Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.     

Differential responses of corticotropin-releasing hormone receptor type 1 variants to protein kinase C phosphorylation

Corticotropin-releasing hormone (CRH) regulates diverse biological functions in mammals, through activation of two types of specific G protein-coupled receptors that are expressed as multiple mRNA spliced variants. In most cells, the type 1alpha CRH receptor (CRH-R1alpha) preferentially activates the G(s)-adenylyl cyclase signaling cascade. CRH-R1alpha-mediated signaling activity is impaired by insertion of 29 amino acids in the first intracellular loop, a sequence modification that is characteristic of the human-specific CRH-R1beta variant. In various tissues, CRH signaling events are regulated by protein kinase C (PKC). The CRH receptors contain multiple putative PKC phosphorylation sites that represent potential targets. To investigate this, we expressed recombinant CRH-R1alpha or CRH-R1beta in human embryonic kidney 293 cells and analyzed signaling events after PKC activation. Agonist (oxytocin) or phorbol 12-myristate 13-acetate-induced activation of PKC led to phosphorylation of both CRH-R1 variants. However, CRH-R1alpha and CRH-R1beta exhibited different functional responses to PKC-induced phosphorylation, with only the CRH-R1beta susceptible to cAMP signaling desensitization. This was associated with a significant decrease of accessible CRH-R1beta receptors expressed on the cell surface. Both CRH-R1 variants were susceptible to homologous desensitization and internalization following treatment with CRH; however, PKC activation increased internalization of CRH-R1beta but not CRH-R1alpha in a beta-arrestin-independent manner. Our findings indicate that CRH-R1alpha and -R1beta exhibit differential responses to PKC-induced phosphorylation, and this might represent an important mechanism for functional regulation of CRH signaling in target cells.     

Stimulation of alpha7 nicotinic acetylcholine receptor inhibits CD14 and the toll-like receptor 4 expression in human monocytes

The lipopolysaccharide (LPS)-receptor complex, CD14/toll-like receptor 4, is known to play a role in the immune responses during sepsis. Excessive inflammation and tumor necrosis factor (TNF)-alpha synthesis have been reported to cause morbidity and mortality in endotoxemia and sepsis. Cell-to-cell interaction through the engagement between intercellular adhesion molecule 1, B7.1, and CD40 on monocytes and their ligands on T cells has been suggested to play a role in the inflammatory response such as TNF-alpha and interleukin 10 production. Nicotine, with the stimulation of the nicotinic acetylcholine receptor alpha7 subunit (alpha7-nAChR), has now become the focus of attention because of its anti-inflammatory effects. However, little is known about the mechanism of the inhibitory effects induced by nicotine on the LPS-induced immune responses. In the present study, we found that nicotine suppressed the expression of CD14, toll-like receptor 4, intercellular adhesion molecule 1, B7.1, and CD40 on monocytes and the production of TNF-alpha, but not interleukin 10, in human peripheral blood mononuclear cells in the presence of LPS. The actions of nicotine were reversed by a nonselective and a selective alpha7-nAChR antagonist, mecamylamine and alpha-bungarotoxin, respectively. Therefore, nicotine might inhibit the LPS receptor complex expression via alpha7-nAChR, thus leading to a decrease in the adhesion molecule expression and TNF-alpha production. Moreover, we demonstrated that a nuclear factor-kappaB and a p38 mitogen-activated protein kinase inhibitor mimicked the actions of nicotine in the presence of LPS. These results suggested that the nuclear factor-kappaB and p38 mitogen-activated protein kinase might be involved in the actions of nicotine.     

Chronic endothelin-1 treatment leads to heterologous desensitization of insulin signaling in 3T3-L1 adipocytes

We recently reported that insulin and endothelin-1 (ET-1) can stimulate GLUT4 translocation via the heterotrimeric G protein G alpha q/11 and through PI3-kinase--mediated pathways in 3T3-L1 adipocytes. Because both hormones stimulate glucose transport through a common downstream pathway, we determined whether chronic ET-1 pretreatment would desensitize these cells to acute insulin signaling. We found that ET-1 pretreatment substantially inhibited insulin-stimulated 2-deoxyglucose uptake and GLUT4 translocation. Cotreatment with the ETA receptor antagonist BQ 610 prevented these effects, whereas inhibitors of G alpha i or G beta gamma were without effect. Chronic ET-1 treatment inhibited insulin-stimulated tyrosine phosphorylation of G alpha q/11 and IRS-1, as well as their association with PI3-kinase and blocked the activation of PI3-kinase activity and phosphorylation of AKT: In addition, chronic ET-1 treatment caused IRS-1 degradation, which could be blocked by inhibitors of PI3-kinase or p70 S6-kinase. Similarly, expression of a constitutively active G alpha q mutant, but not the wild-type G alpha q, led to IRS-1 degradation and inhibited insulin-stimulated phosphorylation of IRS-1, suggesting that the ET-1-induced decrease in IRS-1 depends on G alpha q/11 and PI3-kinase. Insulin-stimulated tyrosine phosphorylation of SHC was also reduced in ET-1 treated cells, resulting in inhibition of the MAPK pathway. In conclusion, chronic ET-1 treatment of 3T3-L1 adipocytes leads to heterologous desensitization of metabolic and mitogenic actions of insulin, most likely through the decreased tyrosine phosphorylation of the insulin receptor substrates IRS-1, SHC, and G alpha q/11.     

