Regulators of G-protein signalling are modulated by bacterial lipopeptides and lipopolysaccharide Sabine Riekenberg1, Katja Farhat1, Jennifer Debarry2, Holger Heine2, Gunther Jung3, Karl-Heinz ă Wiesmuller4 and Artur J Ulmer1 ă Cellular Immunology, Department of Immunology and Cell Biology, Research Center Borstel, Germany Innate Immunity, Department of Immunology and Cell Biology, Research Center Borstel, Germany Institute of Organic Chemistry, University of Tuebingen, Germany EMC microcollections GmbH, Tuebingen, Germany Keywords gene expression; lipopeptides; macrophages; regulator of G-protein signalling; Toll-like receptors Correspondence A J Ulmer, Cellular Immunology and Cell Biology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany Fax: +49 4537 188435 Tel: +49 4537 188448 E-mail: ajulmer@fz-borstel.de (Received 26 August 2008, revised 12 November 2008, accepted 20 November 2008) doi:10.1111/j.1742-4658.2008.06813.x Regulators of G-protein signalling accelerate the GTPase activity of Ga subunits, driving G proteins in their inactive GDP-bound form This property defines them as GTPase activating proteins Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed After stimulation with TLR2 ⁄ or TLR2 ⁄ lipopeptide ligands and the TLR4 ⁄ MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein signalling tested Real-time PCR confirmed modulation of RGS1 and RGS2 In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 after stimulation, followed by downregulation Similar results were also found in the murine macrophage cell line J774 The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway This was confirmed using MyD88) ⁄ ) and TRIF) ⁄ ) bone marrow-derived macrophages Modulation of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection The innate immune system is the first barrier against pathogens and is initiated rapidly after recognition of microbial products by receptors such as the Toll-like receptors (TLR) TLR recognize a broad range of ligands like lipopolysaccharides (LPS) and lipopeptides (LP) representing pathogen-associated molecular patterns [1,2] TLR contain two major domains: the extracellular ligand-binding domain, characterized by leucine-rich repeats and the intracellular Toll ⁄ IL-1 receptor domain (TIR domain) [3] In mammals, 13 TLR homologues recognizing specific bacterial or viral ligands have been identified [4] Bacterial LP and LPS are recognized by the membrane receptors TLR2 and TLR4, respectively Intracellular TLR3 is a receptor for poly(I:C) [5], and CpG oligo-nucleotides are ligands for the intracellular TLR9 [6,7] TLR2 is unique among all TLR, developing heteromers with TLR1 and TLR6 In previous studies we investigated the ligand specificity of different TLR2 dimers in spleen cells from TLR2-, TLR6- and TLR1-deficient mice [8,9] LP have strong TLR2-dependency but differ in their requirement for TLR6 and TLR1, according Abbreviations BMDM, bone marrow-derived macrophages; FSL-1, fibroblast-stimulating lipopeptide-1; GAP, GTPase activating protein; GPCR, G-protein coupled receptor; LP, lipopeptide; LPS, lipopolysaccharide; RGS, regulator of G-protein signalling; TLR, Toll-like receptor FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS 649 RGS are modulated by lipopeptides and LPS S Riekenberg et al to the number and length of their fatty acids and the amino acid sequence of their peptide tail To address TLR2 ⁄ 1- and TLR2 ⁄ 6-mediated signalling we used the lipopeptide Pam3C-SK4 and fibroblast-stimulating lipopeptide-1 (FSL-1), respectively Activation of macrophages by microbes or their cellular components induces the release of different inflammatory mediators Stimulation of TLR leads to activation of a series of signalling proteins, and to the expression of pro- and inflammatory cytokines There is evidence that the heteromeric guanine nucleotidebinding regulatory protein (G protein) is also involved in TLR4 activation