Chemoattractant-stimulated NF- B Activation Is Dependent on The Low Molecular Weight GTPase RhoA

Chemoattractants bind to seven transmembrane-spanning, G-protein-coupled receptors on monocytes and neutrophils and induce a variety of functional responses, including activation of the transcription factor NF-kappaB. The signaling mechanisms utilized by chemoattractants to activate NF-kappaB in human peripheral blood monocytes are poorly defined. We previously demonstrated that fMet-Leu-Phe (fMLP) stimulates NF-kappaB activation, and this function of fMLP requires phosphatidylinositol 3-kinase (PI3K). Here we present evidence that fMLP activates RhoA and that fMLP-induced NF-kappaB activation requires this small GTPase. Stimulation of monocytes with fMLP rapidly activated RhoA as well as NF-kappaB, and their activation was markedly reduced by pertussis toxin treatment. Pretreatment of monocyte with a RhoA inhibitor, C3 transferase from Clostridium botulinum, effectively blocked fMLP-induced NF-kappaB activation as well as interleukin-1beta gene expression. A dominant negative form of RhoA (T19N) also inhibited fMLP-stimulated reporter gene expression in a kappaB-dependent manner. Cotransfection of the monocytic THP1 cells with a constitutively active form of RhoA (Q63L) with the promoter reporter plasmid results in a marked increase in NF-kappaB-mediated reporter gene expression. Furthermore, the PI3K inhibitors wortmannin and LY294002 block RhoA activation induced by fMLP. These results demonstrate that low molecular weight GTPase RhoA is a novel signal transducer for fMLP-induced NF-kappaB activation and Galpha(i) or Galpha(o) class of heterotrimeric G proteins likely mediate RhoA activation via PI3K in human peripheral blood monocytes.


SUMMARY
Chemoattractants bind to seven transmembrane-spanning, G-protein-coupled receptors (GPCR) on monocytes and neutrophils and induce a variety of functional responses, including activation of the transcription factor NF-κB. The signaling mechanisms utilized by chemoattractants to activate NF-κB in human peripheral blood monocytes are poorly defined. We previously demonstrated that fMLP-stimulates NF-κB activation, and this function of fMLP requires phosphatidylinositol 3-kinase (PI3K). Here we present evidence that fMLP activates RhoA, and that fMLP-induced NF-κB activation requires this small GTPase. Stimulation of monocytes with fMLP rapidly activated RhoA as well as NF-κB and their activation was markedly reduced by pertussis toxin treatment.
Pre-treatment of monocyte with a RhoA inhibitor, C3 transferase from Clostridium botulinum, effectively blocked fMLP-induced NF-κB activation as well as IL-1β gene

INTRODUCTION
Leukocytes constitute the first line of host defense against invading microorganisms and are a major cellular component of the inflammatory reaction. When exposed to chemoattractants, such as formyl peptide, leukocytes become rapidly activated.
The bacterial tripeptide fMet-Leu-Phe (fMLP) 1 is able to activate all major functions of neutrophils and is a prototypical ligand for the N-formyl peptide receptor (FPR), which contains 7 putative transmembrane domains characteristic of the G protein-coupled receptor (GPCR) of the rhodopsin family (1,2).
FMLP stimulates peripheral blood mononuclear cells (PBMC) to express a defined set of gene products, including IL-1, IL-6 and IL-8. Pretreatment of the PBMC with pertussis toxin abolishes fMLP-stimulated cytokine synthesis, suggesting that a Gαi containing heterotrimeric G protein may mediate the process (3). Several recent studies have demonstrated activation of the transcription factor NF-κB by G protein-coupled receptors (4)(5)(6)(7)(8). NF-κB is of paramount importance to immune cell function owing to its ability to activate the transcription of many proinflammatory immediate-early genes (9,10). NF-κB is a multiprotein transcription activator originally found to bind a decameric enhancer sequence in the gene for the immunoglobulin κ light chains. In leukocytes, NF-κB activation results in the transcription of immediate-early genes that encode IL-2, IL-6, IL-8, TNFα, MCP-1, GM-CSF, as well as several adhesion molecules (10). Numerous stimuli can activate NF-κB, including the bacterial components LPS, as well as other proinflammatory factors including IL-1 and TNFα.
GTPases of the Rho family exist in both GDP-bound inactive (GDP-Rho) and GTP-bound active (GTP-Rho) forms. When cells are stimulated with different ligands, GDP-Rho is converted to GTP-Rho, which binds to specific targets and then exerts its biological functions. Low molecular weight G proteins of the Rho family (consisting of Cdc42, Rac, and RhoA) have been shown to regulate actin cytoskeletons, focal adhesion complex formation, cell aggregation and cell motility (14)(15)(16). The function of these small G proteins in leukocyte cytokine gene transcription, however, has not been previously addressed. Recent reports indicate that Rho GTPases regulate c-fos transcription activation (17), that constitutively active Rho proteins can activate NF-κB, and that TNFα-induced activation of NF-κB in NIH-3T3 cells is dependent on Cdc42 and RhoA (18). Chang et al demonstrated that Rho activation is also involved in AP-1 mediated transcription in Jurkat cells (19). C. difficile toxin B, which inactivates Rho family proteins including RhoA, Rac, and Cdc42, has been reported to reduce the LPS-induced IL-8 expression in human umbilical vein endothelial cells (20). Despite an apparent role of the Rho GTPases in gene expression, relatively little is known about the molecular mechanisms of Rho signaling in gene transcription.
In this study we therefore investigated the role of Rho GTPase in the signaling events that lead to NF-κB activation in fMLP-stimulated human peripheral blood monocytes. We found that stimulation of monocytes with fMLP rapidly activated RhoA as well as NF-κB in a pertussis toxin-sensitive manner. Furthermore, inhibition of RhoA activity blocked fMLP-induced NF-κB activation. These results indicate that fMLP stimulates RhoA and NF-κB activation and that the RhoA activity is required to mediate this effect in human peripheral blood monocytes. Clostridium botulinum C3 transferase were obtained as previously described (21).

