Osteoclasts and their circulating precursors in rheumatoid arthritis: Relationships with disease activity and bone erosions.

Patients with rheumatoid arthritis (RA) have very different outcomes, particularly with regard to bone erosions. Since osteoclasts are responsible for bone destruction adjacent to rheumatoid synovium, profiling osteoclasts from circulating precursors in RA could help identify patients at risk for bone destruction. In this study, we sought to determine whether the functional characteristics of osteoclasts generated from their blood precursors were modified by RA activity or were intrinsic to osteoclasts and associated with the RA phenotype (erosive or not). Osteoclasts were generated in vitro from peripheral blood mononuclear cells (PBMCs) of subjects with RA (n = 140), as well as sex- and age-matched healthy controls (n = 101). Osteoclastic parameters were analyzed at baseline and during the follow-up for up to 4 years, with regular assessment of RA activity, bone erosions, and bone mineral density (BMD). As a validation cohort, we examined RA patients from the Early Undifferentiated PolyArthritis (EUPA) study (n = 163). The proportion of CD14+ PBMC was higher in RA than in control subjects, but inversely correlated with the 28-joint disease activity score (DAS28). Also surprisingly, in osteoclast cultures from PBMCs, active RA was associated with lower osteoclastogenic capacity, while in vitro bone resorption per osteoclast and resistance to apoptosis were similar in both active and quiescent RA. In a small subgroup analysis, osteoclasts from subjects with recent RA that had progressed at four years to an erosive RA exhibited at baseline greater resistance to apoptosis than those from patients remaining non-erosive. Our findings establish that when RA is active, circulating monocytes have a reduced potential to generate osteoclasts from PBMCs in vitro. In addition, osteoclasts associated with erosive disease had resistance to apoptosis from the start of RA.

Mouse FLRT2 interacts with the extracellular and intracellular regions of FGFR2.

Fibroblast Growth Factor (FGF) signaling is known to be critical in mediating key developmental events during craniofacial development. Recent evidence suggests that members of the Fibronectin (F) Leucine (L) Rich (R) Transmembrane (T), FLRT, family modulate FGF signaling. FLRT2 has a highly specific pattern of expression during craniofacial development, in close relationship with FGFR2. We therefore characterized FLRT2/FGFR2 interactions in the context of craniofacial development and showed, by co-immunoprecipitation and GST pulldown assays with embryonic craniofacial tissue lysates, that FLRT2 interacted with FGFR2. Yeast two-hybrid assays further showed that the intracellular regions of both proteins interacted in addition to the interactions in the extracellular portions. The extracellular Leucine Rich Repeats domain of FLRT2 contributed to the interactions with the extracellular regions of FGFR2. Interactions in the intracellular regions of the 2 proteins were mediated by the C-tail domain in FLRT2. Furthermore, cells stably transfected with FLRT2 shRNAs or FLRT2 cDNA exhibited a concomitant decrease and increase, respectively, in FGFR2 protein, mRNA, and ERK phosphorylation levels, suggesting a positive feedback regulatory loop of FLRT2 on FGF signaling in craniofacial tissues. We propose that FLRT2-FGFR2 interactions represent a potential mechanism for regulation of FGF signaling by FLRT2 during craniofacial development.

The increased in vitro osteoclastogenesis in patients with rheumatoid arthritis is due to increased percentage of precursors and decreased apoptosis - the In Vitro Osteoclast Differentiation in Arthritis (IODA) study.

Increases in local and systemic bone resorption are hallmarks of rheumatoid arthritis (RA). Osteoclasts are implicated in these processes and their enhanced differentiation may contribute to bone destruction. We observed that in vitro osteoclastogenesis varies among healthy individuals and hypothesized that increased osteoclastogenesis could be a marker for the presence of RA. Our objective in the present study was to determine if in vitro osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) was different in patients with RA compared to healthy controls and osteoarthritis (OA) patients. Expression of CD14 in PBMCs was quantified and PBMCs were incubated for 21 days in the presence of the osteoclastogenic cytokines M-CSF and RANKL. Differentiation on cortical bone slices permitted the analysis of bone resorption while apoptotic potential was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. In vitro osteoclastogenesis was higher in PBMCs from RA patients compared to controls, and a similar increase was observed in the percentage of osteoclast precursors in RA patients. Osteoclasts from RA patients showed lower apoptotic rates than osteoclasts from healthy controls. No difference was observed in bone resorption activity between RA patients and controls. Interestingly, the difference in osteoclast number and apoptosis rate allowed the implementation of an algorithm capable of distinguishing patients with RA from controls. In conclusion, our study shows that osteoclast differentiation from PBMCs is enhanced in patients with RA, and this difference can be explained by both a higher percentage of osteoclast precursors in the blood and by the reduced apoptotic potential of mature osteoclasts.