It has been shown that LPS induced TNFa production which can be blocked by pertussis toxin [10] Also, TLR4-induced ERK1 ⁄ phosphorylation is inhibited by dominant-negative Gai protein constructs [11] G proteins are located downstream of G-protein-coupled receptors (GPCR) [12] GPCR represent a large family of cell-surface proteins mediating the effects of a broad spectrum of biological signals After ligand binding, the receptor undergoes a conformational change Ligands include hormones, biogenic amines, histamine, serotonin and lipid derivates, but also immunological and inflammatory mediators such as chemokines Heterotrimeric G proteins are localized on the inner surface of cell membranes They comprise a superfamily of at least 17 distinct Ga, Gb and Gc isoforms [13] Furthermore the a subunits are divided into four main categories: Gai, Gas, Gaq and Ga12 ⁄ 13 [14] In their inactive conformation G proteins consist of a-, b- and c subunits, whereas only the a subunit is bound to GDP GPCR are transmembrane receptor proteins, containing seven membrane-spanning segments After binding of the relevant ligand and activation of the GPCR, the receptor acts as a guanine nucleotide-exchange factor that exchanges GTP for GDP on the a subunit In the active GTP-bound form, the a subunit–GTP complex dissociates from the bc dimer Each of the separated subunits can regulate downstream effectors Signalling is terminated when the a subunit hydrolyses GTP, returns to the GDP-bound state and again associates with bc subunits to give the inactive heterotrimeric form [15] Regulators of G-protein signalling (RGS) interact directly with the G protein a subunit in order to inhibit G-protein signalling [16] RGS proteins belong to a large gene family, whose members are widespread from yeast to mammals [13] RGS proteins differ widely in their size and amino acid identity They were first discovered genetically as negative regulators of G-protein signalling in lower eukaryotic organisms including Aspergillus and Caenorhabditis elegans 650 Currently, more than 25 mammalian RGS proteins have been identified by molecular cloning [17] Each RGS protein contains a conserved sequence of 120 amino acids which is responsible for binding to the Ga subunit [18] The functional effect of most of RGS proteins is unclear Biochemical studies have shown that RGS proteins have GTPase activity and act as a GTPase activating protein (GAP) As a result, RGS proteins enhance GTP hydrolysis rates for purified Gai and Gaq subunits as much as 100- to 300-fold [15,19] They can also modulate the lifetime and kinetics of slow-acting signalling responses like Ca2+ oscillations [20] Different studies have shown that RGS1 stimulates the GTPase activity of several members of the Gai subfamily but is ineffective against Gas [21], whereas RGS2 does not interact with Gai, Gao, Gas and Ga12 ⁄ 13 at all; RGS2 acts selectively as a GAP for Gaq subunits [22,23] In this study, we show using microarray analyses that RGS2 belongs to the most downregulated mRNA after stimulation of murine bone marrow-derived macrophages (BMDM) with LP, whereas RGS1 was upregulated after stimulation with LPS Similar results were found in dendritic cells after activation with LPS These observations led us to investigate the modulation of RGS1 and RGS2 in BMDM after stimulation with LP and LPS in more detail, because regulation of RGS1 and RGS2 after activation of different TLR may modify the effects of G-protein signalling after posterior activation of GPCR Our results indicate that RGS1 and RGS2 have important immunomodulating functions in murine macrophages because these two RGS proteins demonstrate strong modulation of expression after stimulation with LPS and LP LP and LPS mediate immunomodulating functions, at least in part, through regulation of RGS1 and RGS2 expression Results RGS1 and RGS2 mRNA expression is regulated by LP and LPS BMDM were stimulated