Pertussis Toxin-insensitive Pathway Research Articles

Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7

The mGlu(7) receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu(7) monoclonal antibody, MAB1/28, that triggers receptor internalization. MAB1/28's activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [(35) S]-GTPγ binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa. mGlu(7) /mGlu(6) chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu(7) . MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require Gα(i) protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu(7) dimers. We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu(7) function and selective activation of its intracellular trafficking.

Sphingosine 1-Phosphate Triggers Apoptotic Signal for B16 Melanoma Cells via ERK and Caspase Activation

The bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), recently was reported to induce apoptosis of some cancer cells and neurons, although it generally known to exert mitogenic and antiapoptotic effects. In this study, we investigated the effects of S1P on the cell growth, melanogenesis, and apoptosis of cultured B16 mouse melanoma cells. In results, S1P was found to induce apoptosis in B16 melanoma cells in a dose- and time-dependent manner, but exerted minimal effects on melanogenesis. Although receptors of sphingosine 1-phosphate (endothelial differentiation gene 1 [Edg]/S1P1, Edg5/S1P2, Edg3/S1P3) were expressed in B16 melanoma cells, they were shown not to be associated with S1P-induced apoptosis. In addition, pertussis toxin did not block the apoptotic effects of S1P on B16 melanoma cells. S1P induced caspase-3 activation and the extracellular signal-regulated kinase (ERK) activation. Interestingly, the ERK pathway inhibitor, UO126, reversed the apoptotic effects of S1P on B16 melanoma cells. These results suggest that S1P induced apoptosis of B16 melanoma cells via an Edg receptor-independent, pertussis toxin-insensitive pathway, and appears to be associated with the ERK and caspase-3 activation.

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y2 receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y2 receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca2+ mobilization indicating that nucleotides cause Ca2+ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca2+ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca2+ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca2+ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca2+ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca2+ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y2 receptor can cause Ca2+ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.

Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells

Connective tissue growth factor (CTGF) is induced by transforming growth factor-β (TGF-β) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-β does.

D 3 dopamine receptor activates phospholipase D through a pertussis toxin-insensitive pathway

Within the dopamine receptor family, the D 3 dopamine receptor's function remains inadequately described. The D 3 receptor has been shown to couple to inhibition of adenylyl cyclase, stimulation of mitogenesis, and regulation of K + and Ca 2+ currents, all in a pertussis toxin (PTX)-sensitive manner. Here we report D 3 receptor activation of the phospholipase D (PLD) enzyme in HEK 293 cells heterologously expressing the human D 3 receptor. Activation by agonist is dose dependent and displays the pharmacology expected of the D 3 receptor. The D 3 receptor specific antagonists AJ-76 and U99194A ablated the increase in activity by the preferring D 3 agonist (+) 7-OH DPAT. In addition, the D 3 receptor-mediated activation of PLD is not mediated by G-proteins of the G i/G o family, as pretreatment with PTX had no effect. PLD activation is a novel finding for the D 3 receptor, and is the first example of an effector system where D 3 signals without G i/G o protein intermediates.

δ-Opioid peptide receptors activate pertussis toxin-sensitive and pertussis toxin-insensitive pathways during myocardial contraction

δ-Opioid peptide receptors activate pertussis toxin-sensitive and pertussis toxin-insensitive pathways during myocardial contraction

Two Novel Xenopus Homologs of Mammalian LPA1/EDG-2 Function as Lysophosphatidic Acid Receptors inXenopus Oocytes and Mammalian Cells

