Antisense inhibition of osteogenic protein 1 disturbs human articular cartilage integrity.
OBJECTIVE
To delineate the role of endogenous osteogenic protein 1 (OP-1) in human articular cartilage homeostasis via the inhibition of OP-1 gene expression by antisense oligonucleotides.


METHODS
Human adult normal articular cartilage was obtained from the knee and ankle joints of 34 organ donors. Chondrocytes were cultured as tissue explants or isolated cells in alginate or high-density monolayers for 48 hours in the presence of OP-1 antisense or sense oligonucleotides. The effect of OP-1 antisense inhibition was evaluated by reverse transcription-polymerase chain reaction, (35)S incorporation, dimethylmethylene blue assay, histology with Safranin O staining, and immunohistochemistry with anti-proOP-1, anti-mature OP-1, and anti-aggrecan antibodies.


RESULTS
Antisense treatment inhibited OP-1 gene expression by a mean +/- SD of 34 +/- 12% (P < 0.01) in chondrocytes cultured in monolayers and by 77 +/- 27% (P < 0.03) in alginate beads. The inhibition of autocrine OP-1 caused a striking decrease in aggrecan gene expression, in total proteoglycan content accumulated in cartilage matrix, and in the ability of chondrocytes to newly synthesize proteoglycans. OP-1 antisense reduced aggrecan messenger RNA expression by 42 +/- 17% (P < 0.05) and proteoglycan synthesis by 48 +/- 23% (P < 0.01). Histology and immunohistochemistry revealed a dramatic decrease in Safranin O staining and reduced anti-aggrecan staining (primarily in the superficial and middle cartilage layers) with OP-1 antisense treatment.


CONCLUSIONS
Our results suggest that OP-1 is an important endogenous cartilage factor that regulates matrix integrity and possibly needs to be induced or up-regulated to maintain normal cartilage homeostasis. These findings confirm our hypothesis that a lack of autocrine OP-1 may lead to an elevated susceptibility of chondrocytes to the catabolic processes, thus contributing/promoting cartilage degeneration.
Söder
Stephan
S
Rush University Medical Center, Chicago, Illinois 60612, USA.
Hakimiyan
Arnavaz
A
Rueger
David C
DC
Kuettner
Klaus E
KE
Aigner
Thomas
T
Chubinskaya
Susan
S
D030201:Activin Receptors, Type I; D000328:Adult; D053674:Aggrecans; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D002358:Cartilage, Articular / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000032:analysis; D015870:Gene Expression / Q000187:drug effects; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D037181:Lectins, C-Type; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D011509:Proteoglycans / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046509:Tissue Culture Techniques
D053674:Aggrecans; C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D016376:Oligonucleotides, Antisense; D011506:Proteins; D011509:Proteoglycans; C425423:ACVR1 protein, human; D030201:Activin Receptors, Type I