Epithelial sodium channel enhanced osteogenesis via cGMP/PKGII/ENaC signaling in rat osteoblast.
The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis. In addition to epithelial cells, osteoblasts (Obs) express functional ENaCs. Moreover, a correlation between bone Na content and bone disease has been reported, suggesting that ENaC-mediated Na(+) regulation may influence osteogenesis. Obs were isolated and cultured by enzyme digestion. Cell proliferation and differentiation were evaluated by WST-8 assay kit and AKP assay kit respectively. PKGII expression was silenced by siRNA. The mRNA expression was investigated by semi-quantitative PCR and the protein expression was determined by Western-blot. The cell-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) increased α-ENaC channel expression in primary rat Obs as indicated by RT-PCR. In addition, 8-pCPT-cGMP stimulation enhanced expression of the mRNA encoding cGMP-dependent protein kinases II (PKGII). The cGMP analog also promoted osteoblast proliferation, differentiation and induced the expression of several osteogenic genes, including core binding factor al, osteocalcin, alkaline phosphatase, collagen type I, and osteopontin. Furthermore, the expression of α-ENaC, the main functional subunit of ENaC, was reduced when a small interfering RNA specific for PKGII was introduced into Obs. Treatment with 8-pCPT-cGMP in cells transfected with the siRNA for PKGII partially reversed downregulated α-ENaC mRNA expression. Our results suggest that 8-pCPT-cGMP stimulates proliferation, differentiation, and osteogenic gene expression in Obs through cGMP/PKGII-dependent regulation of ENaC channel expression. The cGMP/PKGII signaling pathway is a potential target for pharmaceutical interventions to treat metabolic bone diseases.
School of Life Science and Biopharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, GD, China.
Jun
J
Chen
Hengjie
H
Zhang
Xuling
X
Zhang
Guozhu
G
Yang
Li
L
Lu
Xingyan
X
Lu
Chao
C
Wan
Kosei
K
Ijiri
Honglong
H
Ji
Qingnan
Q
Li
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D062967:Cyclic GMP-Dependent Protein Kinase Type II / Q000235:genetics / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010012:Osteogenesis / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology
D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D034741:RNA, Small Interfering; D013873:Thionucleotides; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D006152:Cyclic GMP