Hypertonic saline attenuates TNF-alpha-induced NF-kappaB activation in pulmonary epithelial cells

Resuscitation with hypertonic saline (HTS) attenuates acute lung injury (ALI) and modulates postinjury hyperinflammation. TNF-alpha-stimulated pulmonary epithelium is a major contributor to hemorrhage-induced ALI. We hypothesized that HTS would inhibit TNF-alpha-induced nuclear factor (NF)-kappaB proinflammatory signaling in pulmonary epithelial cells. Therefore, we pretreated human pulmonary epithelial cells (A549) with hypertonic medium (180 mM NaCl) for 30 min, followed by TNF-alpha stimulation (10 ng/mL). Key regulatory steps and protein concentrations in this pathway were assessed for significant alterations. Hypertonic saline significantly reduced TNF-alpha-induced intercellular adhesion molecule 1 levels and NF-kappaB nuclear localization. The mechanism is attenuated phosphorylation and delayed degradation of IkappaB alpha. Hypertonic saline did not alter TNF-alpha-induced p38 mitogen-activated protein kinase phosphorylation or constitutive vascular endothelial growth factor expression, suggesting that the observed inhibition is not a generalized suppression of protein phosphorylation or cellular function. These results show that HTS inhibits TNF-alpha-induced NF-kappaB activation in the pulmonary epithelium and, further, our understanding of its beneficial effects in hemorrhage-induced ALI.     

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.     

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.     

A p38alpha selective mitogen-activated protein kinase inhibitor prevents periodontal bone loss

In the oral microbial environment, Gram-negative bacterial derived lipopolysaccharide (LPS) can initiate inflammatory bone loss as seen in periodontal diseases. p38 Mitogen-activated protein kinase (MAPK) signaling is critical to inflammatory cytokine and LPS-induced cytokine expression, which may contribute toward periodontal bone loss. The purpose of this proof-of-principle study was to evaluate the ability of an orally active p38alpha MAPK inhibitor (SD-282) to reduce periopathogenic LPS-induced alveolar bone loss in an experimental rat model. Five groups of Sprague-Dawley rats received one of the following treatments: LPS injected to the palatal gingiva adjacent to the maxillary molars three times per week for 8 weeks, LPS plus two doses of SD-282 (15 or 45 mg/kg) twice daily by oral gavage, or control groups given drug vehicle (1% polyethylene glycol) or SD-282 (45 mg/kg) only. Baseline and 8-week alveolar bone loss was assessed by microcomputed tomography (microCT) and histological examination. LPS induced severe bone loss over this time period, whereas control groups were unchanged from baseline measurements. Both doses of SD-282 showed significant protection from LPS-induced bone loss. Bone area and volumetric analysis of maxillas by microCT indicated significant loss of bone volume with LPS treatment, which was blocked with the p38 inhibitor. Histological examination indicated significantly fewer tartate-resistant acid phosphatase-positive osteoclasts and a significant decrease in interleukin (IL)-6, IL-1beta, and tumor necrosis factor alpha expression in p38 inhibitor-treated groups compared with LPS groups by immunostaining. Results from this in vivo study suggest that orally active p38 MAPK inhibitors can reduce LPS-induced inflammatory cytokine production and osteoclast formation and protect against LPS-stimulated alveolar bone loss.     