Benzo[a]pyrene inhibits osteoclastogenesis by affecting RANKL-induced activation of NF-kappaB.

Exposure to polycyclic aryl hydrocarbons is linked to cancer, immunosuppression and other numerous health problems. We previously demonstrated that exposure to benzo[a]pyrene (BaP), an environmental pollutant present in high concentrations in urban smog and cigarette smoke, inhibits osteoclast differentiation and bone resorption. We hypothesized that this inhibition could be due to crosstalk between the receptor activator of NF-kappaB ligand (RANKL) and AhR signaling cascades competing for NF-kappaB, a common transcription factor for both pathways. RAW264.7 cells (a mouse macrophage cell line capable of differentiating into osteoclasts in the presence of RANKL) were exposed to different concentrations of RANKL and BaP and the effect on NF-kappaB activation, nuclear translocation, as well as the effect of NF-kappaB inhibitors on BaP-mediated CYP1B1 gene expression was measured. The results demonstrated that BaP inhibited both RANKL-induced NF-kappaB activation and nuclear translocation. At the same time, BaP-induced CYP1B1 gene expression was inhibited by two NF-kappaB inhibitors in a dose-dependent manner, demonstrating that NF-kappaB is involved in a BaP-mediated signaling pathway. A reporter gene assay showed that both BaP and RANKL-induced luciferase reporter gene transcription under the control of NF-kappaB response elements. Co-immunoprecipitation results demonstrated that AhR interacted with NF-kappaB p65 in RAW cells and BaP appeared to enhance this interaction. However, in the presence of RANKL, we did not observe any interaction between AhR and p65. These results support our hypothesis that BaP-mediated inhibition of osteoclastogenesis is a consequence of crosstalk between AhR and RANKL signaling pathways competing for the common transcription factor NF-kappaB.

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration.

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.

The relative contribution of cysteine proteinases and matrix metalloproteinases to the resorption process in osteoclasts derived from long bone and scapula.

It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.

A novel model system for characterization of phagosomal maturation, acidification, and intracellular collagen degradation in fibroblasts.

Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous alpha(2)beta(1) integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen degradation. There was a time-dependent increase of collagen bead internalization and a time-dependent decrease of bead-associated alpha(2)beta(1) integrin after initial bead binding. beta-Actin and gelsolin associated transiently with beads (0-30 min) followed by LAMP-2 (60-240 min) and cathepsin B (30-240 min). Cytochalasin D prevented phagosome formation and also prevented the sequential fusion of early endosomes with lysosomes. Collagen bead-associated pH was progressively reduced from 7.25 to 5.4, which was contemporaneous with progressive increases in degradation of bead-associated collagen (30-120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation. Phagosomal acidification was partly dependent on elevated intracellular calcium. These studies demonstrate that the cellular machinery required for intracellular collagen degradation in fibroblasts closely resembles the vacuolar system in macrophages.

Evidence that the NH2 terminus of vph1p, an integral subunit of the V0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector.

The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex. V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation. Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1). Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1)-sensitive ATP hydrolysis. Trypsin cleaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation.

Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast.

Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (V-ATPase) is necessary for a variety of essential eukaryotic cellular functions. Nevertheless, yeasts lacking V-ATPase activity (Deltavma) are viable when grown at low pH, suggesting alternative methods of organellar acidification. This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging. When Deltavma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis. Similarly, internalization of the plasmalemmal H(+)-ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the Deltavma phenotype and did not potentiate the vacuolar acidification. To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wild-type and the Deltavma yeasts were subjected to sudden changes in extracellular pH. In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH(4)(+), routinely added as a nitrogen source, was present. The NH(4)(+)-dependent acidification was not due to efflux of NH(3) from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH(4)(+). These findings suggest that although NH(3) can act as a cell-permeant proton scavenger, NH(4)(+) may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concentration of NH(4)(+) frequently added as a nitrogen source to yeast culture media together with effective NH(4)(+) transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.

Substrate- and inhibitor-induced conformational changes in the yeast V-ATPase provide evidence for communication between the catalytic and proton-translocating sectors.