Lysophosphatidic acid (LPA) induces diverse biological responses in many types of cells and tissues by activating its specific G protein-coupled receptors (GPCRs). Previously, three cognate LPA GPCRs (LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7) were identified in mammals. By contrast, an unrelated GPCR, PSP24, was reported to be a high affinity LPA receptor in Xenopus laevis oocytes, raising the possibility that Xenopus uses a very different form of LPA signaling. Toward addressing this issue, we report two novel Xenopus genes, xlp(A1)-1 and xlp(A1)-2, encoding LP(A1) homologs (approximately 90% amino acid sequence identity with mammalian LP(A1)). Both xlp(A1)-1 and xlp(A1)-2 are expressed in oocytes and the nervous system. Overexpression of either gene in oocytes potentiated LPA-induced oscillatory chloride ion currents through a pertussis toxin-insensitive pathway. Injection of antisense oligonucleotides designed to inhibit xlp(A1)-1 and xlp(A1)-2 expression in oocytes eliminated their endogenous response to LPA. Furthermore, retrovirus-mediated heterologous expression of xlp(A1)-1 or xlp(A1)-2 in B103 rat neuroblastoma cells that are unresponsive to LPA conferred LPA-induced cell rounding and adenylyl cyclase inhibition. These results indicate that XLP(A1)-1 and XLP(A1)-2 are functional Xenopus LPA receptors and demonstrate the evolutionary conservation of LPA signaling over a range of vertebrate phylogeny.

Novel Inhibition of Gβγ-activated Potassium Currents Induced by M2 Muscarinic Receptors via a Pertussis Toxin-insensitive Pathway

G(i) protein-coupled receptors such as the M(2) muscarinic acetylcholine receptor (mAChR) and A(1) adenosine receptor have been shown to activate G protein-activated inwardly rectifying K(+) channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M(2) receptors not only activate but also reversibly inhibit these K(+) currents when stimulated with agonist for up to 2 min. The M(2) mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5'-O-(thiotriphosphate) in the pipette. Under these conditions the M(2) mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gbetagamma, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gbetagamma subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M(2) mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M(2) mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M(4) mAChR and alpha(1A) adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A(1) and A(3) adenosine receptors and alpha(2A) and alpha(2C) adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M(2) and M(4) mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gbetagamma-activated GIRK currents in a membrane-delimited fashion.

NF-κB Activation Is Required for C5a-Induced Interleukin-8 Gene Expression in Mononuclear Cells

C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of IL-8 mRNA in the cytosol and by nuclear expression of a kappaB DNA binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less important role in the rapid production of IL-8. The correlation between C5a-induced kappaB binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the kappaB sequence from IkappaBalpha and IL-8 promoter regions. C5a-induced reporter gene expression was abolished by introducing mutations into the kappaB sites and by coexpression of a dominant negative IkappaBalpha construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the Gi proteins known to couple to the C5a receptor, produced minimal inhibition of C5a-induced IL-8 expression and had little effect on C5a-induced calcium mobilization in RAW264.7 cells. These results suggest that NF-kappaB activation is required for C5a-induced IL-8 gene expression and that this response is mediated primarily through a pertussis toxin-insensitive pathway.

Endogenous serotonin-2A and -2C receptors in Balb/c-3T3 cells revealed in serotonin-free medium: Desensitization and down-regulation by serotonin

