[Exploring the detection of MYD88 mutation in patients with Waldenström macroglobulinemia by different methods and specimens].

Objective: To improve the positivity rate and accuracy of MYD88 mutation detection in patients with Waldenström macroglobulinemia (WM) . Methods: MYD88 mutation status was retrospectively evaluated in 66 patients diagnosed with WM in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from June 2017 to June 2021. The positivity rate and accuracy of the different methods and specimens for MYD88 mutation detection were analyzed. Results: MYD88 mutations were detected in 51 of 66 patients with WM, with an overall positivity rate of 77%. The positivity rate of the next-generation sequencing (NGS) or allele-specific polymerase chain reaction (AS-PCR) was significantly higher than that of the first-generation Sanger sequencing (84% vs 71% vs 46%, P<0.05) . For the different specimens, the positivity rate for the lymph nodes or bone marrow was significantly higher than that of peripheral blood (79% vs 84% vs 52%, P<0.05) . The positivity rate of the MYD88 mutation in the lymph nodes, bone marrow, and peripheral blood determined by NGS was 86%, 90%, and 67%, respectively. The positivity rate in the lymph nodes, bone marrow, and peripheral blood detected by AS-PCR was 78%, 81%, and 53%, respectively. Thirty-nine patients with WM underwent ≥ 2 MYD88 mutation detections. The final MYD88 mutational status for each patient was used as the standard to determine the accuracy of the different methods and in different specimens. The accuracy of MYD88 mutation detection in the lymph nodes (n=18) and bone marrow (n=13) by NGS was significantly higher than that in the peripheral blood (n=4) (100% vs 100% vs 75%, P<0.05) . There was no statistically significant difference in the accuracy of MYD88 mutation detection by AS-PCR in the lymph nodes (n=15) , bone marrow (n=11) , or peripheral blood (n=16) (93% vs 91% vs 88%, P>0.05) . Conclusions: In the detection of the MYD88 mutation in patients diagnosed with WM, NGS or AS-PCR is more sensitive than Sanger sequencing. Lymph nodes and bone marrow specimens are better than peripheral blood specimens.

Proteinase-activated receptor 2 expression in breast cancer and its role in breast cancer cell migration.

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor that is activated by trypsin-like proteinases. PAR2 is detected in breast tumor specimens; however, it is not clear how PAR2 level in breast cancer cell/tissues compares with normal cell/tissues. Here, we show the elevation of PAR2 protein level in 76 of 105 breast tumor specimens but only 5 of 24 normal breast tissues. PAR2 level is also higher in breast cancer cell lines than that in normal breast cells and non-cancerous breast cell lines. To determine the role of PAR2 in breast carcinogenesis, we examined the effect of PAR2 agonists on cell proliferation and migration. Our studies show that PAR2 agonists (PAR2-activating peptide and trypsin) are neither potent growth enhancers nor chemoattractants to breast cancer cells. Instead, PAR2 agonists induce significant chemokinesis. PAR2-mediated chemokinesis is G(alphai)-dependent, and inhibiting Src kinase activity or silencing c-Src expression blocks PAR2-mediated chemokinesis. These results suggest that c-Src works downstream of G(alphai) to mediate this PAR2 agonist-induced event. To characterize c-Src effector, we reveal that PAR2 agonists activate JNKs in a Src-dependent manner and that JNK activity is essential for PAR2-mediated chemokinesis. Moreover, PAR2 agonist stimulation leads to paxillin Ser(178) phosphorylation and paxillin(S178A) mutant inhibits PAR2-mediated chemokinesis. In conclusion, our studies show that PAR2 agonists facilitate breast cancer cell chemokinesis through the G(alphai)-c-Src-JNK-paxillin signaling pathway.

Protein kinase C alpha-CARMA3 signaling axis links Ras to NF-kappa B for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells.

