Pro-inflammatory Cytokine Tumor Necrosis Factor-α Induces Bone Morphogenetic Protein-2 in Chondrocytes via mRNA Stabilization and Transcriptional Up-regulation

Takahiro Ochi,Ryuji Suzuki,Shoji Yamane,Linda J. Sandell,Yasuko Ikeda,Naoshi Fukui,Sakae Tanaka,Atsuhiko Hikita,Toshiyuki Ohnuki

doi:10.1074/jbc.m603385200

Abstract

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-alpha induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3'-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3'-untranslated regions, and transient transfection experiments demonstrated that TNF-alpha increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-alpha modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-kappaB.

Highlights

Bone morphogenetic proteins (BMPs)2 are a group of secreted signaling proteins that were originally identified by BMPs are critical in embryonal development and postnatal growth
TNF-␣ and Cycloheximide Induced bone morphogenetic protein-2 (BMP-2) Expression in Differentiated, Not Undifferentiated, ATDC5 Cells—As a preliminary experiment, the time course of chondrogenic differentiation of ATDC5 cells was determined by evaluating the expression of type II procollagen and aggrecan as well as type X procollagen from Day 0 until Day 25, every 5 days after addition of insulin to the media
The induction of BMP-2 by TNF-␣ in ATDC5 cells was investigated between Days 0 and 15 in the following experiments

Summary

Introduction

Bone morphogenetic proteins (BMPs)2 are a group of secreted signaling proteins that were originally identified by BMPs are critical in embryonal development and postnatal growth. Post-transcriptional Levels in ATDC5 Cells—To determine the The experiment with CHX was repeated with human chontranscriptional activity of BMP-2 gene, a nuclear run-off assay drocytes, and a strong induction of BMP-2 was again observed, was performed on differentiated and undifferentiated ATDC5 suggesting the presence of a similar post-transcriptional regucells with or without TNF-␣ stimulation (Fig. 2, A and B).

Results

Conclusion