Cooperative Interactions between Activating Transcription Factor 4 and Runx2/Cbfa1 Stimulate Osteoblast-specific Osteocalcin Gene Expression

Abstract

(2004) Cell 117, 387-398). However, the mechanisms of ATF4 in bone cells are still not clear. In this study, we determined the molecular mechanisms through which ATF4 activates the mouse osteocalcin (Ocn) gene 2 (mOG2) expression and mOG2 promoter activity. ATF4 increased the levels of Ocn mRNA and mOG2 promoter activity in Runx2-containing osteoblasts but not in non-osteoblastic cells that lack detectable Runx2 protein. However, ATF4 increased Ocn mRNA and mOG2 promoter activity in non-osteoblastic cells when Runx2 was co-expressed. Mutational analysis of the OSE1 (ATF4-binding site) and the two OSE2s (Runx2-binding sites) in the 657-bp mOG2 promoter demonstrated that ATF4 and Runx2 activate Ocn via cooperative interactions with these sites. Pull-down assays using nuclear extracts from osteoblasts or COS-7 cells overexpressing ATF4 and Runx2 showed that both factors are present in either anti-ATF4 and anti-Runx2 immunoprecipitates. In contrast, pull-down assays using purified glutathione S-transferase fusion proteins were unable to demonstrate a direct physical interaction between ATF4 and Runx2. Thus, accessory factors are likely involved in stabilizing interactions between these two molecules. Regions within Runx2 required for ATF4 complex formation and activation were identified. Deletion analysis showed that the leucine zipper domain of ATF4 is critical for Runx2 activation. This study is the first demonstration that cooperative interactions between ATF4 and Runx2/Cbfa1 stimulate osteoblast-specific Ocn expression and suggests that this regulation may represent a novel intramolecular mechanism regulating Runx2 activity and, thereby, osteoblast differentiation and bone formation.

Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1

“…The minor defects in ossification contrast with the severe interference with bone development reported for Wwtr1-morphant zebrafish (9), suggesting that other factors may compensate for the loss of Wwtr1 in mammals. ATF4, for example, plays a role in bone formation in mice (12) and interacts like Wwtr1 with Cbfa1 to stimulate osteoblast-specific gene expression (13).…”

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

“…AP-1-Luc and expression constructs for AP-1 family members, including Fra2, Jun, JunB, and JunD, were described previously (8). Expression vectors for ATF2, -3, and -4 were obtained from Dr. Ze'ev Ronai (41), Dr. Michael S. Kilberg (42), and Dr. Guozhi Xiao (43), respectively. Mutant human C/EBP␤ expression plasmids were generated by twostep PCR mutagenesis using hC/EBP␤/pcDNA3.1(ϩ) as template for the first step and the following primer pairs (lowercase letters indicate mutation site): hC/EBP␤ S76A Y78A(SY) for hLAP*SY, forward (CGACTTCgcCCCGTtCCTGGAGCCG-CTG) and reverse (CAGCGGCTCCAGGaACGGGgcGAAG-TCG); hC/EBP␤ F117A L118A(LF) for hLAP*LF, forward (GCACCACGACgcCgcCTCCGACCTCTTCGCCGACGAC-TAC) and reverse (GTAGTCGTCGGCGAAGAGGTCGGAGgcGgcGTCGTGGTGC); hC/EBP␤ T235A, forward (TCCAGCCCGCCCGGCgCGCCGAGCCCCGCT) and reverse (AGC-GGGGCTCGGCGCGCCGGGCGGGCTGGA); hC/EBP␤ Y274F, forward (CAAGCACAGCGACGAGTtCAAGATCC-GGCGCGAGCG) and reverse (CGCTCGCGCCGGATCTTGaACTCGTCGCTGTGCTTG).…”

Overexpression of osteoprotegerin promotes preosteoblast differentiation to mature osteoblasts

“…5,6 Akt1 and Smad1 interact with Runx2 to regulate and control transcriptional levels of osteoblast differentiation. [7][8][9] Alkaline phosphate (ALP) is a key early marker of matrix maturation that is expressed in preosteoblasts during osteoblast differentiation and upregulated expression in mature osteoblasts and is downregulated in osteocytes. 3 Osteoblasts are able to modulate osteoclastogenesis by changing the ratio of OPG and the receptor activator of RANKL during bone remodeling.…”