We reported previously that a signaling pathway consisting of G(i)-Ras-NF-kappaB mediates lysophosphatidic acid (LPA)-induced urokinase plasminogen activator (uPA) upregulation in ovarian cancer cells. However, it is not clear what signaling components link Ras to nuclear factor (NF)-kappaB for this LPA-induced event. In the present study, we found that treatment of protein kinase C (PKC) inhibitors including conventional PKC (cPKC) inhibitor Gö6976 abolished LPA-induced uPA upregulation in ovarian cancer cell lines tested, indicating the importance of cPKC activity in this LPA-induced event. Indeed, LPA stimulation led to the activation of PKCalpha and Ras-PKCalpha interaction. Although constitutively active mutants of PKCalpha (a cPKC), PKCtheta (a novel PKC (nPKC)) and PKCzeta (an atypical PKC (aPKC)) were all able to activate NF-kappaB and upregulate uPA expression, only dominant-negative PKCalpha mutant attenuated LPA-induced NF-kappaB activation and uPA upregulation. These results suggest that PKCalpha, rather than PKC isoforms in other PKC classes, participates in LPA-induced NF-kappaB activation and uPA upregulation in ovarian cancer cells. To determine the signaling components downstream of PKCalpha mediating LPA-induced uPA upregulation, we showed that forced expression of dominant-negative CARMA3 or silencing CARMA3, Bcl10 and MALT1 with specific siRNAs diminished these LPA-induced events. Furthermore, we demonstrated that PKCalpha/CARMA3 signaling axis is important in LPA-induced ovarian cancer cell in vitro invasion.

The G12/13-RhoA signaling pathway contributes to efficient lysophosphatidic acid-stimulated cell migration.

The membrane redistribution and phosphorylation of focal adhesion kinase (FAK) have been reported to be important for cell migration. We previously showed that Lysophosphatidic acid (LPA) induced FAK membrane redistribution and autophosphorylation in ovarian cancer SK-OV3 cells and the signaling pathway consisting of Gi-Ras-MEKK1 mediated LPA-induced FAK membrane redistribution but not FAK autophosphorylation. We also showed that the disruption of the Gi-Ras-MEKK1 pathway led to a significant reduction in LPA-stimulated cell migration. These findings raised the question of whether LPA-induced FAK autophosphorylation was required for LPA-stimulated cell migration and what signaling mechanism was involved in LPA-induced FAK autophosphorylation. In this study, we expressed the membrane anchored wild-type FAK (CD2-FAK) in SK-OV3 cells and found that the expression of CD2-FAK greatly rescued LPA-stimulated cell migration in Gi or Ras-inhibited cells. However, Gi inhibitor pertussis toxin or dominant-negative H-Ras still significantly inhibited LPA-stimulated cell migration in cells expressing the membrane anchored FAK containing a mutation in the autophosphorylation site [CD2-FAK(Y397A)]. These results suggest that FAK autophosphorylation plays a role in LPA-stimulated cell migration. With the aid of p115RhoGEF-RGS, G12 and G13 minigenes to inhibit G12/13, we found that the G12/13 pathway was required for LPA-induced FAK autophosphorylation and efficient cell migration. Moreover, LPA activated RhoA and Rho kinase (ROCK) in a G12/13-dependent manner and their activities were required for LPA-induced FAK autophosphorylation. However, Rho or ROCK inhibitors displayed no effect on LPA-induced FAK membrane redistribution although they abolished LPA-induced cytoskeleton reorganization. Our studies show that the G12/13-RhoA-ROCK signaling pathway mediates LPA-induced FAK autophosphorylation and contributes to LPA-stimulated cell migration.

Biological significance of MAPK, AKT and JAK-STAT protein activation by various erythropoietic factors in normal human early erythroid cells.