Preparation of monocytes from peripheral blood Heparinized human peripheral blood
from health donors was fractionated on Percoll (Pharmacia) density gradients.
Mononuclear cells and neutrophils were initially separated by centrifugation through a 55%/74% discontinuous Percoll gradient. Monocytes were further prepared from the mononuclear cell population with gelatin/plasma coated flasks as described (7). The purity of monocytes was greater than 85-90% as determined by staining with an anti-CD14 monoclonal antibody (Coulter Immunology, Miami, FL), and cell viability was greater than 95% as measured by trypan blue exclusion. Monocytes were resuspended in RPMI-6 bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM; Irvine Scienfic, Santa Ana, CA). Electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared from human peripheral blood monocytes using a modified method of Dignam et al. (25), and EMSA were performed using 2.5 µg of the nuclear extract as described previously (7).

Chloramphenicol acetyltransferase (CAT) assay
The promoter:reporter plasmid pIκB-CAT (WT-IκB-CAT) contains a κB-like enhancer from the promoter region of the IκB gene. This enhancer was deleted in the plasmid MU-IκB-CAT. Both constructs were previously described in (21,26

FMLP-induced B binding is blocked by a RhoA inhibitor
To assess the role of Rho GTPase in fMLP-induced NF-κB activation, we examined the consequences of pre-incubating cells with a specific Rho inhibitor. The C3 transferase is an exotoxin produced by Clostridium botulinum that specifically inhibits the Rho small GTP binding proteins (Rho A, B, and C) but does not inhibit Rac or Cdc42 (28). These results suggest that RhoA is required for fMLP-but not TNFα-induced NF-κB activation. We next examined the dose-response of the inhibitory effect of C3 transferase on fMLP-induced NF-κB activation. As shown in figure 1B, C3 transferase at doses greater than or equal to 5 µg/ml significantly inhibited fMLP-induced NF-κB activation.

fMLP stimulates a rapid but transient increase in RhoA activity
The results presented above demonstrate that inhibition of RhoA activity abrogates fMLP-induced NF-κB activation. We next examined whether fMLP would induce increased RhoA activity in human peripheral blood monocytes.
At various times following stimulation, monocytes were lysed and total cell lysates incubated with GST or GST-RBD beads. Immunoblotting of the bound proteins was performed as described in Experimental Procedures. Both fMLP and other tested leukocyte chemoattractants (PAF, C3a, and C5a) stimulated increased RhoA activity ( Fig.   2A). fMLP stimulated a time-dependent increase in RhoA activity (Fig. 2B). The fMLP induced increase of RhoA activity was seen within 5 minutes of stimulation and peaked at 5-10 minutes. The kinetics of fMLP-induced RhoA activity preceded that of fMLPinduced NF-κB activation (13), consistent with a role for RhoA in the activation of NF-κB.

fMLP-induced RhoA and NF-B activation involves fMLP receptors coupled to pertussis sensitive heterotrimeric G proteins
We previously demonstrated that fMLP-induced NF-κB activation in peripheral blood mononuclear cells (PBMC) is mediated through the fMLP receptor (11), a member of the seven transmembrane G protein coupled receptor (GPCR) superfamily. The identity of the heterotrimeric G proteins coupling the fMLP receptor to RhoA activity and NF-κB activation in monocytes has not been elucidated. We therefore examined the effect of pertussis toxin and cholera toxin on fMLP-induced NF-κB activation and RhoA GTPase.

