NATURE MEDICINE • VOLUME 5 • NUMBER 8 • AUGUST 1999 913 ARTICLES The central nervous system (CNS) is one of the first organ sys- tems affected by sepsis. Sepsis is associated with behavioral changes that include increased sleep, hypomotility, hypophagia and decreased libido. Sepsis-induced CNS dysfunctions have been attributed to changes in mean arterial blood pressure and body temperature, endothelial cell dysfunction, and intravascu- lar coagulopathy. Various cytokines, including TNF-α, inter- leukin (IL)-1β, IL-6 and IL-8, are released during the onset of endotoxic shock; compelling evidence indicates TNF-α and IL- 1β are central mediators of this syndrome 1–4 . Treatment with TNF-α and IL-1β elicits characteristic somnogenic, pyrogenic and anorectic effects in rodents, indicating that these proteins are directly involved in sepsis-induced complications of the central nervous system 5–7 . The ‘regulator of G protein signaling’ (RGS) proteins stimulate the intrinsic GTPase activity of activated Gα subunits and thereby accelerate G-protein inactivation. By stabilizing acti- vated Gα i and Gα q subunits in a transition state that facilitates the release of phosphate from bound GTP, RGS proteins inhibit signaling downstream of many G protein-coupled receptors 8–11 . Several RGS proteins, including RGS4, RGS7, RGS8, RGS9 and RGS10, are densely expressed in the mammalian brain 12–19 . RGS7 is the closest mammalian homolog of EGL-10, a protein that in the nematode Caenorhabditis elegans regulates the frequency of behaviors such as locomotion, foraging and egg laying in a dose- dependent manner 12 . RGS7 is an unstable protein that is rapidly degraded by the ubiquitin–proteasome pathway 20 . Here, we report that TNF-α augments RGS7 protein levels through the inhibition of protea- somal degradation. This effect is mediated by p38, a stress-acti- vated protein kinase. Furthermore, injection of mice with endotoxin, an inducer of TNF-α release and a sepsis-like syn- drome, rapidly increases RGS7 protein levels in the brain, an up- regulation that is substantially curtailed in mice lacking tumor necrosis factor receptor 1 (TNFR1). TNF-α inhibits the degradation of RGS7 RGS7 was poorly expressed compared with RGS9 and EGL-10 after transient transfection in HEK 293T cells (Fig. 1a); however, its expression was increased considerably by treatment with TNF-α, a cytokine involved in many inflammatory and apop- totic pathways induced by sepsis. To demonstrate that this effect was mediated through the stabilization of RGS7 rather than an increase in protein synthesis, we assessed the effect of TNF-α on the half-life of RGS7 while blocking de novo protein synthesis. In the presence of the protein synthesis inhibitor cycloheximide, TNF-α increased the half-life of RGS7, but not that of green fluo- rescent protein (GFP), used to confirm transfection efficiency and protein loading, at each time point (Fig. 1b). RGS7 is subject to ubiquitin-dependent degradation by the proteasome complex and is ubiquitinated in the presence of the proteasome inhibitor MG132 and HA-tagged ubiquitin. In contrast, TNF-α did not fa- cilitate the accumulation of ubiquitinated RGS7 molecules (Fig. 1c). These findings indicate that TNF-α may induce a post-trans- lational modification of RGS7 that prevents ubiquitination of RGS7. To demonstrate that the effect of TNF-α was specific for RGS7, we assessed the effect of TNF-α on β-catenin, a protein also subject to proteasome degradation 21,22 . Degradation of β-catenin involves activation of the protein kinase GSK-3β (ref. 21); thus, lithium, a GSK-3β inhibitor, and the proteasome inhibitor MG132 increased steady-state protein levels of β-catenin, whereas TNF-α did not (Fig. 1d). The onset of TNF-α upregula- tion of RGS7 protein levels was detectable after 2 hours with a progressive decrease after 6 hours (Fig. 1e). Endogenous RGS7 in Upregulation of RGS7 may contribute to tumor necrosis factor-induced changes in central nervous function THOMAS BENZING 1 , RALF BRANDES 2 , LORENZ SELLIN 1 , BERNHARD SCHERMER 1 , STEWART LECKER 3 , GERD WALZ 1 & EMILY KIM 4 1 Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215, USA 2 Institute for Cardiovascular Physiology, University Hospital of Frankfurt, D-60590 Frankfurt, Germany 3 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA 4 Department of Psychiatry, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215, USA Correspondence should be addressed to E.K.; email: ekim@caregroup.harvard.edu The central nervous dysfunctions of lethargy, fever and anorexia are manifestations of sepsis that seem to be mediated by increased cytokine production. Here we demonstrate that tumor necrosis factor (TNF)-α, an essential mediator of endotoxin-induced sepsis, prevents the protea- some-dependent degradation of RGS7, a regulator of G-protein signaling. The stabilization of RGS7 by TNF-α requires activation of the stress-activated protein kinase p38 and the presence of candidate mitogen-activated protein kinase phosphorylation sites. In vivo, RGS7 is rapidly upreg- ulated in mouse brain after exposure to either endotoxin or TNF-α, a response that is nearly ab- rogated in mice lacking TNF receptor 1. Our findings indicate that TNF-mediated upregulation of RGS7 may contribute to sepsis-induced changes in central nervous function. © 1999 Nature America Inc. • http://medicine.nature.com © 1999 Nature America Inc. • http://medicine.nature.com