PKR plays a positive role in osteoblast differentiation by regulating GSK-3β activity through a β-catenin-independent pathway
Graphical abstract
Highlights
► LiCl, a GSK-3β inhibitor, attenuates osteoblastogenesis in vitro and in vivo. ► Loss of PKR activity phosphorylates GSK-3β in osteoblasts. ► Inactivation of PKR also inhibits osteoblast differentiation. ► PKR-regulated GSK-3β is involved in osteoblast differentiation. ► β-catenin does not participate in PKR-mediated osteoblast differentiation.
Introduction
Double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase constitutively expressed in mammalian cells, although at a low basal level, and its expression is induced by dsRNA during viral infection. PKR is activated by dimerization and autophosphorylation, and subsequently, it phosphorylates the eukaryotic translation initiation factor 2 (eIF2). This leads to the inhibition of protein synthesis, indicating a central role for PKR in the response against viral infection. PKR, activated by various additional stimuli, participates in other cellular functions such as cell growth, tumorigenesis, and apoptosis (García et al., 2007). We previously reported that PKR positively regulates osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells (Yoshida et al., 2005). We also showed that aberrant accumulation of STAT1 protein induced by the loss of PKR activity was implicated in PKR-mediated osteoblast differentiation (Yoshida et al., 2007). However, the detailed mechanisms involved in PKR-mediated osteoblast differentiation remain unclear.
Glycogen synthase kinase 3 (GSK-3) is another ubiquitous serine/threonine kinase involved in a multitude of cellular processes. Further, GSK-3 is recognized to regulate the development of various diseases (e.g., diabetes, cancer, inflammation, and Alzheimer’s disease) although it was first isolated as an enzyme which inactivates glycogen synthase (Rayasam et al., 2009).
Many studies have suggested that GSK-3 performs critical functions in osteoblast differentiation. Accordingly, GSK-3 is known to regulate disparate pathways, including Indian hedgehog (Lanske et al., 1996, Jia et al., 2002), the nuclear factor of activated T-cells (NFAT) (Tomita et al., 2002, Okamura et al., 2002), and canonical Wnt signaling, that are important for osteoblast differentiation. It is also well known that GSK-3β regulates bone formation by mediating both phosphatidylinositol 3-kinase (PI3 K)/Akt and canonical Wnt/β-catenin signaling pathways (Krishnan et al., 2006).
The involvement of PKR in GSK-3β signaling has received less attention. PKR activates the PI3 K/Akt pathway, which is able to phosphorylate and inactivate GSK-3β (Desbois-Mouthon et al., 2001, Sunters et al., 2010). In contrast, it has also been reported that PKR activates GSK-3β and promotes p53 functions (Baltzis et al., 2007) PKR also inhibits GSK-3α/β phosphorylation and positively regulates cell growth in acute leukemic cells (Blalock et al., 2009). Moreover, it has been shown that PKR directly associates with GSK-3β and modulates GSK-3β activation in the brains of patients with Alzheimer’s disease (Bose et al., 2010). These facts lead us to hypothesize that PKR may promote osteoblast differentiation by regulating GSK-3β activity.
To test our hypothesis, we examined the role of PKR in GSK-3β signaling in osteoblast differentiation. First, to confirm the relationship between the activity of GSK-3β and osteoblast differentiation, we treated MC3T3-E1 cells with lithium chloride (LiCl), a pharmacological inhibitor of GSK-3β. LiCl was also injected into the calvarial region of mice. We observed that the inhibition of GSK-3β activity by LiCl suppressed osteoblastogenesis in vitro and in vivo. Next, we examined whether PKR affects the activity of GSK-3β in MC3T3-E1 cells that constitutively expressed kinase-deficient PKR (KR cells). The loss of PKR activity increased GSK-3β phosphorylation, whereas it did not affect phosphorylation of Akt. Moreover, GSK-3β phosphorylation induced by the loss of PKR activity resulted in the inhibition of osteoblast differentiation. These results showed that PKR is a critical factor in the regulation of GSK-3β activity during the terminal differentiation of osteoblasts.
Section snippets
Materials
LiCl was purchased from Kanto Chemical (Tokyo, Japan). 2-Aminopurine (2-AP) was obtained from Sigma Chemicals (St. Louis, MO, USA). Antibody for β-catenin was obtained from R&D Systems (Minneapolis, MN, USA), while those for GSK-3β (27C10), phosphor-GSK-3β (Ser9), and phosphor-Akt (Ser473, Thr308) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies for β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other materials used were of the highest
Treatment with LiCl suppresses osteoblast differentiation
To examine the influence of GSK-3β on osteoblast differentiation in vitro, we treated mouse osteoblastic MC3T3-E1 cells and human osteosarcoma MG-63 cells with LiCl, an inhibitor of GSK-3β. In both cell lines, LiCl induced the phosphorylation of GSK-3β at serine 9, a modification known to inactive GSK-3β (Fig. 1A).
Next, we examined whether the phosphorylation of GSK-3β induced by LiCl affects the differentiation of osteoblasts in vitro. We cultured MC3T3-E1 cells for 7 days with or without 50 mM
PKR-regulated GSK-3β activity is involved in osteoblast differentiation
In the present study, we first investigated the effects of GSK-3β on osteoblast differentiation, as many conflicting observations about its roles in ossification have been reported. For example, Kugimiya et al. reported that GSK-3β acts a negative regulator of bone formation after analyzing the phenotypes of heterozygous GSK-3β-deficient (Gsk-3β+/−) mice (Kugimiya et al., 2007). They showed evidence that GSK-3β, as well as LiCl, inhibits the level of transcriptional activity of Runx2. On the
Conclusion
In summary, we have shown that loss of PKR activity induced the phosphorylation and inactivation of GSK-3β, leading to the inhibition of osteoblast differentiation (Fig. 6). Further, this regulation of osteoblastogenesis by PKR is independent of β-catenin signaling. These findings suggest that the ability of PKR to maintain GSK-3β activity is required to promote osteoblast differentiation.
Acknowledgements
We thank Dr. Bukasa Kalubi (The University of Tokushima Graduate School) for helpful editorial advices. This work was supported by Grant-in-Aid-for Scientific Research (B), the Ministry of Education, Science, Sports and Culture of Japan (MEXT) (HY, 20390536).
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