The vacuolar-type H(+)-ATPases (V-ATPases) are composed of two distinct sectors, a catalytic complex (V(1)) involved in ATP hydrolysis and a membrane-associated complex (V(0)) mediating proton translocation across a lipid bilayer. To date, little is known about the mechanism by which these two functions are coupled. We sought to examine the impact of nucleotide and cation binding on the structure of the core components of the catalytic complex and to determine whether conformational changes within the catalytic complex impact subunits of the membrane-associated complex. Nucleotide- and cation- induced changes in the catalytic core of the V-ATPase were investigated by monitoring changes in the rate and pattern of tryptic digests. ATP.Mg-induced changes were detected in both the catalytic (Vma1p or 69 kDa) and the regulatory subunits (Vma2p or 60 kDa) of the V(1) sector. ATP alone increased the rate of trypsinization of the regulatory subunit, but did not have any effect on Vma1p. Surprisingly, ATP also had an impact on the 95-kDa subunit, a component of the V(0) sector of the V-ATPase. Although the presence of divalent cations had no impact on the V(1) sector, the rate of trypsinization of the 95-kDa subunit was greatly enhanced. The effect of divalent cations on the structure of the 95-kDa subunit was abrogated when trypsinization was performed in the absence of the catalytic sector. Addition of bafilomycin A(1), a V-ATPase inhibitor that putatively binds to the 95-kDa subunit, increased the rate of trypsinization of the catalytic subunit. These data suggest that structural alterations within the V(1) sector result in alterations within the V(0) sector and vice versa. Clearly, a structural link must exist to couple the two sectors. The 95-kDa subunit is ideally suited to fulfill this role. Hydropathy analysis suggests a bipartite structure, with the NH(2)-terminal portion predicted to lie in an aqueous environment and the C-terminal portion predicted to contain 6 transmembrane segments. Tryptic digests of sealed vacuolar vesicles and immunofluorescence studies revealed that the large hydrophilic NH(2)-terminal domain of the 95-kDa subunit is localized toward the cytosol. This region therefore is ideally positioned to interact with components of the V(1) complex, potentially functioning as the elusive link between the two sectors of the V-ATPase.

Function of the COOH-terminal domain of Vph1p in activity and assembly of the yeast V-ATPase.

We have previously shown that mutations in buried charged residues in the last two transmembrane helices of Vph1p (the 100-kDa subunit of the yeast V-ATPase) inhibit proton transport and ATPase activity (Leng, X. H., Manolson, M., Liu, Q., and Forgac, M. (1996) J. Biol. Chem. 271, 22487-22493). In this report we have further explored the function of this region of Vph1p (residues 721-840) using a combination of site-directed and random mutagenesis. Effects of mutations on stability of Vph1p, assembly of the V-ATPase complex, 9-amino-6-chloro-2-methoxyacridine quenching (as a measure of proton transport), and ATPase activity were assessed. Additional mutations were analyzed to test the importance of Glu-789 in TM7 and His-743 in TM6. Although substitution of Asp for Glu at position 789 led to a 50% decrease in 9-amino-6-chloro-2-methoxyacridine quenching, substitution of Ala at this position gave a mutant with 40% quenching relative to wild type, suggesting that a negative charge at this position is not absolutely essential for proton transport. Similarly, a positive charge is not essential at position His-743, since the H743Y and H743A mutants retain 20 and 60% of wild-type quenching, respectively. Interestingly, H743A approaches wild-type ATPase activity at elevated pH while the E789D mutant shows a slightly lower pH optimum than wild type, suggesting that these residues are in a location to influence V-ATPase activity. The low pumping activity of the double mutant (E789H/H743E) suggests that these residues do not form a simple ion pair. Random mutagenesis identified a number of additional mutations both inside the membrane (L739S and L746S) as well as external to the membrane (H729R and V803D) which also significantly inhibited proton pumping and ATPase activity. By contrast, a cluster of five mutations were identified between residues 800 and 814 in the soluble segment just COOH-terminal to TM7 which affected either assembly or stability of the V-ATPase complex. Two mutations (F809L and G814D) may also affect targeting of the 100-kDa subunit. These results suggest that this segment of Vph1p plays a crucial role in organization of the V-ATPase complex.

Chronic extracellular acidosis induces plasmalemmal vacuolar type H+ ATPase activity in osteoclasts.

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H+ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na+/H+ antiport activity. In contrast, chronic acidosis activated vacuolar H+ pumps in the osteoclasts. Although only approximately 5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6. 5 for 4-6 h were able to recover from the acute acid load by means of bafilomycin A1-sensitive proton extrusion. Conversely, the H+-selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H+ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.

Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.