The aim of this study was to identify signal transduction pathways activated by erythropoietin (EpO) and erythropoietin co-stimulatory factors (kit ligand), insulin-like growth factor, thrombopoietin, interleukin 3 and granulocyte-macrophage colony-stimulating factor) in normal human bone marrow CD34(+) cells and d 11 erythroid burst forming unit derived glycophorin+ cells. The activation of these signal transduction pathways was further correlated with various biological effects such as (i) cell proliferation, (ii) inhibition of apoptosis, (iii) activation of adhesion and (iv) secretion of the matrix metalloproteinases (MMPs) MMP-9 and MMP-2, and vascular endothelial growth factor (VEGF). We found that in human CD34(+) cells and erythroblasts erythropoietic factors may activate similar but different signalling pathways, and that activation of each of the JAK-STAT, MAPK p42/44 or PI-3K-AKT axes alone is not sufficient either to stimulate cell proliferation or inhibit apoptosis, suggesting that these processes are regulated by orchestrated activation of multiple signalling cascades. Accordingly, we found that although cell proliferation was more related to simultaneous activation of JAK-STAT and MAPK p42/44, the effect on cell survival correlated with activation of PI-3K-AKT, MAPK p42/44 and JAK-STAT proteins. We also demonstrated that differentiating normal human erythroid cells lose their adhesive properties and secrete angiopoietic factors such as MMP-9, MMP-2 and VEGF, and we postulate that this secretion by early erythroid cells may play a role in their maturation and egress from the haematopoietic niches of the bone marrow.

Two Listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16.

Two recombinant Listeria monocytogenes (rLm) strains were produced that secrete the human papilloma virus-16 (HPV-16) E7 protein expressed in HPV-16-associated cervical cancer cells. One, Lm-E7, expresses and secretes E7 protein, whereas a second, Lm-LLO-E7, secretes E7 as a fusion protein joined to a nonhemolytic listeriolysin O (LLO). Lm-LLO-E7, but not Lm-E7, induces the regression of the E7-expressing tumor, TC-1, established in syngeneic C57BL/6 mice. Both recombinant E7-expressing rLm vaccines induce measurable anti-E7 CTL responses that stain positively for H-2D(b) E7 tetramers. Depletion of the CD8+ T cell subset before treatment abrogates the ability of Lm-LLO-E7 to impact on tumor growth. In addition, the rLm strains induce markedly different CD4+ T cell subsets. Depletion of the CD4+ T cell subset considerably reduces the ability of Lm-LLO-E7 to eliminate established TC-1 tumors. Surprisingly, the reverse is the case for Lm-E7, which becomes an effective anti-tumor immunotherapeutic in mice lacking this T cell subset. Ab-mediated depletion of TGF-beta and CD25+ cells improves the effectiveness of Lm-E7 treatment, suggesting that TGF-beta and CD25+ cells are in part responsible for this suppressive response. CD4+ T cells from mice immunized with Lm-E7 are capable of suppressing the ability of Lm-LLO-E7 to induce the regression of TC-1 when transferred to tumor-bearing mice. These studies demonstrate the complexity of L. monocytogenes-mediated tumor immunotherapy targeting the human tumor Ag, HPV-16 E7.

Alpha(v) integrin, p38 mitogen-activated protein kinase, and urokinase plasminogen activator are functionally linked in invasive breast cancer cells.

We reported previously that endogenous p38 MAPK activity is elevated in invasive breast cancer cells and that constitutive p38 MAPK activity is important for overproduction of uPA in these cells (Huang, S., New, L., Pan, Z., Han, J., and Nemerow, G. R. (2000) J. Biol. Chem. 275, 12266-12272). However, it is unclear how elevated endogenous p38 MAPK activity is maintained in invasive breast cancer cells. In the present study, we found that blocking alpha(v) integrin functionality with a function-blocking monoclonal antibody or down-regulating alpha(v) integrin expression with alpha(v)-specific antisense oligonucleotides significantly decreased the levels of active p38 MAPK and inhibited cell-associated uPA expression in invasive breast cancer MDA-MB-231 cells. These results suggest a function link between alpha(v) integrin, p38 MAPK activity, and uPA expression in invasive tumor cells. We also found that vitronectin/alpha(v) integrin ligation specifically induced p38 MAPK activation and uPA up-regulation in invasive MDA-MB-231 cells but not in non-invasive MCF7 cells. Finally, using a panel of melanoma cells, we demonstrated that the cytoplasmic tail of alpha(v) integrin subunit is required for alpha(v) integrin ligation-induced p38 MAPK activation.