RhoA activity is required for fMLP-induced NF-B activation
Further demonstration of the necessity for RhoA activity in fMLP-induced NF-

Effects of PI3K inhibitors on fMLP-induced RhoA activation in human monocytes
We have previously demonstrated that fMLP stimulated phosphatidylinositol 3kinase (PI3K) activity and this activity is required for fMLP-induced NF-κB activity in human monocytes (13). Based on our current results and our previous report, the relationship between PI3K activation and activation of the RhoA appears to be an transduction is a pertussis toxin substrate, since treatment of neutrophils with the pertussis toxin blocks the majority of FPR-mediated responses (29,30). To assess the type of heterotrimeric G protein coupling the FPR to RhoA and NF-κB, we analyzed the effects of pertussis and cholera toxins. Pertussis toxin ADP-ribosylates Gαi and Gαo proteins, while cholera toxin ADP-ribosylates Gαs proteins (31). FMLP stimulated RhoA activity and NF-κB activation were both inhibited by pertussis toxin but not by cholera toxin, indicating that both responses are transduced through Gαi or Gαo class of heterotrimeric G proteins. The mechanism for the potentiation of fMLP induced RhoA activity by cholera toxin is currently unknown. It is possible that Gs and adenylyl cyclase might also function in the signaling mechanism governing RhoA activity in these system, but this will require further investigation.
We previously demonstrated that fMLP induced NF-κB activation required PI3K activity in human monocytes (13) and here we present evidence that RhoA activity is essential for fMLP-induced NF-κB activation. To test the possibility that PI3K is a component of the fMLP stimulated signaling pathway leading to RhoA activation, we assessed the effect of inhibiting PI3K activity on subsequent fMLP-induced RhoA activation. Wortmannin and LY294002 have been shown to be specific PI3K inhibitors.
Wortmannin irreversibly inactivates PI3K by binding to its p110 catalytic subunit (32); LY294002 is a competitive inhibitor, binding to the ATP-binding site of the PI3K (33).
A more recent study suggests that Toll-like receptor-2 (TLR2)-mediated NF-κB activation requires Rac1 and PI3K is involved as a downstream effector (41). Other studies, however, have suggested that PI3K may activate the small Rho GTPases. Expression of a constitutively active PI3K mutant in Swiss 3T3 cells induced a subset of Rac and Rhomediated cellular responses (42). The PI3K product PtdIns (3,4,5)P3 has been shown to bind the pleckstrin homology (PH) domain of guanine nucleotide exchange factors (GEF), providing a potential mechanism for PI3K-mediated regulation of Rho activation (42,43).
Additionally, the p85 regulatory subunit of PI3K contains a breakpoint cluster regionhomology domain (BH) that has been shown to have GTPase activating protein (GAP) activity (44).
In summary, we have shown that fMLP rapidly activates the RhoA GTPase in human peripheral blood monocytes. FMLP stimulated RhoA activity and NF-κB activation were both inhibited by pertussis toxin but not cholera toxin, suggesting that both FPR-mediated responses are the results of coupling to the G i /G o class of Gα proteins.
Utilizing both a specific inhibitor and transient expression of a dominant-negative RhoA mutant, we further showed that fMLP-induced NF-κB activation required RhoA activity.
We have previously reported that RhoA is also involved in NF-κB activation through the Gq-coupled B2 bradykinin receptor (21)   (A) Human monocytes were incubated for 4 h with either pertussis toxin (0.5 µg/ml) or cholera toxin (5 µg/ml) and then for 60 min with 100 nM fMLP (F) or 100 ng/ml TNFα (T), before NF-κB activation was determined as described for Fig. 1, bracket marks the DNA-protein complex. (B) Activity of RhoA, measured as described for Fig. 2  A PhosphoImager screen was exposed, and the autoradiograph is shown. These results are representative of two separate experiments.