The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide. After receptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport. We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact live cells. Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy. After 1 h at 37 degrees C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including alpha-mannosidase II, beta-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex. Imaging of the emission of fluorescein-labeled VT1B was used to measure intra-Golgi pH (pHG), which was calibrated in situ with ionophores. In intact Vero cells, pHG averaged 6.45 +/- 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar-type ATPases, pHG remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H+ antiport. Similarly, pHG was unaffected by acute changes in cytosolic calcium. Furthermore, pHG recovered quickly toward the basal level after departures imposed with weak bases. These findings suggest that pHG is actively regulated, despite the presence of a sizable H+ "leak" pathway. The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.

Site-directed mutagenesis of the 100-kDa subunit (Vph1p) of the yeast vacuolar (H+)-ATPase.

Vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for acidification of intracellular compartments in eukaryotic cells. V-ATPases possess a subunit of approximate molecular mass 100 kDa of unknown function that is composed of an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain. To test whether the 100-kDa subunit plays a role in proton transport, site-directed mutagenesis of the VPH1 gene, which is one of two genes that encodes this subunit in yeast, has been carried out in a strain lacking both endogenous genes. Ten charged and twelve polar residues located in the seven putative transmembrane helices in the COOH-terminal domain of the molecule were individually changed, and the effects on proton transport, ATPase activity, and assembly of the yeast V-ATPase were measured. Two mutations (R735L and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly reduced levels of the 100-kDa subunit in the vacuolar membrane, suggesting that these mutations affected stability of the 100-kDa subunit. Two mutations, D425N and K538A, in transmembrane helix 1 and at the border of transmembrane helix 3, respectively, showed reduced assembly of the V-ATPase, with the D425N mutation also reducing the activity of V-ATPase complexes that did assemble. Two mutations, H743A and K593A, in transmembrane helix 6 and at the border of transmembrane helix 4, respectively, have significantly greater effects on activity than on assembly, with proton transport and ATPase activity inhibited 40-60%. One mutation, E789Q, in transmembrane helix 7, virtually completely abolished proton transport and ATPase activity while having no effect on assembly. These results suggest that the 100-kDa subunit may be required for activity as well as assembly of the V-ATPase complex and that several charged residues in the last four putative transmembrane helices of this subunit may play a role in proton transport.

Characterization and subcellular localization of target membrane soluble NSF attachment protein receptors (t-SNAREs) in macrophages. Syntaxins 2, 3, and 4 are present on phagosomal membranes.

Phagosomes formed during ingestion of microorganisms by leukocytes undergo a rapid maturation, generating an acidic, microbicidal organelle. Maturation requires interactions with intracellular vesicles that dock and fuse preferentially with the phagosomal membrane. The basis of specificity of vesiculo-phagosomal interaction has not been elucidated. By contrast, the molecular basis of vesicular fusion in other systems is better understood. At neural synapses, vesicular docking and fusion to the plasma membrane are mediated by a protein complex including syntaxin 1. We explored whether macrophages contain syntaxins, and whether selective fusion of vesicles with the phagosome results from the accumulation of syntaxins in the phagosomal membrane. Isoform-specific Abs were utilized to demonstrate utilized to demonstrate that syntaxins 2, 3, and 4, but not syntaxin 1, are present in murine and human macrophages. Biochemical characterization demonstrated the presence of these syntaxins on microsomes, where they are integral membrane proteins. Subcellular localization using confocal immunofluorescence microscopy demonstrated that syntaxins 3 and 4 are present on the plasma membrane as well as on intracellular vesicles. Importantly, phagosomes isolated by fractionation were shown by immunoblotting to contain syntaxins 2, 3, and 4, suggesting that they may participate in phagosomal maturation. The density of the syntaxins on the phagosomal membrane was found to be comparable with that on the surface membrane. This suggests that preferential fusion of vesicles with the phagosomal membrane is not the result of segregation of the syntaxins to this organelle. Instead, local generation of second messengers in the vicinity of the phagosomal membrane may trigger focal fusion.

Interleukin-1 increases vacuolar-type H+-ATPase activity in murine peritoneal macrophages.

