated NF-κB levels and suppressed osteoclast differentiation. Left panel: western blot showed that Tak1 inhibitor down-regulated NF-κB levels. Middle panel: Tak1 inhibitor suppressed osteoclast differentiation. Right panel: Quantitation data. N = 3. (F) Western blot results revealed that p38α deficient monocyte cultures at high densities showed a decrease in c-Fos protein levels. (G) p38α deficiency did not affect the resorbing activity of osteoclasts on dentine slices. WT and p38 deficient monocytes were induced to differentiate into osteoclasts by M-CSF and RANKL for 2 days and then counted and the same numbers of cells were plated onto dentine slices. After 7 days, the dentine slices were sonicated and stained with Gill’s hematoxylin. Right panel: quantitation data. Scale bar, 200 μm. N = 3. For all results in Fig. 2, P-values are based on Student’s t-test. *p < 0.05, **p < 0.01 when the value of mutant mice or cells was compared to that of control mice or cells, or the drugs-treated group compared to control group.
Mentions: We then compared the differentiation potential of p38α deficient monocytes isolated from 2.5-month-old mice. We found that in response to M-CSF and RANKL, p38α deficiency either stimulated or impeded osteoclast differentiation depending on the cell densities. When plated at a low density, p38α deficient monocyte cultures showed an increase in the number of TRAP positive osteoclasts (Fig. 2A), and an increase in the mRNA levels of PU.1, c-Fos, and NFATc1, transcription factors required for osteoclastogenesis (Fig. 2B). However, when plated at a high density, p38α deficient monocyte cultures showed a modestly decreased number of TRAP positive osteoclasts and a decrease in the mRNA levels of c-Fos and NFATc1 (Fig. 2A and C). Note that at high cell densities, osteoclasts appeared to be smaller than the ones in low-density cultures, suggesting that the limit of space may have interfered with osteoclast growth and/or differentiation, which warrants further investigation. Moreover, since cells at high densities do not usually have space to proliferate, these results also suggest that p38α’s roles in regulating early osteoclast differentiation may be linked to its effects on cell proliferation.

View Article: PubMed Central - PubMed

ABSTRACT

Bone mass is determined by the balance between bone formation, carried out by mesenchymal stem cell-derived osteoblasts, and bone resorption, carried out by monocyte-derived osteoclasts. Here we investigated the potential roles of p38 MAPKs, which are activated by growth factors and cytokines including RANKL and BMPs, in osteoclastogenesis and bone resorption by ablating p38&alpha; MAPK in LysM+monocytes. p38&alpha; deficiency promoted monocyte proliferation but regulated monocyte osteoclastic differentiation in a cell-density dependent manner, with proliferating p38&alpha;&minus;/&minus; cultures showing increased differentiation. While young mutant mice showed minor increase in bone mass, 6-month-old mutant mice developed osteoporosis, associated with an increase in osteoclastogenesis and bone resorption and an increase in the pool of monocytes. Moreover, monocyte-specific p38&alpha; ablation resulted in a decrease in bone formation and the number of bone marrow mesenchymal stem/stromal cells, likely due to decreased expression of PDGF-AA and BMP2. The expression of PDGF-AA and BMP2 was positively regulated by the p38 MAPK-Creb axis in osteoclasts, with the promoters of PDGF-AA and BMP2 having Creb binding sites. These findings uncovered the molecular mechanisms by which p38&alpha; MAPK regulates osteoclastogenesis and coordinates osteoclastogenesis and osteoblastogenesis.

No MeSH data available.


Related in: MedlinePlus