We studied the endogenous expression of the serotonin-2A (5-hydroxytryptamine 2A, 5-HT 2A) 5-HT 2C, and a splice-variant of the 5-HT 2C receptor in murine Balb/c-3T3 fibroblast cells that is revealed when these cells are maintained in medium containing 5-HT-free serum. RNA editing of the 5-HT 2C receptor was exclusively at a single brain-specific site. Addition of 5-HT ( ec 50 = 23 ± 2.9 nM) induced an immediate release of calcium from an ionomycin-sensitive intracellular store by coupling to a pertussis toxin-insensitive pathway. The 5-HT-induced calcium mobilization displayed a 5-HT- 2-like pharmacology, and ligand binding analyses indicated the presence of specific binding sites (27.5 ± 2 fmol/mg protein) with a 5-HT 2A-like pharmacology. Although the 5-HT 2A receptor site was predominant, the smaller component of 5-HT 2C receptors alone was sufficient to mediate a maximal calcium response. The 5-HT-induced increase in [Ca 2+] i was reversibly inhibited by >75% following a 12-hr pretreatment (T 1/2 = 2 hr) with 5-HT ( ec 50 = 400 nM). Extended treatment (24–96 hr) with 5-HT induced a complete functional desensitization that was associated with a partial (60%) reduction in 5-HT 2 receptor number, implicating both receptor down-regulation and post-receptor mechanisms in 5-HT-induced desensitization. Long-term (hours to days) treatment with 5-HT did not modulate DNA synthesis, cell proliferation, or transformation in Balb/c-3T3 cells. These results demonstrate that Balb/c-3T3 cells express endogenous 5-HT 2 receptors that are desensitized by the 5-HT present in normal serum, illustrating the importance of growth conditions in the identification of receptor responsiveness. The lack of proliferative response to 5-HT in Balb/c-3T3 suggests a putative role of desensitization as a “safety valve” to prevent abnormal cell growth during sustained 5-HT 2 receptor activation.

Identification of an endogenous 5-hydroxytryptamine2A receptor in NIH-3T3 cells: agonist-induced down-regulation involves decreases in receptor RNA and number.

NIH-3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5-hydroxytryptamine (5-HT) when cultured in 5-HT-free medium, became responsive to 5-HT, which induced an increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH-3T3 cells endogenously express a 5-HT2A receptor that, when activated, mobilizes calcium from ionomycin-sensitive intracellular stores via coupling to a pertussis toxin-insensitive pathway. Using reverse transcriptase-PCR cloning and northern blot analysis, the presence of 5-HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH-3T3 cells. Responsiveness of the endogenous 5-HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half-time = 2 h) with 1 microM 5-HT and resensitized on removal of 5-HT. In contrast to NIH-3T3 cells transfected with 5-HT2A receptor cDNA under control of a viral promoter, the long-term agonist-induced functional desensitization in nontransfected NIH-3T3 cells was paralleled by a decrease in both 5-HT2A receptor density and RNA level. These results show that NIH-3T3 cells express an endogenous 5-HT2A receptor that is desensitized by agonist via down-regulation of both receptor number and mRNA. The NIH-3T3 cells provide a novel system for understanding 5-HT2A receptor regulation.

G-Protein Modulation of Neuronal Class E (α1E) Calcium Channel Expressed in GH3Cells

GH3cell lines stably expressing α1Echannel were established and the modulation of this channel by G-protein through membrane-delimited pathways was studied. α1Echannel expressed in GH3cells showed slowing of activation and reduction of current amplitude by the application of carbachol or somatostatin. Both of these effects caused by these agents were pertussis toxin (PTX) sensitive and voltage dependent. Dialysis of the cell interior with GTPγS mimicked the action of these externally applied neurotransmitters, indicating that the α1Echannel is modulated by the PTX sensitive G-protein(s) through the membrane-delimited pathway but not by the PTX insensitive pathway that has been observed in α1Achannel expressed in GH3cells. Thus different types of neuronal Ca2+channels can be modulated not only by a similar mechanism but also by a different mechanism conferring a multilateral regulation of Ca2+entry through these channels.

G-Protein Modulation of α1A (P/Q)-Type Calcium Channel Expressed in GH3 Cells

GH(3) cell lines stably expressing α1A channel were established and the modulation of this channel by G-protein through membrane-delimited pathways were studied. Wild type GH(3) cells were found to express ω-conotoxin MVIIC (MVIIC) sensitive Ca2+ current but this component was different from the α1A channel because of its susceptibility to G-protein modulation, suggesting MVIIC also blocks channels other than P/Q type. α1A channel expressed in GH3 cells showed slowing of activation and reduction of current amplitude by the application of carbachol. Both of these effects were pertussis toxin (PTX) sensitive and voltage dependent. α1A channels were also found to be modulated through a PTX insensitive pathway, the modulations observed were similar to those in the PTX sensitive pathway. The results further suggest that these two effects are governed by a different mechanism in both PTX sensitive and insensitive pathways.