Maintenance of cytoplasmic pH (pHi) within a narrow physiological range is crucial to normal cellular function. This is of particular relevance to phagocytic cells within the acidic inflammatory microenvironment where the pHi tends to be acid loaded. We have previously reported that a vacuolar-type H(+)-ATPase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism within the inflammatory milieu. Since this microenvironment is frequently characterized by the influx of cells known to release inflammatory cytokines, we performed studies to examine the effect of one such mediator molecule, interleukin-1 (IL-1), on pHi regulation in peritoneal macrophages. IL-1 caused a time- and dose-dependent increase in macrophage pHi recovery from an acute acid load. This effect was specific to IL-1 and was due to enhanced plasmalemmal V-ATPase activity. The increased V-ATPase activity by IL-1 occurred following a lag period of several hours and required de novo protein and mRNA synthesis. However, Northern blot analysis revealed that IL-1 did not exert its effect via alterations in the levels of mRNA transcripts for the A or B subunits of the V-ATPase complex. Finally, stimulation of both cAMP-dependent protein kinase and protein kinase C was required for the stimulatory effect of IL-1 on V-ATPase activity. Thus, cytokines present within the inflammatory milieu are able to modulate pHi regulatory mechanisms. These data may represent a novel mechanism whereby cytokines may improve cellular function at inflammatory sites.

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.

Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal macrophages.

The role of protein kinase C in the regulation of vacuolar-type H(+)-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na(+)- and HCO(3-)-free K(+)-medium containing Zn2+, a H(+)-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 +/- 0.04 pH/min (n = 9). Pretreatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonist was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.

STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p.

The Saccharomyces cerevisiae gene, VPH1 (Vacuolar pH 1), encodes a 95-kDa integral membrane subunit of the vacuolar-type H(+)-ATPase (V-ATPase) that is required for enzyme assembly; disruption of the VPH1 gene impairs vacuolar acidification (Manolson, M.F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. Chem. 267, 14294-14303). Here we show that STV1 (Similar To VPH1) encodes an integral membrane polypeptide of 102 kDa with 54% identity with the peptide sequence of Vph1p. High copy expression of STV1 partially restores vacuolar acidification in a delta vph1 mutant strain; solubilization and fractionation of membrane proteins from these vacuoles show that Stv1p co-purifies with bafilomycin A1-sensitive ATPase activity and with the 60- and 69-kDa V-ATPase subunits. Immunofluorescence microscopy of strains bearing a single copy of epitope-tagged STV1 reveals punctate staining of the cytoplasm; overexpression of epitope-tagged Stv1p reveals both punctate cytoplasmic staining and vacuolar membrane staining. Northern analysis shows that disruption of STV1 does not affect the level of transcription of VPH1 and that disruption of VPH1 does not affect the level of transcription of STV1. Strains bearing disruption of genes encoding other V-ATPase subunits (VMA1, VMA2, VMA3, and VMA4) fail to grow on media supplemented with 100 mM CaCl2 or 4 mM ZnCl2, media buffered to pH 7.5, or media with a glycerol carbon source. On the same types of media only a delta vph1 delta stv1 double disruption mutant has growth phenotypes equivalent to strains bearing a single disruption of the VMA1, VMA2, VMA3, and VMA4 genes; a delta vph1 strain has only moderate growth inhibition while a delta stv1 strain has wild type growth on the conditions listed above. We conclude that Stv1p is a functional homologue of Vph1p and suggest that Stv1p and Vph1p may be equivalent subunits for V-ATPases located on different organelles. The function of these 100-kDa homologues may be to target or regulate other common V-ATPase subunits for two distinct cellular locations.

The plant vacuolar Na+/H+ antiport.

Salt stress imposes severe limitations on plant growth, however, the extent of growth reduction depends upon the soil salinity level and the plant species. One of the mechanisms employed by salt tolerant plants is the effective vacuolar compartmentalization of sodium. The sequestration of sodium into the vacuole occurs by the operation of a Na+/H+ antiport located at the tonoplast. Evidence for a plant vacuolar Na+/H+ antiport has been demonstrated in tissues, intact vacuoles and isolated tonoplast vesicles. In sugar beet cell suspensions, the activity of the vacuolar Na+/H+ antiport increased with increasing NaCl concentrations in the growth medium. This increased activity was correlated with the increased synthesis of a 170 kDa tonoplast polypeptide. In vivo labelling of tonoplast proteins showed the enhanced synthesis of the 170 kDa polypeptide not only upon exposure of the cells to salt, but also when the cells were grown in the presence of amiloride. Exposure of the cells to amiloride also resulted in increased vacuolar Na+/H+ antiport activity. Polyclonal antibodies raised against the 170 kDa polypeptide almost completely inhibited the antiport activity, suggesting the association of this protein with the plant vacuolar Na+/H+ antiport. Antibodies against the Na+/H+ antiport-associated polypeptide were used to screen a Beta lambda ZAP expression library. A partial clone of 1.65 kb was sequenced and found to encode a polypeptide with a putative transmembrane domain and a large hydrophilic C terminus. This clone showed no homology to any previously cloned gene at either the nucleic acid or the amino acid level.