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

Abstract

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) 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-␣ 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-␣ increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-␣ 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-B.

αvβ5 Integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes

“…Human articular cartilage samples were obtained from the macroscopically intact areas within osteoarthritic (OA) knee joints during prosthetic surgery, and were processed for experiments within 4 hours after the surgery. Primary cultured human articular chondrocytes were prepared from those cartilage samples by serial enzymatic digestion using Pronase (Merck Bioscience) and Collagenase P (Roche Diagnostics) (26). Following digestion, chondrocytes were plated onto plastic culture dishes (Iwaki) at a density of 2 ϫ 10 5 /cm 2 , and maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 containing 10% fetal bovine serum (FBS) and 25 g/ml ascorbic acid.…”

“…Bovine articular chondrocytes were prepared from the cartilage of metacarpophalangeal joints of 18-24-month-old steers. The chondrocytes were prepared and cultured in monolayers as described for human chondrocytes (26). For some experiments, cell morphology was observed under an inverted microscope, and the circularity of the cells was determined by ImageJ image analysis software, version 1.43 (NIH Image, National Institutes of Health; online at: http://rsbweb.nih.gov/ij/).…”

“…All small interfering RNA (siRNA) were obtained from Qiagen. The siRNA were introduced into primary cultured chondrocytes by electroporation using a Nucleofector, according to the recommendations of the manufacturer (Lonza) with some modifications (26). Briefly, immediately after enzymatic digestion, 1 ϫ 10 6 chondrocytes were suspended in 100 l of electroporation buffer containing 20 pmoles of siRNA, and the siRNA was introduced into the cells by electroporation.…”

“…Recombinant adenoviruses were constructed using a ViraPower Adenoviral Expression System (Invitrogen). For this, human MAP2K2, H-ras, R-ras, rap1a, rap1b, and CDC42 complementary DNA (cDNA) were cloned into the adenoviral-generating constructs after the introduction of constitutively active or dominant-negative mutations (26). These constructs were then transfected into 293A cells (Invitrogen) using FuGENE 6 (Roche Diagnostics), and the cells were subcultured to generate recombinant adenoviruses carrying these genes under the control of the human cytomegalovirus immediate-early enhancer/promoter.…”

“…Type II collagen contained in the cell matrix layer was quantified by enzyme-linked immunosorbent assay (Chondrex), which was normalized to the DNA content determined with a Quant-iT double-stranded DNA (dsDNA) assay kit (Invitrogen). Quantitative assessment of proteoglycan synthesis was performed by a previously described method (26). Briefly, the culture medium was replaced with a fresh medium containing 0.1% FBS and 10 Ci/ml 35 S-sulfate.…”