Effect of interleukin-1beta on osteogenic protein 1-induced signaling in adult human articular chondrocytes.
OBJECTIVE
Two major receptor-activated Smad (R-Smad) signaling pathways, bone morphogenetic protein (BMP) and MAPK, were examined in a model of interleukin-1beta (IL-1beta)-induced cartilage degeneration to investigate the effect of IL-1beta on osteogenic protein 1 (OP-1) signaling in adult human articular chondrocytes.


METHODS
Chondrocytes from the ankles of 26 normal human donors were cultured in high-density monolayers in serum-free medium. The effect of IL-1beta on BMP receptors was studied by reverse transcription-polymerase chain reaction and flow cytometry. Phosphorylation of R-Smads was tested in cells treated with IL-1beta (10 ng/ml), OP-1 (100 ng/ml), or the combination of IL-1beta and OP-1. Cell lysates were analyzed by Western blotting with polyclonal antibodies against 2 R-Smad phosphorylation sites (BMP- and MAPK-mediated) or with total, nonphosphorylated R-Smad as a control. To identify which MAPKs play a role in IL-1beta activation of the linker region, chondrocytes were preincubated with specific MAPK inhibitors (PD98059 for MAP/ERK, SP600125 for JNK, and SB203580 for p38).


RESULTS
IL-1beta reduced the number of activin receptor-like kinase 2 (ALK-2) and ALK-3 receptors, inhibited expression of Smad1 and Smad6, delayed and prematurely terminated the onset of OP-1-mediated R-Smad phosphorylation, and affected nuclear translocation of R-Smad/Smad4 complexes. The alternative phosphorylation of R-Smad in the linker region via the MAPK pathway (primarily p38 and JNK) was observed to be a possible mechanism through which IL-1beta offsets OP-1 signaling and the response to OP-1. Conversely, OP-1 was found to directly inhibit phosphorylation of p38.


CONCLUSIONS
These findings describe new mechanisms of the crosstalk between OP-1 and IL-1beta in chondrocytes. The study also identifies potential targets for therapeutic interventions in the treatment of cartilage-degenerative processes.
Elshaier
Amel M
AM
Rush University Medical Center, Chicago, Illinois 60612, USA.
Hakimiyan
Arnavaz A
AA
Rappoport
Lev
L
Rueger
David C
DC
Chubinskaya
Susan
S
D000328:Adult; D055419:Bone Morphogenetic Protein 7 / Q000378:metabolism* / Q000494:pharmacology; D052004:Bone Morphogenetic Protein Receptors / Q000235:genetics / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism* / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051898:Smad1 Protein / Q000378:metabolism; D051901:Smad4 Protein / Q000378:metabolism; D051905:Smad6 Protein / Q000378:metabolism
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D053583:Interleukin-1beta; C497399:SMAD1 protein, human; C497796:SMAD4 protein, human; C497802:SMAD6 protein, human; D051898:Smad1 Protein; D051901:Smad4 Protein; D051905:Smad6 Protein; D052004:Bone Morphogenetic Protein Receptors