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: Subtype-specific coupling to ion channels

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

Molecular cloning, sequencing, and functional expression of a cDNA encoding the human V1a vasopressin receptor.

Vasopressin (AVP), the antidiuretic hormone, is a cyclic nonapeptide that acts through binding to G protein-coupled specific membrane receptors pharmacologically divided into three subtypes (V1a, V1b, and V2) linked to distinct second messengers. Within the family of human AVP receptors, the V2 AVP receptor has been cloned, but the structure of the human V1a and V1b AVP receptors remains unknown. We report here the structure and functional expression of a human V1a AVP receptor complementary DNA isolated from human liver cDNA libraries. Cloning and sequencing of a full-length clone isolated a 1472-nucleotide sequence encoding a 418-amino acid polypeptide with seven putative transmembrane domains typical of G protein-coupled receptors. Amino acid sequence identity with the rat liver V1a AVP receptor, the human and rat V2 AVP receptors, and the human oxytocin receptor was 72, 36, 37, and 45%, respectively. Functional characterization of the cloned receptor was done by transient expression in COS-7 cells and stable expression in Chinese hamster ovary cells. Localization of the expressed receptor at the cellular surface was illustrated by using the fluorescent linear analog phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 coupled to fluorescein-avidin by dodecabiotin. Competition binding experiments with phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-[125I]Tyr-NH2 and AVP analogs revealed high affinity specific binding sites of the V1a subtype. Saturation binding experiments with [3H]AVP confirmed the presence of a single class of high affinity binding sites. Measurement of AVP-induced inositol phosphate production and calcium mobilization confirmed that the expressed V1a AVP receptor is coupled to phospholipase C via a pertussis toxin-insensitive pathway. Thus, the human V1a AVP receptor belongs to the superfamily of seven-transmembrane segment receptors with a significant sequence identity with the other members of the AVP-oxytocin family of receptors.

G 12 and G 13 α-Subunits Are Immunochemically Detectable in Most Membranes of Various Mammalian Cells and Tissues

The cDNAs of two putatively pertussis toxin-insensitive G-protein α-subunits, α 12 and α 13 were recently cloned. mRNA analyses based on the reverse transcriptase polymerase chain reaction indicated a widespread distribution of both mRNAs [Strathmann, M. P., and Simon, M. 1. (1991) Proc. Nati. Acad. Sci. USA 88, 5582-5586]. Generating specific antibodies directed against internal and C-terminal peptide sequences, we identified α 12 protein in all and α 13 protein in most tissues and cell lines tested. No species differences were observed, indicating a high degree of identity between mammalian species. Strong immunoreactive signals of both proteins were obtained in neuronal cell membranes of various species. Our results support the hypothesis that G 12 and G 13 are involved in pertussis toxin-insensitive pathways of signal transduction common to most tissues.

Tissue-type plasminogen activator is a potent mitogen for human aortic smooth muscle cells.

Tissue-type plasminogen activator (t-PA) is a potent and efficacious mitogen for growth-arrested cultured human aortic smooth muscle cells, stimulating an increase in cell number at 0.3-30 nM concentration. Double-chain t-PA is as efficient as single-chain t-PA in stimulating smooth muscle cell mitogenesis, whereas single-chain urokinase-type plasminogen activator (u-PA) or u-PA and plasmin or plasminogen are ineffective. Plasminogen activator inhibitor-1, Pefabloc-TPA, diisopropyl fluorophosphate or alpha 1-anti-trypsin inhibit the mitogenic effect of t-PA for smooth muscle cells in a dose-dependent manner, showing that it is dependent on the enzymatic activity. t-PA activated phosphoinositide turnover in smooth muscle cells through a pertussis toxin-insensitive pathway and stimulated proto-oncogene c-fos and c-jun mRNA levels. These findings indicate that t-PA stimulates vascular human smooth muscle cell proliferation and suggest for the first time that it may contribute to intimal smooth muscle cell proliferation after vascular injury as a result of angioplasty or vascular compromise during atherogenesis.

