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. 2011 Feb 11;286(6):4809-18.
doi: 10.1074/jbc.M110.152900. Epub 2010 Dec 6.

Endoplasmic reticulum stress response mediated by the PERK-eIF2(alpha)-ATF4 pathway is involved in osteoblast differentiation induced by BMP2

Atsushi Saito  1 , Kimiko Ochiai, Shinichi Kondo, Kenji Tsumagari, Tomohiko Murakami, Douglas R Cavener, Kazunori Imaizumi
Affiliations

Endoplasmic reticulum stress response mediated by the PERK-eIF2(alpha)-ATF4 pathway is involved in osteoblast differentiation induced by BMP2

Atsushi Saito et al. J Biol Chem. .

Abstract

To avoid excess accumulation of unfolded proteins in the endoplasmic reticulum (ER), eukaryotic cells have signaling pathways from the ER to the cytosol or nucleus. These processes are collectively termed the ER stress response. Double stranded RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) is a major transducer of the ER stress response and directly phosphorylates eIF2α, resulting in translational attenuation. Phosphorylated eIF2α specifically promotes the translation of the transcription factor ATF4. ATF4 plays important roles in osteoblast differentiation and bone formation. Perk(-/-) mice are reported to exhibit severe osteopenia, and the phenotypes observed in bone tissues are very similar to those of Atf4(-/-) mice. However, the involvement of the PERK-eIF2α-ATF4 signaling pathway in osteogenesis is unclear. Phosphorylated eIF2α 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, were also down-regulated. Treatment of wild-type primary osteoblasts with BMP2, which is required for osteoblast differentiation, induced ER stress, leading to an increase in ATF4 protein expression levels. In contrast, the level of ATF4 in Perk(-/-) osteoblasts was severely diminished. The results indicate that PERK signaling is required for ATF4 activation during osteoblast differentiation. Perk(-/-) osteoblasts exhibited decreased alkaline phosphatase activities and delayed mineralized nodule formation relative to wild-type cultures. These abnormalities were almost completely restored by the introduction of ATF4 into Perk(-/-) osteoblasts. Taken together, ER stress occurs during osteoblast differentiation and activates the PERK-eIF2α-ATF4 signaling pathway followed by the promotion of gene expression essential for osteogenesis, such as Ocn and Bsp.