Chemoattractant-stimulated NF-kappaB 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.

Regression of established human papillomavirus type 16 (HPV-16) immortalized tumors in vivo by vaccinia viruses expressing different forms of HPV-16 E7 correlates with enhanced CD8(+) T-cell responses that home to the tumor site.

Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-D(b)-specific tetramer-positive CD8(+) T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8(+) T cells but may also increase the number of antigen-specific CD8(+) T cells in the tumor, the principle site of antigen expression.

Therapeutic potential of protein and adjuvant vaccinations on tumour growth.

Over 90% of cervical cancers are associated with HPV infection, the commonest being the HPV-16 subtype. Two early viral genes, E6 and 7, play major roles in the development and maintenance of the malignant phenotype. The vaccine potential of a recombinant HPV16 E7 protein was examined in two murine models of E7-expressing tumours. Formulations including the immunostimulants MPL and QS21 induced therapeutically active immune responses leading to regression of pre-established TC1 tumour lesions, associated with induction of IgG antibodies, lymphoproliferation and CTL. Our data provide a clear incentive to investigate the clinical application of this approach in cancer immunotherapy.

The tumor recall response of antitumor immunity primed by a live, recombinant Listeria monocytogenes vaccine comprises multiple effector mechanisms.

Listeria monocytogenes, a facultative intracellular bacterium, can induce a potent antitumor immune response if engineered to express a model tumor antigen also expressed by the tumor cells. The effectiveness of this approach is dependent on L. monocytogenes-induced tumor-specific CD4(+) and CD8(+) T-cells. CD8(+) T-cells may mediate tumor eradication largely through direct CTL activity, but the role of CD4(+) T-cells and other cells of the immune system is less clear. Here we investigate their role and the role of the cytokines they produce in the ability of L. monocytogenes-induced antitumor immunity to protect against tumor challenge. Our results suggest that a complex cytokine response, involving type 2 as well as type 1 cytokines, is responsible for the ability of Lm-NP-immunized mice to resist tumor challenge, potentially mediating tumor cell killing through multiple effector pathways.

Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis.

The role of the chemokine binding stromal-derived factor 1 (SDF-1) in normal human megakaryopoiesis at the cellular and molecular levels and its comparison with that of thrombopoietin (TPO) have not been determined. In this study it was found that SDF-1, unlike TPO, does not stimulate alpha(IIb)beta(3)(+) cell proliferation or differentiation or have an antiapoptotic effect. However, it does induce chemotaxis, trans-Matrigel migration, and secretion of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) by these cells, and both SDF-1 and TPO increase the adhesion of alpha(IIb)beta(3)(+) cells to fibrinogen and vitronectin. Investigating the intracellular signaling pathways induced by SDF-1 and TPO revealed some overlapping patterns of protein phosphorylation/activation (mitogen-activated protein kinase [MAPK] p42/44, MAPK p38, and AKT [protein kinase B]) and some that were distinct for TPO (eg, JAK-STAT) and for SDF-1 (eg, NF-kappa B). It was also found that though inhibition of phosphatidyl-inositol 3-kinase (PI-3K) by LY294002 in alpha(IIb)beta(3)(+) cells induced apoptosis and inhibited chemotaxis adhesion and the secretion of MMP-9 and VEGF, the inhibition of MAPK p42/44 (by the MEK inhibitor U0126) had no effect on the survival, proliferation, and migration of these cells. Hence, it is suggested that the proliferative effect of TPO is more related to activation of the JAK-STAT pathway (unique to TPO), and the PI-3K-AKT axis is differentially involved in TPO- and SDF-1-dependent signaling. Accordingly, PI-3K is involved in TPO-mediated inhibition of apoptosis, TPO- and SDF-1-regulated adhesion to fibrinogen and vitronectin, and SDF-1-mediated migration. This study expands the understanding of the role of SDF-1 and TPO in normal human megakaryopoiesis and indicates the molecular basis of the observed differences in cellular responses. (Blood. 2000;96:4142-4151)

fMet-Leu-Phe stimulates proinflammatory cytokine gene expression in human peripheral blood monocytes: the role of phosphatidylinositol 3-kinase.