Comparison of actin changes and calcium metabolism in plastic- and fibronectin-adherent human neutrophils.

Abstract Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.

Binding of IgG containing immune complexes to human neutrophil Fc gamma RII and Fc gamma RIII induces actin polymerization by a pertussis toxin-insensitive transduction pathway.

The ability of a phagocytic stimulus, rabbit IgG anti-BSA/BSA immune complexes, to increase the F-actin content of human polymorphonuclear leukocytes was quantitated by flow cytometry following staining with nitrobenzoxadiazole-phallacidin. A significant rise in F-actin assembly was induced by addition of 5 micrograms/ml immune complex. Concentrations of immune complex of more than 200 micrograms/ml caused a maximal (approximately twofold) increase in F-actin content. After a delay of 5 s, the F-actin levels rose and reached maximum levels by 60 s after adding immune complexes. The twofold elevation in F-actin persisted for up to 60 min. Both anti-Fc gamma RII and anti-Fc gamma RIII mAb blocked immune complex stimulated actin polymerization. Exposure to pertussis toxin failed to affect the rate or extent of immune complex-induced actin polymerization. Cells incubated with immune complexes and then lysed with Triton had an increased number of sites able to nucleate actin polymerization. These findings suggest that immune complex binding to both polymorphonuclear leukocytes Fc gamma RII and Fc gamma RIII is required for actin filament assembly and that the induction of assembly occurs via transduction pathways that differ from those used by chemoattractants. As with adhesion this phagocytic stimulus induces actin assembly by a pertussis toxin insensitive pathway and produces a rise in actin filament content that persists for prolonged periods of time.

Aluminum-Induced Mitogenesis in MC3T3-E1 Osteoblasts: Potential Mechanism Underlying Neoosteogenesis*

We recently reported that aluminum administration to beagles stimulates uncoupled bone formation in the marrow cavity which increases trabecular bone volume and generates new osseous networks within the axial skeleton. To investigate whether this osteogenic process results from direct stimulation of bone cell replication, we examined the mitogenic effects of aluminum on undifferentiated osteoblasts derived from the MC3T3-E1 clonal cell line. Addition of AlCl3 (1-50 microM) to serum-free culture medium of quiescent osteoblasts resulted in a dose-dependent increase in [3H]thymidine incorporation into DNA and a concordant increase in cell number to 48% of the density achieved at the maximum replicative rate induced by fetal bovine serum (FBS). The time course of aluminum-induced mitogenesis was similar to that of FBS, with onset of DNA synthesis detectable by 12 h and progressive increases in replicative rates observed over a 48-h study period. Moreover, maximal stimulation of DNA synthesis by AlCl3 and that by FBS were not additive, whereas aluminum exerted additional effects on cell replication when combined with low FBS concentrations. Analysis of cell cycle kinetics indicated that aluminum, analogous to FBS, influences the osteoblast replicative activity by inducing transition from the G0 to the S phase of the cell cycle. In addition, exposure of cells to aluminum resulted in rapid accumulation of c-fos mRNA by 30 min, indicating that aluminum, like fetal bovine serum, induced expression of growth-regulating genes. Deferoxamine mesylate, a chelator of aluminum, blocked the replicative actions of aluminum in a dose-dependent fashion. In contrast, pertussis toxin, a specific inhibitor of certain G-proteins, had no effect on the mitogenic effects of aluminum, indicating that aluminum-induced mitogenesis occurs by a pertussis toxin-insensitive pathway. Though the particular cellular pathway remains to be defined, these data provide initial evidence that aluminum-induced neoosteogenesis may depend upon direct stimulation of osteoblast replication.