with FSL-1, a ligand for the TLR2 ⁄ heteromer, and LPS, a ligand for TLR4 ⁄ MD2 After various culture times, mRNA was isolated and the expression of multiple probe sets was analysed by microarray analysis According to the microarray data, only 11 of 18 tested RGS mRNAs are expressed in LP-stimulated BMDM, and of 16 tested RGS mRNAs were expressed in LPS-stimulated BMDM We did not detect the mRNA of several of the tested RGS genes in control or stimulated cells FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS S Riekenberg et al RGS are modulated by lipopeptides and LPS Table Gene regulation of different RGS in BMDM Macrophages were stimulated with 10 ngỈmL)1 LPS (A) or 100 nM FSL-1 (B) and mRNA was determined by microarray analysis The results are expressed as relative fluorescence and fold induction compared with control A Stimulation h Gene Control relative fluorescence LPS relative fluorescence (fold) RGS1 RGS2 RGS3 RGS4a RGS5 RGS6a RGS7a RGS9a RGS10 RGS11a RGS14 RGS16 RGS17a RGS18 RGS19 RGS20a 168 1532 153 8 63 2161 20 247 10 407 382 1366 773 149 8 68 1485 15 323 16 249 212 a B (8.13) ()2.0) ()1.0) (1.0) (1.0) ()1.0) (1.0) (1.1) ()1.5) ()1.4) (1.3) (1.3) (1.1) ()1.9) ()1.8) ()1.0) Stimulation h Stimulation h Gene Control relative fluorescence FSL-1 relative fluorescence (fold) Control relative fluorescence FSL-1 relative fluorescence (fold) RGS1 RGS2 RGS3 RGS4a RGS5a RGS6a RGS7a RGS8a RGS9 RGS10 RGS11 RGS12 RGS13a RGS14 RGS16 RGS18 RGS19 RGS20a 492 1872 172 13 8 11 10 105 1535 14 39 224 26 292 691 420 149 152 11 9 107 1191 17 33 158 53 176 412 574 1540 206 14 9 10 108 1790 23 25 311 20 742 990 10 190 22 188 14 9 114 605 27 19 240 23 316 832 ()1.2) ()12.6) ()1.1) ()1.2) (1.0) ()1.1) ()1.2) ()1.1) (1.0) ()1.3) (1.2) ()1.2) (1.2) ()1.4) (2.0) ()1.7) ()1.7) (1.1) ()3.0) ()70.0) ()1.1) (1.0) (1.3) (1.0) ()1.1) ()1.1) (1.1) ()3.0) (1.2) ()1.3) (1.0) ()1.3) (1.2) ()2.3) ()1.2) ()1.1) RGS mRNA not expressed in BMDM (Table 1) We gave special regard to RGS2, because we observed that the mRNA of RGS2 was the strongest downregulated mRNA of 45 101 probe sets after h of stimulation with FSL-1 Interleukin-6, by contrast, was the strongest upregulated gene (data not given) In addition, RGS1 and RGS10, but none of the other listed RGS mRNAs, were also found to be modulated Stimulation with Pam2C-SK4 (TLR2 ⁄ and TLR2 ⁄ ligand) and PamOct2C(VPGVG)4VPGKG (TLR2 ⁄ ligand) showed similar results (data not shown) Interestingly, after microarray analysis with LPS-stimulated BMDM strong upregulation of RGS1 was found, but no modulation (more than twofold) of other RGS mRNAs (Table 1A) These finding led us to investigate the modulation of RGS1 and RGS2 after stimulation with LP and LPS in more detail To confirm modulation of RGS1 and RGS2 mRNA determined by microarray analysis, real-time PCR was performed with BMDM as described in Materials and methods To control stimulation of BMDM, the TNFa release in the supernatant by ELISA was measured (Fig 1B) Expression levels of RGS1 and RGS2 after real-time PCR were referred to the housekeeping gene HPRT After activation of BMDM with LPS or LP (FSL-1 and Pam3C-SK4) there was an increase in RGS1 mRNA at a very early period (15 min) of stimu- lation After 1–2 h, the expression decreased, was found to be at control levels  12 h after stimulation (Fig 1A), and was further decreased at 24 h of culture For RGS2, no upregulation but rather a decrease in mRNA expression in BMDM after stimulation with LPS and LP could be detected, which was seen after 30–60 of stimulation Expression of RGS2 mRNA was further reduced after stimulation with LP up to a culture period of 24 h Similar expression of RGS1 and RGS2 mRNA was also found after stimulation of the macrophages cell line J774 with LPS and LP at and 14 h (Fig 2) The control of this stimulation is given by the relative expression of TNFa (Fig 2) Cytokine mRNA expression can be increased by LP and LPS