The fMLP-stimulated release of proinflammatory cytokines such as IL-1 by human peripheral blood monocytes is an important component of the inflammatory process. The signaling mechanisms used by fMLP to stimulate the release of cytokines are still incompletely understood. We previously demonstrated that fMLP-stimulated NF-kappaB activation in PBMC and now we present evidence that the lipid products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for fMLP-stimulated activation of NF-kappaB. Pretreatment with the PI 3-kinase inhibitors, wortmannin and LY294002, effectively blocked fMLP-induced IL-1beta gene expression as well as NF-kappaB activation. Transient transfection of THP1 cells with a dominant-negative mutant of the PI 3-kinase p85 subunit also abrogated fMLP-induced kappaB activity. These results suggest a potential role of fMLP in the transcription of proinflammatory cytokines and provide the first evidence that such regulation may occur through PI 3-kinase activity.

Regression of established B16F10 melanoma with a recombinant Listeria monocytogenes vaccine.

We have previously shown that Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting tumor-specific antigens to the immune system. In the present study, we extend these studies to the highly tumorigenic mouse melanoma B16F10, transduced with a model tumor antigen. We are able to induce the regression of primary tumors and established lung metastases by parenteral immunization with a L. monocytogenes recombinant that expresses the same antigen. Adjunctive therapy with granulocyte macrophage colony-stimulating factor or a vaccinia-based vaccine does not result in an improved cure rate over the L. monocytogenes vaccine alone. Tumor regression is accompanied by the expression of inflammatory cytokines in the tumor.

Requirement of phosphatidylinositol 3-kinase activity for bradykinin stimulation of NF-kappaB activation in cultured human epithelial cells.

The signaling mechanisms utilized by bradykinin (BK) to activate the transcription factor nuclear factor kappaB (NF-kappaB) are poorly defined. We previously demonstrated that BK-stimulated NF-kappaB activation requires the small GTPase RhoA. We present evidence that BK-induced NF-kappaB activation both activates and requires phosphatidylinositol 3-kinase (PI 3-kinase) in A549 human epithelial cells. Pre-treatment with the PI 3-kinase-specific inhibitors, wortmannin, and LY294002 effectively blocked BK-induced PI 3-kinase activity. Wortmannin and LY294002 also abolished BK-induced NF-kappaB activation, as did transient transfection with a dominant negative mutant of the p85 subunit. BK-stimulated PI 3-kinase activity and NF-kappaB activation were sensitive to pertussis but not cholera toxin, suggesting that the B2 BK receptors transducing the response were coupled to Galphai or Galphao heterotrimeric G proteins. Tumor necrosis factor alpha (TNFalpha) also stimulated increased PI 3-kinase activity, however TNFalpha-stimulated NF-kappaB activation was not affected by the PI 3-kinase inhibitors or the p85 dominant negative mutant. These findings provide evidence that BK-induced NF-kappaB activation utilizes a signaling pathway that requires activity of both RhoA and PI 3-kinase and is distinct from the signaling pathway utilized by TNFalpha. Furthermore, we show that the p85 regulatory subunit is required for activation of PI 3-kinase activity by this G protein-coupled receptor.

Anaphylatoxins C5a and C3a induce nuclear factor kappaB activation in human peripheral blood monocytes.

