General Transcription Factor IIA-γ Increases Osteoblast-specific Osteocalcin Gene Expression via Activating Transcription Factor 4 and Runt-related Transcription Factor 2

Abstract

ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIA␥ (TFIIA␥) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA␥ interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIA␥ is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258 -286) was found to be required for TFIIA␥ binding. Although TFIIA␥ interacts with Runx2, it does not activate Runx2. Instead, TFIIA␥ binds to and activates ATF4. Furthermore, TFIIA␥ together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA␥ markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA␥ increases levels of ATF4 protein. Finally, TFIIA␥ significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA␥, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.

Activating transcription factor 4 regulates osteoclast differentiation in mice

“…PKA phosphorylation of ATF4 at its Ser254 residue mediates β-adrenergic induction of Rankl mRNA expression in OBLs (42). Additionally, ATF4 increases OBL function and bone formation through interactions with the OBL-specific transcription factor Runx2 (57)(58)(59)(60). In contrast, in OCLs, ATF4 is regulated by M-CSF and the PI3K/AKT pathway, mediates M-CSF induction of RANK, and facilitates RANKL induction of MAPKs and expression of NFATc1.…”

“…pCMV/β-gal, pCMV/ ATF4, and pCMV/Runx2 were described previously (41,57,58,67). NFATc1-luc, containing a mouse Nfatc1 promoter element (-847/+66) driving a firefly luciferase reporter gene, was constructed in the project laboratory by PCR subcloning promoter fragment using mouse tail DNA as a template into pGL3-luc vector (Promega).…”

“…ChIP assays were performed as described previously (41,58). The equivalent of 10 μg DNA was used as starting material (input) in each ChIP reaction with 2 μg of the appropriate antibody (ATF4, c-Fos, or NFATc1).…”

“…Western blot analysis was performed as previously described (53,58). Antibodies used were as follows: antibodies against NFATc1, c-Fos, TRAF6, RANK, and CSFR1 and anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology Inc.; antibodies recognizing phosphorylated and total ERK1/2, p38 MAPK, JNK, AKT, Src, IκBα, and PU.1 from Cell Signaling Technology Inc.; and mouse monoclonal antibody against β-actin from Sigma-Aldrich.…”

MAPK phosphatase–3 promotes hepatic gluconeogenesis through dephosphorylation of forkhead box O1 in mice

“…The immune complexes were collected by addition of 30 μl of protein A/G-agarose beads and incubation for 4 hours at 4°C, followed by centrifugation. Precipitates were washed 4 times with 1× wash buffer (20 mM HEPES pH 7.6, 50 mM KCl, 1 mM DTT, 0.25% Nonidet P40, 5 mM NaF, 1 mM EGTA, 5 mM MgCl2, 0.25 mM PMSF) and 2 times with 1× PBS, and the immunoprecipitated complexes were suspended in SDS sample buffer and analyzed by SDS-PAGE followed by Western blot analysis (22).…”