Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.
In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression.
Yoshida
Kaya
K
Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto, Tokushima 770-8504, Japan. kaya@dent.tokushima-u.ac.jp
Okamura
Hirohiko
H
Amorim
Bruna Rabelo
BR
Ozaki
Akiko
A
Tanaka
Hiroaki
H
Morimoto
Hiroyuki
H
Haneji
Tatsuji
T
D000469:Alkaline Phosphatase; D000818:Animals; D001842:Bone and Bones / Q000502:physiology*; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D005799:Genes, Dominant; D066298:In Vitro Techniques; D050832:Interferon-Stimulated Gene Factor 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D012330:RNA, Double-Stranded / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D015534:Trans-Activators; D014158:Transcription, Genetic; D019892:eIF-2 Kinase / Q000502:physiology*
D050659:Core Binding Factor Alpha 1 Subunit; D050832:Interferon-Stimulated Gene Factor 3; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; C493716:Runx2 protein, mouse; D015534:Trans-Activators; C079868:gamma interferon activation factor; D019892:eIF-2 Kinase; D000469:Alkaline Phosphatase