The anaphylatoxins C5a and C3a are involved in the regulation of cytokine production. In this study the capability of C5a and C3a to induce transcription factor activation was examined. C5a and C3a stimulation of human peripheral blood monocytes resulted in nuclear expression of a DNA binding activity with specificity to the kappaB sequence. The p50 and p65 proteins, constituents of the prototypic nuclear factor kappaB, were identified as components of the DNA-protein complexes by anti-peptide antibodies in gel supershift assays. C5a induced kappaB binding activity was detected 15 min after agonist stimulation, peaked at 30-40 min, and remained detectable at 2 h. Binding to kappaB sequence was accompanied by an initial decrease and subsequent increase in the cytoplasmic IkappaBalpha levels, as detected by Western blotting using an anti-IkappaBalpha antibody. Pertussis toxin treatment markedly decreased kappaB binding activities induced by both C5a and C3a, whereas cholera toxin displayed no inhibitory effect. Neither of the two toxins affected kappaB binding activity induced by TNFalpha in the same cells. These results imply a potential role of the anaphylatoxins C5a and C3a in regulating leukocytes gene expression through G protein-coupled transcription factor activation.

Role of the Rho GTPase in bradykinin-stimulated nuclear factor-kappaB activation and IL-1beta gene expression in cultured human epithelial cells.

Recent evidence suggests a novel role of bradykinin (BK) in stimulating gene transcription. This study examined the effect of BK on nuclear factor kappaB (NF-kappaB) activation and IL-1beta synthesis in human epithelial cells. Stimulation of A549 cells and primary bronchial epithelial cells with BK rapidly activated NF-kappaB. BK also increased the level of secreted immunoreactive IL-1beta in A549 culture supernatants, an effect that was blocked by actinomycin D and the B2 BK receptor antagonist HOE-140. The role of NF-kappaB activation in BK-induced IL-1beta synthesis was demonstrated by the ability of BK to stimulate increased chloramphenicol acetyltransferase (CAT) activity in A549 cells transfected with a reporter plasmid containing three kappaB enhancers from the IL-1beta gene, while deletion of the kappaB enhancer sequences eliminated BK-stimulated CAT activity. C3 transferase exoenzyme, an inhibitor of Rho, abolished BK-induced NF-kappaB activation at 10 microg/ml and significantly inhibited BK-stimulated IL-1beta synthesis at 5 microg/ml. A dominant-negative form of RhoA (T19N) inhibited BK-stimulated reporter gene expression in a dose-dependent and kappaB-dependent manner. Cotransfection of A549 cells with an expression vector encoding a constitutively active form of RhoA (Q63L) along with the IL-1beta promoter-CAT reporter plasmid resulted in a marked increase in NF-kappaB activity compared with transfection with the IL-1beta promoter-CAT reporter plasmid alone. These results demonstrate that BK stimulates NF-kappaB activation and IL-1beta synthesis in A549 cells, and that RhoA is both necessary and sufficient to mediate this effect.

Identification of an N-formyl peptide receptor ligand binding domain by a gain-of-function approach.

Replacement of N-formyl peptide receptor (FPR) domains with those from a homologous receptor, FPR2, resulted in chimeric receptors displaying low binding affinity to fMet-Leu-Phe (fMLF). To characterize fMLF binding domain, we adopted a "gain-of-function" approach by selective replacement of non-conserved residues in the FPR2 portion of the chimeric receptors with those from the FPR. This led to the identification of 3 clusters of residues required for high-affinity fMLF binding. Introduction of 2 positively charged amino acids, Arg84 and Lys85, dramatically improved binding affinity of one chimeric receptor (Kd from 105 nM to 1.6 nM). Similarly, restoration of either Gly89/His90 or Phe102/Thr103 improved the binding affinity of another chimeric receptor from a Kd of 275 nM to a 2.3 Kd and 3.3 nM, respectively. Increased ligand binding affinity was accompanied by a gain in calcium mobilization capability, suggesting functional coupling to G proteins. These results demonstrate the presence of structural determinants in the first extracellular loop and its adjacent transmembrane domains that are essential for high affinity fMLF binding.