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1 Department of Cellular and Molecular Physiology, Pennsylvania State University, Hershey, PA, USA
* To whom correspondence should be addressed. E-mail: rfrost{at}psu.edu.
The inducible form of nitric oxide synthase (NOS2) catalyzes the synthesis of nitric oxide (NO) from arginine in response to injury and infection. NOS2 is expressed predominantly by macrophages and lymphocytes. However, skeletal muscle also expresses NOS2 in response to inflammatory stimuli. The present study determined whether lipopolysaccharide (LPS) stimulates NOS2 in skeletal muscle via Toll-like receptor-4 (TLR4). Secondly, we investigated whether the clonal C2C12 mouse skeletal muscle cell line also responds to LPS and determined which signal transduction pathways are activated in this response. Intraperitoneal injection of LPS in wild-type mice (C3H/HeSnJ) increased NOS2 mRNA 4-fold in skeletal muscle, while no change in NOS2 mRNA was seen in C3H/HeJ mice that harbor a mutation in the LPS receptor. NOS2 co-immunprecipitated with the muscle-specific caveolin -3 protein suggesting that myofibers per se respond to LPS in vivo. LPS stimulated NOS2 mRNA expression in C2C12 myocytes in a dose- and time-dependent manner. The regulation of NOS2 mRNA was comparable in myoblasts and differentiated myotubes. Pretreatment of C2C12 cells with 5,6-dichloro-D-ribofuranosyl-benzimidazole (a transcriptional inhibitor) or cycloheximide (an inhibitor of new protein synthesis) completely blocked the induction of NOS2 mRNA expression. LPS transiently stimulated the phosphorylation of the interleukin-1 receptor associated kinase (IRAK-1) in C2C12 cells and decreased the total amount of IRAK-1 both in vitro and in vivo over time. LPS stimulated the expression of an NF
B reporter plasmid and this was inhibited by the proteasomal inhibitor MG132. Both myoblasts and myotubes expressed TLR2 and TLR4 mRNA. Expression of a dominant negative form of the TLR4 receptor in C2C12 cells blocked LPS-induced NFB reporter activity but not IL-1
-induced NFB activity. SP600125 (a Jun N-terminal kinase [JNK] inhibitor) also prevented LPS-stimulation of NOS2 expression. Moreover, the JNK inhibitor prevented the LPS-induced increase in NO synthesis as evidenced by the decreased accumulation of nitrate in myocyte conditioned media. Exogenous NO, provided by sodium nitroprusside, attenuated the expression of selective LPS-induced mRNAs including NOS2 and interleukin-6. These data indicate that LPS increases NOS2 mRNA expression in muscle via a TLR4-dependent mechanism. NOS2 mRNA expression is subject to transcriptional regulation that requires de novo protein synthesis and JNK activity.
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