Thus, these results demonstrate that both BMDM and J774 cells express RGS1 and RGS2 mRNA and modulate expression after stimulation with LP and LPS Expression patterns of RGS1 and RGS2 after activation of TLR3 and TLR9 To investigate the expression levels of RGS1 and RGS2 after activation of other TLR, BMDM were stimulated with ODN1826 for 0–24 h to activate TLR9 signalling After real-time PCR we found a slight increase in RGS1 mRNA ( 2.6-fold) 30 after stimulation After 12–24 h expression decreased FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS 651 RGS are modulated by lipopeptides and LPS S Riekenberg et al RGS1 10 100 nM Pam3C-SK4 100 nM FSL-1 0.25 0.5 12 24 0.25 0.5 12 24 0.25 0.5 12 24 (h) RGS2 1.4 100 nM FSL-1 1.2 100 ng·mL–1 LPS 100 nM Pam3C-SK4 1.0 0.8 0.6 0.4 0.2 FSL-1 20 000 30 000 25 000 TNF-α Pam3C-SK4 20 000 15 000 15 000 10 000 10 000 Ctr 0.25 0.5 12 24 5000 Ctr 0.25 0.5 12 24 5000 3500 3000 2500 2000 1500 1000 500 (h) LPS Ctr 0.25 0.5 12 24 25 000 0.25 0.5 12 24 0.25 0.5 12 24 0.0 B TNF-α (pg·mL–1) 100 ng·mL–1 LPS 0.25 0.5 12 24 Relative expression of RGS2 mRNA Relative expression of RGS1 mRNA A (h) Fig Modulation of RGS1 and RGS2 mRNA Expression was measured after stimulation of BMDM with 100 ngỈmL)1 LPS, 100 nM FSL-1 and 100 nM Pam3C-SK4 for 0–24 h (A) Specific mRNA expression was determined by real-time PCR The release of TNFa into the culture supernatants was determined by ELISA (B) For real-time PCR, similar data were obtained in three independent experiments Data for ELISA are the mean ± SE from two experiments and was found at control level (Fig 3), similar to the modulation after activation of TLR2 and TLR4 by LP and LPS In contrast to RGS1, RGS2 showed only a decrease in mRNA expression in BMDM after stimulation with ODN1826 We measured mRNA expression in BMDM treated with poly(I:C) to activate TLR3 signalling in a 652 kinetic manner Strong upregulation of RGS1 mRNA was found only after 12 and 24 h (Fig 3) Treatment with poly(I:C) increased the mRNA level of RGS1  150-fold compared with the control after 24 h Surprisingly, in contrast to the other TLR ligands, we detected an upregulation for RGS2 mRNA (approximately fivefold changes) after 12 and FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS S Riekenberg et al RGS are modulated by lipopeptides and LPS Relative expression of RGS1 mRNA Fig Expression of RGS1, RGS2 and TNFa mRNA in J774 After stimulation with 100 ngỈmL)1 LPS, 100 nM FSL-1 and 100 nM Pam3C-SK4 for 0–14 h, specific mRNA expression was determined by realtime PCR Similar data were obtained in three independent experiments 1.2 Relative expression of TNF-α mRNA α Relative expression of RGS2 mRNA RGS1 1.2 100 nM Pam3C-SK4 100 nM FSL-1 100 ng·mL–1 LPS 1.0 0.8 0.6 0.4 0.2 0.0 14 14 14 (h) RGS2 100 nM FSL-1 100 ng·mL–1 LPS 100 nM Pam3C-SK4 1.0 0.8 0.6 0.4 0.2 0.0 14 14 14 (h) TNF-α 100 nM FSL-1 100 ng·mL–1 LPS 100 nM Pam3C-SK4 0 24 h Therefore poly(I:C) was a strong stimulator of RGS1 mRNA production, and of RGS2 mRNA, suggesting that regulation of RGS1 and RGS2 after stimulation with poly(I:C) is due to the TRIF-dependent pathway Poly(I:C) induced upregulation of RGS1 and RGS2 mRNA expression via a TRIF-dependent pathway To further analyse the regulation of RGS1 and RGS2 mRNA after activation of TLR3 signalling we measured mRNA expression in wild-type and TRIF) ⁄ ) BMDM after stimulation with poly(I:C) Fig shows that poly(I:C) induced a 180-fold increase in RGS1 mRNA in cells from wild-type mice A slight increase 14 14 14 (h) in expression occurred as early as 0.5 h and reached a peak after 24 h (Fig 4) As shown in Fig 3, there was also strong upregulation of RGS2 mRNA after stimulation with poly(I:C) We detected a 17-fold increase in RNA expression after 24 h As expected in BMDM of TRIF) ⁄ ) mice, we found no regulation of RGS1 and RGS2 mRNA, indicating that poly(I:C) can only activate genes via a TRIF-dependent pathway