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Adseverin knockdown inhibits osteoclastogenesis in RAW264.7 cells.

Qi W, Gao Y, Tian J, Jiang H - Int. J. Mol. Med. (2014)

Bottom Line: Osteoclast differentiation was morphologically examined via cell staining with osteoclast specific markers and light microscopy.The results showed that Ads expression was significantly increased in response to receptor activator of nuclear factor-κB ligand during osteoclastogenesis, and Ads was highly expressed in mature osteoclasts.Ads-knockdown macrophages showed major osteoclastogenesis defects, most likely caused by a pre-osteoclast fusion defect.

View Article: PubMed Central - PubMed

Affiliation: Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China.

ABSTRACT
Osteoclastogenesis is a complex process that is highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin (Ads), a member of the gelsolin superfamily of actin-binding proteins, regulates actin remodeling by severing and capping actin filaments. The objective of the present study was to characterize the role of Ads during osteoclastogenesis by assessing Ads expression and using a knockdown strategy. Immunoblot analyses were used to examine Ads expression during osteoclastogenesis. A stable Ads knockdown macrophage cell line was generated using a retroviral shRNA construct. Osteoclast differentiation was morphologically examined via cell staining with osteoclast specific markers and light microscopy. The results showed that Ads expression was significantly increased in response to receptor activator of nuclear factor-κB ligand during osteoclastogenesis, and Ads was highly expressed in mature osteoclasts. Ads-knockdown macrophages showed major osteoclastogenesis defects, most likely caused by a pre-osteoclast fusion defect. These results indicate that Ads deficiency in monocytes inhibits osteoclastogenesis. Thus, in future studies it could be noteworthy to investigate the function of Ads in bone marrow monocytes during osteoclastogenesis.

No MeSH data available.


Related in: MedlinePlus

Adseverin (Ads)-knockdown cells lack the ability to fuse even at high cell densities. Monocytes were plated at three initial plating densities, low (1×104 cells/well), normal (5×104 cells/well), and high (1×105 cells/well), to observe the effects of cell density on osteoclastogenesis (OCG). Representative photomicrographs from Ads-knockdown and Luc-knockdown cultures are shown on days 0 and 4 of OCG, and representative TRAcP-stained images of day 4 cultures are shown on the right. At the 2-fold-higher normal plating density, Ads-knockdown cells cannot form more multinucleated osteoclasts, suggesting a fusion defect.
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f4-ijmm-34-06-1483: Adseverin (Ads)-knockdown cells lack the ability to fuse even at high cell densities. Monocytes were plated at three initial plating densities, low (1×104 cells/well), normal (5×104 cells/well), and high (1×105 cells/well), to observe the effects of cell density on osteoclastogenesis (OCG). Representative photomicrographs from Ads-knockdown and Luc-knockdown cultures are shown on days 0 and 4 of OCG, and representative TRAcP-stained images of day 4 cultures are shown on the right. At the 2-fold-higher normal plating density, Ads-knockdown cells cannot form more multinucleated osteoclasts, suggesting a fusion defect.

Mentions: At low, normal and high densities, no osteoclasts were formed from Ads-knockdown cells (Fig. 4). As density increased, the control cells formed larger osteoclasts, whereas Ads-knockdown macrophages never formed osteoclasts with >3 nuclei. The results of cell seeding density rescue assays indicate a fusion defect in Ads-knockdown cells.


Adseverin knockdown inhibits osteoclastogenesis in RAW264.7 cells.

Qi W, Gao Y, Tian J, Jiang H - Int. J. Mol. Med. (2014)

Adseverin (Ads)-knockdown cells lack the ability to fuse even at high cell densities. Monocytes were plated at three initial plating densities, low (1×104 cells/well), normal (5×104 cells/well), and high (1×105 cells/well), to observe the effects of cell density on osteoclastogenesis (OCG). Representative photomicrographs from Ads-knockdown and Luc-knockdown cultures are shown on days 0 and 4 of OCG, and representative TRAcP-stained images of day 4 cultures are shown on the right. At the 2-fold-higher normal plating density, Ads-knockdown cells cannot form more multinucleated osteoclasts, suggesting a fusion defect.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214352&req=5

f4-ijmm-34-06-1483: Adseverin (Ads)-knockdown cells lack the ability to fuse even at high cell densities. Monocytes were plated at three initial plating densities, low (1×104 cells/well), normal (5×104 cells/well), and high (1×105 cells/well), to observe the effects of cell density on osteoclastogenesis (OCG). Representative photomicrographs from Ads-knockdown and Luc-knockdown cultures are shown on days 0 and 4 of OCG, and representative TRAcP-stained images of day 4 cultures are shown on the right. At the 2-fold-higher normal plating density, Ads-knockdown cells cannot form more multinucleated osteoclasts, suggesting a fusion defect.
Mentions: At low, normal and high densities, no osteoclasts were formed from Ads-knockdown cells (Fig. 4). As density increased, the control cells formed larger osteoclasts, whereas Ads-knockdown macrophages never formed osteoclasts with >3 nuclei. The results of cell seeding density rescue assays indicate a fusion defect in Ads-knockdown cells.

Bottom Line: Osteoclast differentiation was morphologically examined via cell staining with osteoclast specific markers and light microscopy.The results showed that Ads expression was significantly increased in response to receptor activator of nuclear factor-κB ligand during osteoclastogenesis, and Ads was highly expressed in mature osteoclasts.Ads-knockdown macrophages showed major osteoclastogenesis defects, most likely caused by a pre-osteoclast fusion defect.

View Article: PubMed Central - PubMed

Affiliation: Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China.

ABSTRACT
Osteoclastogenesis is a complex process that is highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin (Ads), a member of the gelsolin superfamily of actin-binding proteins, regulates actin remodeling by severing and capping actin filaments. The objective of the present study was to characterize the role of Ads during osteoclastogenesis by assessing Ads expression and using a knockdown strategy. Immunoblot analyses were used to examine Ads expression during osteoclastogenesis. A stable Ads knockdown macrophage cell line was generated using a retroviral shRNA construct. Osteoclast differentiation was morphologically examined via cell staining with osteoclast specific markers and light microscopy. The results showed that Ads expression was significantly increased in response to receptor activator of nuclear factor-κB ligand during osteoclastogenesis, and Ads was highly expressed in mature osteoclasts. Ads-knockdown macrophages showed major osteoclastogenesis defects, most likely caused by a pre-osteoclast fusion defect. These results indicate that Ads deficiency in monocytes inhibits osteoclastogenesis. Thus, in future studies it could be noteworthy to investigate the function of Ads in bone marrow monocytes during osteoclastogenesis.

No MeSH data available.


Related in: MedlinePlus