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Figures

FIGURE 1.
FIGURE 1.
Morphological changes in bone tissues and gene expression in Perk−/− mice. A, hematoxylin-eosin staining of femurs in P4 wild-type (WT) and Perk−/− (−/−) mice. Right panels are a higher magnification of the boxed areas in the left panels. Scale bars, 100 μm (left), 20 μm (right). B, the panel shows the quantification of the thickness of cortical bones. Note that the cortical bone of Perk−/− mice is extremely thin (mean ± S.D., n = 3, ***, p < 0.001; Student's t test). C, electron microscopy images of osteoblasts in femur cortical bones of P4 WT (upper) and Perk−/− (lower) mice. In Perk−/− osteoblasts, the rough ER was abnormally expanded. Scale bar, 1 μm. D, Western blotting analysis of procollagen Type I in primary osteoblasts prepared from WT and Perk−/− calvariae. E, Trap staining of femurs in P4 WT (top) and Perk−/− (bottom) mice. Scale bar, 100 μm. F, the number of osteoclasts did not significantly change in Perk−/− mice when compared with WT mice. G, quantitative real-time PCR (QRT-PCR) analysis of Perk in primary osteoblasts and osteoclasts prepared from P4 WT mice calvariae. Primary osteoblasts showed high levels of Perk mRNA expression, whereas osteoclasts did not show high expression levels. H, RT-PCR analysis of type I collagen (a marker of osteoblast) and Trap (a marker of osteoclast) in primary osteoblasts and osteoclasts prepared from P4 WT mice calvariae. I, QRT-PCR analysis using mRNA extracted from P4 WT and Perk−/− calvariae. The expression levels of Ocn, Bsp, Asns, and Glyt1 mRNAs were suppressed in Perk−/− calvariae. J, Western blotting using proteins extracted from P4 WT and Perk−/− calvariae. The expression levels of ATF4, OCN, BSP, and phosphorylated eIF2α proteins were suppressed in Perk−/− calvariae. The lower panel shows the quantitative analysis of the protein expression levels (mean ± S.D., n = 3, **, p < 0.01; ***, p < 0.001; Student's t test).
FIGURE 2.
FIGURE 2.
The PERK-eIF2α-ATF4 signaling pathway was impaired in Perk−/− osteoblasts treated with BMP2. A, QRT-PCR analysis of Bip mRNA in primary osteoblasts prepared from P4 WT and Perk−/− calvariae. The osteoblasts were treated with BMP2 (100 ng/ml) for the indicated times. The expression levels of Bip mRNA were almost equal in WT and Perk−/− osteoblasts. B, RT-PCR analysis of Xbp1 in primary osteoblasts prepared from P4 WT and Perk−/− calvariae. The osteoblasts were treated with BMP2 (100 ng/ml) for the indicated times. The expression levels of unspliced forms of Xbp1 mRNA (uXbp1) and spliced forms of Xbp1 mRNA (sXbp1) mRNA were essentially equal in WT and Perk−/− osteoblasts. Lower panels show the quantitative analysis of the mRNA expression levels (mean ± S.D. n = 3, ***, p < 0.001; Student's t test). C, QRT-PCR analysis of Asns mRNA in primary osteoblasts prepared from P4 WT and Perk−/− calvariae treated with BMP2 for the indicated times. The expression levels of Asns were suppressed in Perk−/− osteoblasts. D, Western blotting analysis of primary osteoblasts prepared from P4 WT and Perk−/− calvariae. The osteoblasts were treated with BMP2 (100 ng/ml) for the indicated times. The expression levels of ATF4, phosphorylated eIF2α, OCN, and BSP proteins were gradually up-regulated after treatment with BMP2 in WT primary osteoblasts. In contrast, the induction of these proteins was severely inhibited in Perk−/− primary osteoblasts. The lower panel shows the quantitative analysis of the protein expression levels (mean ± S.D., n = 3, **, p < 0.01, ***, p < 0.001; Student's t test).
FIGURE 3.
FIGURE 3.
Impaired activation of the PERK-eIF2α-ATF4 pathway by ER stress in Perk−/− osteoblasts. A, RT-PCR analysis of UPR-related genes in primary osteoblasts prepared from P4 WT and Perk−/− calvariae. The osteoblasts were exposed to thapsigargin (TG; 1 μm), which is an ER stressor, for the indicated times. The expression levels of Bip and spliced forms of Xbp1 mRNA (sXbp1) were almost equal in WT and Perk−/− osteoblasts. The lower panel shows the quantitative analysis of the mRNA expression levels (mean ± S.D., n = 3, ***, p < 0.001; Student's t test). uXbp1, unspliced forms of Xbp1 mRNA. B, Western blotting analysis of primary osteoblasts prepared from P4 WT and Perk−/− calvariae. The osteoblasts were exposed to TG (1 μm) for the indicated times. The expression levels of ATF4, OCN, BSP, and phosphorylated eIF2α proteins were suppressed in Perk−/− osteoblasts when compared with WT osteoblasts. The lower panel shows the quantitative analysis of the protein expression levels (mean ± S.D., n = 3, *, p < 0.05, **, p < 0.01, ***, p < 0.001; Student's t test).
FIGURE 4.
FIGURE 4.
The activation of the OSE1 promoter region mediated by ATF4 is inhibited in Perk−/− osteoblasts. A, scheme of the promoter region of mouse OG2 (Ocn promoter) that includes the OSE2 (Runx2 binding site) and OSE1 (ATF4 binding site) regions. B, reporter assays using the OG2 promoter. Primary osteoblasts were transfected with the OG2 reporter construct and treated with or without BMP2 (100 ng/ml). In Perk−/− osteoblasts treated with BMP2, the reporter activities were reduced when compared with WT osteoblasts. Data are presented as the ratio of firefly luciferase activities to Renilla luciferase activities (mean ± S.D., n = 3, *, p < 0.05, ***, p < 0.001; Student's t test). C, reporter assays using OG2 promoter and indicated constructs. The reporter activities were observed to increase upon exposure to TG (1 μm) in WT primary osteoblasts but not in Perk−/− osteoblasts. Note that the reporter activities increased significantly when ATF4 was introduced to Perk−/− osteoblasts. The introduction of other CREB/ATF family members did not induce the reporter activities (mean ± S.D., n = 3, **, p < 0.01; Student's t test). D, reporter assays using OG2-OSE1 and OG2-OSE2. The reporter activities of WT primary osteoblasts transfected with the OG2-OSE1 reporter were induced by exposure to TG (1 μm) but not with OSE2 (mean ± S.D., n = 3, *, p < 0.05, **, p < 0.01; Student's t test). OASIS (Old Astrocyte Specifically Induced Substance).
FIGURE 5.
FIGURE 5.
The PERK-eIF2α-ATF4 pathway is involved in osteoblast differentiation. A, Western blotting of primary osteoblasts infected with adenovirus expressing ATF4. The expression levels of OCN and BSP were restored by the introduction of ATF4 to Perk−/− osteoblasts. mock is an empty vector. B, mineralization nodule formation in WT and Perk−/− osteoblasts. WT and Perk−/− primary osteoblasts were treated with BMP2 for 0 or 4 days, and cells were stained with alizarin red for visualizing mineralization. The delayed mineralized nodule formation in Perk−/− osteoblasts was restored by the infection with an adenovirus expressing ATF4. The lower panel shows the quantitative analysis of alizarin red staining (mean ± S.D., n = 4, *, p < 0.05, **, p < 0.01; Student's t test). C, alkaline phosphatase activity in WT and Perk−/− primary osteoblasts. Cells were stained with 5-bromo-4-chloro-3-indolyl phosphate for measuring ALP activities. Although ALP activities were reduced in Perk−/− osteoblasts, they were restored by the introduction of ATF4 into Perk−/− osteoblasts. The lower panel shows the quantitative analysis of ALP activities (mean ± S.D., n = 4, **, p < 0.01, ***, p < 0.001; Student's t test). D, proposed model for the induction of ATF4 target osteoblast markers mediated by the PERK-eIF2α-ATF4 pathway during osteoblast differentiation.
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