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Endoplasmic Reticulum Stress Response Mediated by the PERK-eIF2α-ATF4 Pathway Is Involved in Osteoblast Differentiation Induced by BMP2*
Douglas R. Cavener¶, , and
From the Department of Biochemistry, Division of Genome Radiobiology and Medical Science, Graduate School of Biomedical Science, University of Hiroshima, Hiroshima 734-8553, Japan,
the Division of Molecular and Cellular Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan, and
the ¶Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802
To avoid excess accumulation of unfolded proteins in the endoplasmicreticulum (ER), eukaryotic cells have signaling pathways fromthe ER to the cytosol or nucleus. These processes are collectivelytermed the ER stress response. Double stranded RNA activatedprotein kinase (PKR)-like endoplasmic reticulum kinase (PERK)is a major transducer of the ER stress response and directlyphosphorylates eIF2α, resulting in translational attenuation.Phosphorylated eIF2α specifically promotes the translationof the transcription factor ATF4. ATF4 plays important rolesin osteoblast differentiation and bone formation. Perk–/–mice are reported to exhibit severe osteopenia, and the phenotypesobserved in bone tissues are very similar to those of Atf4–/–mice. However, the involvement of the PERK-eIF2α-ATF4signaling pathway in osteogenesis is unclear. PhosphorylatedeIF2α and ATF4 protein levels were attenuated in Perk–/–calvariae, and the gene expression levels of osteocalcin (Ocn)and bone sialoprotein (Bsp), which are targets for ATF4, werealso down-regulated. Treatment of wild-type primary osteoblastswith BMP2, which is required for osteoblast differentiation,induced ER stress, leading to an increase in ATF4 protein expressionlevels. In contrast, the level of ATF4 in Perk–/–osteoblasts was severely diminished. The results indicate thatPERK signaling is required for ATF4 activation during osteoblastdifferentiation. Perk–/– osteoblasts exhibited decreasedalkaline phosphatase activities and delayed mineralized noduleformation relative to wild-type cultures. These abnormalitieswere almost completely restored by the introduction of ATF4into Perk–/– osteoblasts. Taken together, ER stressoccurs during osteoblast differentiation and activates the PERK-eIF2α-ATF4signaling pathway followed by the promotion of gene expressionessential for osteogenesis, such as Ocn and Bsp.
Key Words: Bone Differentiation ER Stress Transcription Factors Transcription Target Genes Osteoblast
Received for publication June 8, 2010.
Revision received October 26, 2010.