Looking at downstream signalling events after stimulation, the involvement of different MAP kinases was determined Use of PD98059, an inhibitor of Erk, or SB203580, an inhibitor of p38, had no effect on RGS1 or RGS2 regulation after 0.5 and h of stimulation with a TLR2, TLR3 or TLR4 ligand (data not shown) Also, the inhibition of the Gai subunit by pertussis toxin [24] FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS 653 200 ODN 2.5 2.0 1.5 1.0 0.5 0.0 Ctr 0.5 12 24 24 (h) (h) Rel expression of mRNA Rel expression of mRNA Rel expression of mRNA RGS1 160 140 Poly(I:C) 120 100 80 60 40 20 Ctr 0.5 12 Rel expression of mRNA RGS1 3.0 S Riekenberg et al 1.2 Rel expression of mRNA RGS are modulated by lipopeptides and LPS Poly (I:C) Wild-type Trif –/– 100 50 Ctr 0.5 12 24 (h) Rel expression of mRNA RGS1 150 RGS2 ODN 1.0 0.8 0.6 0.4 0.2 0.0 Ctr 12 24 (h) RGS2 Poly(I:C) Ctr 0.5 12 RGS2 18 16 Poly (I:C) 14 Wild-type Trif –/– 12 10 Ctr 0.5 has no influence on RGS1 modulation after stimulation with LPS and FSL-1 Involvement of TRIF in the upregulation of RGS1 and RGS2 mRNA The findings obtained from activation of TLR3 by stimulation with poly(I:C) indicate a different modulation of RGS1 and RGS2 mRNA by the MyD88- or TRIF-dependent signalling pathway To confirm this we stimulated wild-type, TRIF) ⁄ ) and MyD88) ⁄ ) BMDM with LP, which induce only the MyD88dependent signalling pathway, or with LPS, which induced the MyD88- and TRIF-dependent signalling pathways Kinetic studies showed that RGS1 mRNA was found to be first upregulated and then downregulated to the same degree after stimulation of wild-type and TRIF) ⁄ ) mice with LP, indicating that the TRIF signalling pathway is not involved The same kinetics of RGS1 modulation was found after stimulation of 654 0.5 24 12 (h) 24 (h) Fig Verification of RGS1 and RGS2 mRNA after activation of TLR9 and TLR3 BMDM were stimulated with lM ODN1826 or 50 lgỈmL)1 poly(I:C) RNA level was detected by real-time PCR Data were representative for three independent experiments Fig Modulation of RGS1 and RGS2 mRNA in wild-type or TRIF) ⁄ ) BMDM after stimulation with poly(I:C) Specific mRNA expression was determined by real-time PCR Similar data were obtained in three independent experiments the cells with LPS in the absence of the TRIF pathway, indicating that LP and LPS regulate RGS1 in the same manner via the MyD88 pathway In the absence of the MyD88-dependent signalling pathway in cells of MyD88) ⁄ ) mice, there is no modulation of RGS1 mRNA expression after stimulation with LP but a strong upregulation after stimulation of the cells with LPS This indicates that activation of the TRIF pathway resulted in a different modulation of RGS1 mRNA than after activation of the MyD88 pathway following stimulation with LPS This differential response of the BMDM resulted in prolonged upregulation of RGS1 mRNA after stimulation with LPS, depending on whether the MyD88- or TRIF pathway was activated Downregulation of RGS2 mRNA by FSL-1 was seen only in wild-type and TRIF) ⁄ ) BMDM, whereas in MyD88) ⁄ ) BMDM no modulation of RGS2 was found By contrast, LPS downregulates RGS2 mRNA expression in wild-type and TRIF) ⁄ ) cells but strongly FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS S Riekenberg et al upregulates this RGS mRNA in MyD88) ⁄ ) macrophages This indicates a different modulation of RGS mRNA via the MyD88 and TRIF pathways Discussion RGS1 and RGS2 are proven to be the main RGS mRNA modulated in murine macrophages after stimulation with LP, LPS, poly(I:C) and ODN1826 Microarray analysis identified RGS2 mRNA as the most downregulated gene after h of stimulation (Table 1), whereas interleukin-6 was the strongest upregulated gene within 45 101 probe sets [25] These findings suggest that RGS2 plays an important role in the biological consequences after activation of TLR by different ligands However, little is known about the involvement