A role for IL-18 in human neutrophil apoptosis

Decreased neutrophil apoptosis is associated with persistent inflammation, the severity of which correlates with serum IL-18 levels. IL-18 receptors as well as Toll-like receptors, including Toll-like receptor 4, a receptor for LPS, possess a highly conserved intracellular domain called "Toll-IL-1R domain" and activate overlapping signaling pathways. Here, we show that IL-18 modulates neutrophil apoptosis and compare its mechanism of action with LPS. We found that both IL-18 and LPS decreased neutrophil apoptosis in a similar dose- and time-dependent fashion. However, pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 increased apoptosis more effectively in IL-18- than in LPS-stimulated cells, whereas the ERK inhibitor PD98059 had the same effect in both. In contrast, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 had no influence on apoptosis at all. Neutrophils constitutively expressed mRNA for IL-18 receptor beta, but little or no receptor alpha, both of which increased during coculture with either IL-18 or LPS in a time- and dose-dependent manner. Of the Bcl-2 family, antiapoptotic A1/Bfl-1 tended to increase on IL-18 and LPS stimulation, but was further increased despite increased apoptosis in the presence of MAPK inhibitors. Thus, human neutrophils can express mRNA for IL-18 receptors alpha and beta, and IL-18, like LPS, inhibits neutrophil apoptosis by activating PI3K and ERK pathways but not p38MAPK. However, PI3K may play more important role(s) in IL-18- than in LPS-induced inhibition of apoptosis. Mitogen-activated protein kinases seem to mediate antiapoptotic signals through factors other than Bcl-2 gene family expression.     

alpha1-Adrenergic receptor stimulation of mitogenesis in human vascular smooth muscle cells: role of tyrosine protein kinases and calcium in activation of mitogen-activated protein kinase.

Signaling pathways of many G protein-coupled receptors overlap with those of receptor tyrosine kinases. We have found previously that alpha1-adrenergic receptors stimulate DNA synthesis and cell proliferation in human vascular smooth muscle cells; these effects were attenuated by the tyrosine protein kinase (TPK) inhibitor genistein and the mitogen-activated protein kinase (MAPK) antagonist 2-aminopurine. Experiments were designed to determine if activation of alpha1 receptors directly stimulated TPKs and MAPKs in human vascular smooth muscle cells. Norepinephrine stimulated time- and concentration-dependent tyrosine phosphorylation of multiple proteins, including p52-, 75-, 85-, 120-, and 145-kDa proteins. Increased TPK activity was demonstrated in proteins precipitated by an antiphosphotyrosine antibody, both in autophosphorylation assays and with a peptide substrate. These effects of norepinephrine were completely blocked by alpha1 receptor antagonists. A membrane-permeable Ca2+ chelator [1,2-bis(o-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid tetra(acetoxymethyl)ester], completely blocked norepinephrine stimulation of phosphorylation of tyrosine proteins, suggesting that intracellular Ca2+ plays a critical role in alpha1 receptor stimulation phosphorylation of tyrosine proteins. Of the tyrosine-phosphorylated proteins, the results suggest that two of them are PLCgamma1 and adapter protein Shc. Also, alpha1 receptor stimulation caused a time-dependent increase in MAPK activity due to increased phosphorylation of p42/44(ERK1/2). The alpha1 receptor-mediated activation of MAPK was also attenuated by TPK inhibitors and intracellular Ca2+ chelator [1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester]. These results suggest that phosphorylation of tyrosine proteins and intracellular Ca2+ plays a critical role in alpha1 receptor-stimulated MAPK signaling pathways, potentially contributing to increased DNA synthesis and cell proliferation.     

Lipopolysaccharide (LPS) recognition in macrophages. Participation of LPS-binding protein and CD14 in LPS-induced adaptation in rabbit peritoneal exudate macrophages.

Exposure of rabbit peritoneal exudate macrophages (PEM) or whole blood to picomolar concentrations of LPS induces adaptation or hyporesponsiveness to LPS. Because of the importance of plasma LPS-binding protein (LBP) and the macrophage cell membrane protein CD14 in recognition of LPS, we examined the effect of LBP on LPS-induced adaptation in PEM. PEM exposed to LPS in the presence of LBP for 8 h were markedly less responsive to subsequent stimulation by LPS than monocytes/macrophages (M phi) adapted in the absence of LBP. LPS-induced expression of TNF was sharply reduced in LBP-LPS-adapted PEM, but in contrast these cells remained fully responsive to Staphylococcus aureus peptidoglycan. We considered that specific hyporesponsiveness in LPS-adapted M phi or in blood monocytes could be due to decreased expression of CD14 or diminished binding of LBP-LPS complexes to CD14. However, flow cytometry analysis revealed only minimal reduction of CD14 expression or CD14-dependent binding of a fluorescent LPS derivative when normo- and hyporesponsive cells were compared. These results show that complexes of LPS and LBP are more effective than LPS alone in inducing adaptation to LPS, and LPS-induced hyporesponsiveness probably results from changes in cellular elements distinct from CD14 that are involved in either LPS recognition or LPS-specific signal transduction.