Further insights in the mechanisms of interleukin-1beta stimulation of osteoprotegerin in osteoblast-like cells.
The mechanisms of IL-1beta stimulation of OPG were studied in more detail. Whereas p38 and ERK activation was confirmed to be needed, NF-kappaB was not necessary for this regulation. We also found that OPG production after IL-1beta stimulation was not sufficient to block TRAIL-induced apoptosis in MG-63 cells.


BACKGROUND
Osteoprotegerin (OPG) plays a key role in the regulation of bone resorption and is stimulated by interleukin (IL)-1beta. Herein, we defined the mechanisms of IL-1beta stimulation of OPG focusing on the potential involvement of MAPK and NF-kappaB. We also examined whether OPG production in response to IL-1beta influences TRAIL-induced apoptosis in MG-63 cells.


METHODS
OPG mRNA levels in MG-63 cells were quantified by real-time RT-PCR and protein levels of OPG and IL-6 by ELISA. Cell viability was assessed using the methyltetrazidium salt (MTS) reduction assay. The role of the MAPK pathway was studied by both Western blotting and the use of specific chemical inhibitors. NF-kappaB function was studied using BAY 11-7085 and by siRNA transfection to inhibit p65 synthesis. Transcription mechanisms were analyzed by transiently transfecting MG-63 cells with OPG promoter constructs. Post-transcriptional effects were examined by using cycloheximide and actinomycin D.


RESULTS
MG-63 cells treatment with IL-1beta resulted in the phosphorylation of c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). The use of the specific inhibitors showed that p38 and ERK but not JNK were needed for IL-1beta-induced OPG production. In contrast, NF-kappaB was not essential for IL-1beta induction of OPG. We also showed a small transcriptional and a possible post-transcriptional or translational regulation of OPG by IL-1beta. Exogenous OPG blocked TRAIL-induced apoptosis, but IL-1beta induction of OPG did not influence TRAIL-induced cell death.


CONCLUSIONS
IL-1beta stimulates OPG production by mechanisms dependent on p38 and ERK. In contrast, NF-kappaB was not essential for this regulation. Although the relevance of IL-1beta stimulation of OPG is still not fully understood, our data showed that IL-1beta stimulation of OPG does not modify TRAIL-induced cell death.
Lambert
Cécile
C
Department of Rheumatology, Center for Biomedical Intergrative Genoproteomics, University of Liège, Liège, Belgium.
Oury
Cécile
C
Dejardin
Emmanuel
E
Chariot
Alain
A
Piette
Jacques
J
Malaise
Michel
M
Merville
Marie-Paule
MP
Franchimont
Nathalie
N
D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D017931:DNA Primers; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D053244:Osteoprotegerin / Q000096:biosynthesis* / Q000494:pharmacology; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D053583:Interleukin-1beta; D053244:Osteoprotegerin; D034741:RNA, Small Interfering; D020928:Mitogen-Activated Protein Kinases