of RGS2 proteins in the context of inflammation Under all RGS proteins, RGS2 contains a unique function, because it is the only RGS protein that does not interact with Gai subunits, but selectively regulates the function of Gaq [23] These findings are supported by unique structural features of its G-protein-binding interface [26] RGS2 inhibits Gaq-induced activation of phospholipase C in cell membranes [23] After downregulation of RGS2 the Gaq subunit stays active, with the consequence that phospholipase C can cleave phosphatidylinositol 4,5-bisphoshate into two second messengers, inositoltriphosphate and diacylglycerol [27] These secondary messengers can themselves mediate, for example, Ca2+ flux and activate protein kinase C [28] This activation leads then to further downstream effects like changes in gene transcription or morphological and cytoskeletal changes Another function of RGS2 proteins is to bind directly to certain subtypes of adenylyl cyclases [29] This interaction between the cyclases and RGS2 leads to an inhibition of the cAMP production [20] After downregulation of RGS2 it is likely that the inhibition of the adenylyl cyclase is compensated Taken together, downregulation of RGS2 mRNA prohibits the deactivation of phospholipase C and adenylyl cyclases, followed by different signal cascades to counteract against microbial lipids It is interesting to see the strong upregulation of RGS1 mRNA between 30 and 60 after stimulation with LPS and also with LP (Fig 1) and ODN1826 (Fig 3) in BMDM Similar results were obtained in J774 cells (Fig 2) The fast kinetics of RGS regulation indicates a primary effect due to the TLR activation and not a secondary effect due to G-protein signalling Using microarray analysis we found a relevant modulation of RGS1 after h of stimulation with LPS (Table 1) The gene was upregulated eightfold and represents the only upregulated RGS gene tested using RGS are modulated by lipopeptides and LPS this microarray approach Confirming the data by real-time PCR, strong upregulation of RGS1 mRNA after 30 of stimulation was observed However, the real-time PCR assay does not show strong regulation at or h of stimulation in several experiments This effect may be due to the peculiarity of this single gene array experiment indicating that such an experiment should be confirmed by real-time PCR Nevertheless such early RGS1 modulation is likely to participate in appropriate cellular responses like RGS2 Comparable results were found in dendritic cells after stimulation with LPS RGS16, a RGS protein similar to RGS1 and RGS2, was strongly upregulated [30] and the regulation of different RGS proteins in murine macrophages are discussed, but no function is known to date [31] RGS1 proteins stimulate the intrinsic GTPase activity of Gai subunits These subunits are responsible for the activation of different ion channels, several phospholipases and for the inhibition of the cAMP production Upregulation of RGS1 accelerates the GTP hydrolyse of the Gai subunits and thereby inhibits the Gai subunit signalling, which presumably results in compensation of the inhibition of the adenylyl cyclases and Ca2+ channels as well as the activation of K+ channels or phosphodiesterases [32] Upregulation of RGS1 leads to a higher cAMP level and this second messenger activates protein kinase A Protein kinase A phosphorylation leads to an increased expression of cyclo-oxygenase-2, also known as prostaglandin synthase-2 in HEC-1B cells [33] We found also strong upregulation of Cox-2 in BMDM after stimulation with different lipopeptides in our microarray analysis [25] That means that upregulation of RGS1 mRNA may lead to modulation of cyclo-oxygenase-2 transcription involved in inflammation [34] Another surprising point was the strong upregulation of RGS1 and RGS2 mRNA after activation of the TLR3 signalling pathway with poly(I:C) (Fig 4) Upregulation in this dimension ( 180-fold