AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes.
OBJECTIVE
AG-041R, a novel indolin-2-one derivative, has recently been demonstrated to induce systemic hyaline cartilage hyperplasia in rats. The aim of this study was to characterize its anabolic actions on chondrocytes.


METHODS
Chondrocytes were isolated from knee joints of 5-week-old SD rats. Effects of AG-041R on cartilage matrix synthesis were examined by measuring [(35)S]sulfate incorporation into proteoglycans, Alcian blue staining, and Northern blotting of cartilage matrix genes. ALP activity, mineral deposition and the expression of markers for hypertrophic chondrocytes, were assessed for terminal differentiation of chondrocytes. Roles of endogenous TGF-beta/BMPs and MEK1/Erk signaling in the action of AG-041R were investigated using the neutralizing soluble receptors and the MEK1 inhibitor.


RESULTS
AG-041R accelerated proteoglycan synthesis assessed by both [(35)S]sulfate incorporation and Alcian blue stainable extracellular matrix accumulation. It also up-regulated the gene expression of type II collagen and aggrecan, as well as tenascin, a marker for articular cartilage. In contrast, AG-041R suppressed ALP activity, mineralization, and the gene expression of type X collagen and Cbfa1, indicating that AG-041R prevents chondrocyte terminal differentiation. AG-041R increased in BMP-2 mRNA, and the neutralizing soluble receptor for BMPs reversed the stimulatory effects of AG-041R on cartilage matrix synthesis. Moreover, AG-041R activated MEK1/Erk pathway, which was revealed to prevent chondrocyte terminal differentiation.


CONCLUSIONS
AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes, which is mediated at least in part by endogenous BMPs and Erk. The data demonstrates that AG-041R has a potential to be a useful therapeutic agent for articular cartilage disorders.
Okazaki
M
M
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. okazakimkt@chugai-pharm.co.jp
Higuchi
Y
Y
Kitamura
H
H
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000032:analysis / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D003094:Collagen / Q000032:analysis; D050659:Core Binding Factor Alpha 1 Subunit; D005109:Extracellular Matrix / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology*; D009363:Neoplasm Proteins; D014176:Protein Biosynthesis; D011509:Proteoglycans / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction; D014157:Transcription Factors / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000378:metabolism
C109755:AG-041R; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D007211:Indoles; D009363:Neoplasm Proteins; D011509:Proteoglycans; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D003094:Collagen; D000469:Alkaline Phosphatase