MKK3/6-p38 MAPK signaling is required for IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.
Coupled bone turnover is directed by the expression of receptor-activated NF-kappaB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1beta treatment and subsequently reduced approximately 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1beta or TNF-alpha treatment. IL-1beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.
Rossa
Carlos
C
Department of Diagnosis and Surgery, State University of Sao Paulo (UNESP), Araraquara, SP, Brazil.
Ehmann
Kathryn
K
Liu
Min
M
Patil
Chetan
C
Kirkwood
Keith L
KL
D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002454:Cell Differentiation; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology*; D017930:Genes, Reporter; D007093:Imidazoles / Q000494:pharmacology; D007375:Interleukin-1 / Q000037:antagonists & inhibitors / Q000494:pharmacology; D048371:MAP Kinase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048669:MAP Kinase Kinase 6 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009154:Mutation; D010010:Osteoclasts / Q000166:cytology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D017154:Stromal Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002352:Carrier Proteins; D016207:Cytokines; D007093:Imidazoles; D007375:Interleukin-1; D008562:Membrane Glycoproteins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D020929:Mitogen-Activated Protein Kinase Kinases; C093642:SB 203580