of RGS1 mRNA) has an enormous effect in BMDM, because the modulation of RGS1 and therefore the regulation of different proteins is enforced by RGS2 mRNA after 12 h of stimulation The upregulation of both RGS mRNA was found only after activation of the TLR3 signalling pathway This upregulation of RGS1 and RGS2 mRNA is due to the TRIF pathway We verified the data by stimulation experiments with LPS in wild-type versus TRIF) ⁄ ) and MyD88) ⁄ ) BMDM (Fig 5) It is known that LPS can signal via TLR4 in a MyD88- and TRIF-dependent manner Stimulation of MyD88) ⁄ ) mice with LPS activates the TRIFdependent pathway The effect on RGS modulation FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS 655 14 FSL-1 12 10 Rel expresssion of mRNA Rel expresssion of mRNA RGS are modulated by lipopeptides and LPS S Riekenberg et al RGS1 LPS Wild-type MyD88–/– TRIF–/– Wild-type MyD88–/– TRIF–/– Ctr 0.5 12 24 Ctr 0.5 12 24 (h) RGS2 FSL-1 LPS Wild-type MyD88–/– TRIF–/– Wild-type MyD88–/– TRIF–/– Ctr 0.5 12 24 Ctr 0.5 resembles the results we obtained after poly(I:C) stimulation, thus proving the responsibility of the TRIF activation for the upregulation of both RGS mRNA Stimulation of TLR3 and activation of the TRIF pathway leads to interferon-b production [35] Takaoka et al [36] demonstrated that interferon-b can induce the transcription of p53 and this is critical for an antiviral defence of the host In addition, T cells with a lack of RGS2 impair antiviral immunity [37] In conclusion, after activation of TLR3 by poly(I:C) RGS2 is necessary for an adequate antiviral immune response After stimulation of distinct TLR pathways different MAP kinases and several transcription factors like Nf-jB are activated and the induction of proinflammatory cytokines are found [38] The participation of these signal transduction molecules in RGS1 and RGS2 modulation is not obvious, because usage of different inhibitors (PD98059 an inhibitor of Erk, SB203580 an inhibitor of p38) had no influence on modulating RGS1 and RGS2 mRNA This indicates that modulation of both RGS transcripts is regulated by a pathway independent of these two MAP kinases It is possible that the modulation is due to the activation of JNK Other MAP kinase inhibitors as well as G-protein inhibitors should be investigated to find out the participating proteins in RGS1 and RGS2 modulation In conclusion, our results show strong modulation of RGS1 and RGS2 mRNA induced by different TLR 656 6 12 24 (h) Fig Modulation of RGS1 and RGS2 mRNA in wild-type, TRIF) ⁄ ) and MyD88) ⁄ ) BMDM Expression after stimulation was measured by real-time PCR Data were obtained in three independent experiments ligands After stimulation with bacterial LP, LPS and ODN we detected strong upregulation and afterwards downregulation of RGS1 and a decrease in RGS2 because of the MyD88-dependent pathway Stimulation with poly(I:C) only leads to upregulation of both RGS1 and RGS2 mRNA, as a result of the TRIFdependent pathway, without involvement of MyD88 (Fig 6) We suggest that the inflammatory and the adjuvant activities of TLR-ligands are at least partially mediated through modulation of RGS1 and RGS2 The molecular mechanisms, leading to this modulation and the consequences of the modulation of RGS1 and RGS2 remain to be investigated Materials and methods Reagents Dulbecco’s modified Eagles medium, RPMI-1640, penicillin-streptomycin, l-glutamine, sodium pyruvate and Hepes buffer were obtained from Invitrogen (Karlsruhe, Germany) Fetal calf serum (Linaris, Wertheim-Bettingen, Germany) was heat-inactivated before use LPS from Salmonella enterica serovar Friedenau was a gift from H Brade (Research Center Borstel, Germany) Poly(I:C) and ODN1826 was received from InvivoGen (San Diego, CA, USA) Pertussis toxin, SB203580 and PD98059 were obtained from Calbiochem (San Diego, CA, USA) All FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel Journal compilation ª 2008 