Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells

Ni Qiu, Honghao Zhou, Zhousheng Xiao

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARg and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 mM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1mM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.

Original languageEnglish (US)
Pages (from-to)967-976
Number of pages10
JournalJournal of Cellular Biochemistry
Volume113
Issue number3
DOIs
StatePublished - Mar 1 2012

Fingerprint

Small Interfering RNA
Down-Regulation
Osteoblasts
Calcium
Cell proliferation
Bone
Cell Proliferation
Adipogenesis
Lentivirus
Calcium Signaling
Sequence Deletion
Colforsin
Bromodeoxyuridine
Adipocytes
Osteogenesis
Alkaline Phosphatase
Shear stress
Flow of fluids
Genes
Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells. / Qiu, Ni; Zhou, Honghao; Xiao, Zhousheng.

In: Journal of Cellular Biochemistry, Vol. 113, No. 3, 01.03.2012, p. 967-976.

Research output: Contribution to journalArticle

@article{3097ac2c09504f6bb70f6b0a5d9084d8,
title = "Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells",
abstract = "Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53{\%} reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARg and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 mM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1mM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.",
author = "Ni Qiu and Honghao Zhou and Zhousheng Xiao",
year = "2012",
month = "3",
day = "1",
doi = "10.1002/jcb.23426",
language = "English (US)",
volume = "113",
pages = "967--976",
journal = "Journal of Cellular Biochemistry",
issn = "0730-2312",
publisher = "Wiley-Liss Inc.",
number = "3",

}

TY - JOUR

T1 - Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells

AU - Qiu, Ni

AU - Zhou, Honghao

AU - Xiao, Zhousheng

PY - 2012/3/1

Y1 - 2012/3/1

N2 - Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARg and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 mM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1mM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.

AB - Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARg and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 mM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1mM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.

UR - http://www.scopus.com/inward/record.url?scp=84863269754&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84863269754&partnerID=8YFLogxK

U2 - 10.1002/jcb.23426

DO - 10.1002/jcb.23426

M3 - Article

VL - 113

SP - 967

EP - 976

JO - Journal of Cellular Biochemistry

JF - Journal of Cellular Biochemistry

SN - 0730-2312

IS - 3

ER -