FEBS S Riekenberg et al CpG-DNA TLR9 RGS are modulated by lipopeptides and LPS Pam3C-SK4 TLR2/X LPS TLR4 Poly(I:C) TLR3 ELISA After stimulation, cell-free supernatants were collected and assayed for TNFa measurement using commercial ELISA (Biosource, Solingen, Germany) according to the manufacturer’s protocol RNA isolation MyD88 Response TRIF RGS1 RGS2 RGS1 RGS2 RGS1 RGS2 Early Late Fig Schematic of modulation of RGS1 and RGS2 mRNA due to the MyD88 and ⁄ or TRIF pathway after activation of different TLR lipopeptides were synthesized and characterized by EMC microcollections (Tuebingen, Germany) Cell culture J774 macrophages were cultured at 37 °C, 5% CO2 in Dulbecco’s modified Eagles medium supplemented with 10% fetal calf serum and 100 mL)1 penicillin–streptomycin Bone marrow-derived macrophages of C57N BL ⁄ mice were differentiated by incubation with macrophage colonystimulating factor as described elsewhere [39] All animal experiments were approved by the Ministerium fur Umwelt, ă Naturschutz und Landwirtschaft, Schleswig-Holstein (Germany) For stimulation, 2.5 · 105 cells were seeded in 48-well cell culture dishes for h and stimulated with 100 ngỈmL)1 LPS, 100 nm LP, 50 lgỈmL)1 poly(I:C) or lm ODN1826 Affymetrix gene chip analysis BMDM were stimulated with 100 nm LP for and h or 10 ngỈmL)1 LPS for h Control samples were treated only with medium and gene chip analyses were performed for each experiment Total RNA (3 lg) was processed and hybridized to mouse expression array MOE430 2.0 according to manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA) Arrays were scanned and fluorescence intensities were analyzed using affymetrix gcos software CEL files were processed for global normalization and generation of expression values using the robust multi-array analysis algorithm implemented in the R-affy package (http:// www.bioconductor.org/) [40] Data from 11 oligis for each probe set were statistically analysed by s-score test Total RNA was isolated using Absolutely RNA Miniprep kit (Stratagene, Amsterdam, the Netherlands), including DNase treatment, in accordance with the manufacture’s instructions The integrity of RNA was examined by gel electrophoresis before real-time PCR analysis cDNA synthesis and real-time PCR First-strand cDNA were synthesized from lg RNA by using SuperScript III reverse transcriptase (Invitrogen) Amplification was performed in a fluorescence temperature cycler (Light Cycler 2.0 system, Roche Diagnostics, Mannheim, Germany) cDNA (20 ng) was used as template in a 10 lL reaction volume containing 0.5 lm of each primer, 1· LightCyclerÒ Fast Start DNA MasterPlus SYBR Green I mix (Roche Diagnostics) The following primers were used: muRGS1 5¢-TCTGCTAGCCCAAAGGATTC-3¢ (sense), 5¢TTCACGTCCATTCCAAAAGTC-3¢ (anti-sense); muRGS2 5¢-GAGAAAATGAAGCGGACACTCT-3¢ (sense), 5¢-TTG CCAGTTTTGGGCTTC-3¢ (antisense); muHPRT as housekeeping gene 5¢-ACTTTGCTTTCCCTGGTTA-3¢ (sense), 5¢-CAAAGTCTGGCCTGTATCC-3¢ (antisense); muTNF-a 5¢-GACCCTCACACTCAGATCATCTTC-3¢ (sense), 5¢-CC ACTTGGTTTGCTACGA-3¢ (antisense) Acknowledgements We appreciate the excellent technical assistance of Suhad Al-Badri and Franziska Daduna We thank Roland Lang and Jorg Mages (Technical University ă Munich, Institute of Medical Microbiology) for microarray analysis This work was supported by the Deutsche Forschungsgemeinschaft UL68 ⁄ 3-2 References Janeway CA & Medzhitov R (2002) Innate immune recognition Annu Rev Immunol 20, 197–216 Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, Carroll JD, Espevik T, Ingalls RR, Radolf JD et al (1999) Toll-like receptor 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RGS1 and RGS2 after activation of different TLR may modify the effects of G-protein signalling. .. et al RGS are modulated by lipopeptides and LPS Relative expression of RGS1 mRNA Fig Expression of RGS1, RGS2 and TNFa mRNA in J774 After stimulation with 100 ngỈmL)1 LPS, 100 nM FSL-1 and 100