Department of Medicine, McGill University Health Centre, McGill University, Montreal, Quebec, Canada H3A 1A1
Submitted 22 July 2003 ; accepted in final form 21 November 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
inflammation; lipid mediators; protein kinases; signal transduction
In a number of cells, cPLA2 is regulated by [Ca2+]i and phosphorylation (11, 13). It has been proposed that an increase in [Ca2+]i into the submicromolar range induces translocation of cPLA2 from the cytosol to an intracellular membrane, where cPLA2 would bind via its NH2-terminal Ca2+-dependent lipid binding or C2 domain, gaining access to phospholipid substrate. Phosphorylation on Ser505 increases the catalytic activity of cPLA2. In GEC, cPLA2 is the major endogenous PLA2 isoform. C5b-9 increases free AA, and the release of AA is amplified by overexpression of cPLA2 (23). In GEC, cPLA2 localizes and hydrolyzes phospholipids at the plasma membrane, the membrane of the ER, and the nuclear envelope, but not at mitochondria or the Golgi apparatus (18). Thus the activation of cPLA2 and release of AA are compartmentalized to specific organelles. In GEC, complement enhances cPLA2 phosphorylation and catalytic activity via PLC activation, production of 1,2-diacylglycerol (DAG), and activation of the PKC pathway (23). Mutation of the cPLA2 ERK phosphorylation site (Ser505 to Ala) or pharmacological inhibition of ERK did not reduce cPLA2-mediated AA release, but cPLA2-Ser505-to-Ala mutation nevertheless required the action of PKC (6). Thus in GEC, the role of PKC in complement-dependent cPLA2 activation is essential, but ERK appears to be redundant.
Our earlier studies indicate that the stimulation of cPLA2 activity and AA release is dependent, at least in part, on subcellular localization or compartmentalization (18). However, relatively little is known about the organization of signaling cascades activated by C5b-9. Such organization/compartmentalization of phospholipases and protein kinases may be dependent on the cytoskeleton, specifically the actin filament network. The purpose of this study was to examine the role of the actin cytoskeleton in transmission of signals by the C5b-9 complex. We demonstrate that complement-mediated activation of cPLA2 in GEC is regulated by the actin cytoskeleton. The cytoskeleton modulates upstream protein kinase pathways involved in regulating cPLA2 catalytic activity, whereas the membrane association of cPLA2 is unrelated to cytoskeletal integrity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and transfection. Rat GEC culture and characterization have been published previously (6, 9, 10, 23). GEC were cultured in K1 medium, and studies were done with cells between passages 8 and 60. GEC that stably overexpress cPLA2 were employed in all experiments, except those involving L63RhoA. Production and characterization of the GEC that stably overexpress cPLA2 were described previously (23). GEC that express L63RhoA were produced by stable transfection using a method analogous to that for cPLA2.
Incubation of GEC with complement. The standard protocol involved incubation of GEC in monolayer culture with rabbit anti-GEC or sheep anti-Fx1A antiserum (5% vol/vol) in modified Krebs-Henseleit buffer containing (in mM) 145 NaCl, 5 KCl, 0.5 MgSO4, 1 Na2HPO4, 0.5 CaCl2, 5 glucose, and 20 HEPES, pH 7.4, for 40 min at 22°C (6, 9, 10, 23). GEC were then incubated with sublytic normal human serum (NS; diluted in Krebs-Henseleit buffer) or heat-inactivated (decomplemented) human serum (HIS; 56°C, 30 min) in control for 40 min at 37°C. In some experiments, antibody-sensitized GEC were incubated with C8-deficient human serum, or C8-deficient serum supplemented with purified C8 (80 µg/ml undiluted serum). As in previous studies, we have generally used heterologous complement to minimize possible signaling via complement-regulatory proteins, although we have demonstrated that homologous complement induces AA release as well (6, 9, 10, 23). Previous studies have shown that, in GEC, complement is not activated in the absence of antibody (6, 9, 10, 23).
Induction of PHN in rats. PHN was induced by a single intravenous injection of 0.4 ml of sheep anti-Fx1A antiserum, as described previously (8, 9). Urine was collected on day 14, and rats were then killed and glomeruli were isolated by differential sieving. All studies were approved by the McGill University Animal Care Committee.
Measurement of free [3H]AA, [3H]DAG, cPLA2 activity, and PKC activity. GEC phospholipids were labeled to isotopic equilibrium with [3H]AA for 48-72 h, as detailed previously (23). Lipids were extracted from 1 x 106 cells and cell supernatants. Methods of extraction and separation of radiolabeled lipids (e.g., [3H]AA) by thin-layer chromatography are published elsehwere (23). cPLA2 activity was measured using an in vitro assay that monitors release of [14C]AA from [14C]phosphatidylethanolamine (23). PKC activity was determined by measuring phosphorylation of myelin basic protein(4-14) peptide, as described previously (6).
Immunoprecipitation and immunoblotting. Preparation of GEC and glomerular lysates and cell fractions was described previously (18, 23). After incubation with antibody and complement, 6 x 106 GEC were lysed, and proteins were immunoprecipitated with primary antiserum, as described previously (6, 9). Immune complexes were incubated with agarose-coupled protein A. For analysis of the EGF-R interaction with PLC-
1,
2 x 107 GEC were lysed, and incubated with agarose-conjugated GST-PLC-
1-SH2-SH2-SH3 fusion protein (4 µg) for 3 h at 4°C. Complexes were boiled in Laemmli sample buffer and subjected to SDS-PAGE under reducing conditions. Proteins were then electrophoretically transferred onto nitrocellulose paper, blocked with 3% BSA/2% ovalbumin, and incubated with primary antibody and then with horseradish peroxidase-conjugated secondary antibody. The blots were developed using the enhanced chemiluminescence technique (Amersham Pharmacia Biotech). Protein content was quantified by scanning densitometry, using National Institutes of Health Image software. Preliminary studies demonstrated that there was a linear relationship between densitometric measurements and the amounts of protein loaded onto gels.
Immunofluorescence microscopy. Cells adherent to glass coverslips were fixed with 3% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 (18). After being washed, cells were incubated with rhodamine-phalloidin (1 µg/ml) or with primary and secondary antibodies (diluted in 3% BSA in PBS) for 30 min (18). Coverslips were mounted onto glass slides and photographed using a Nikon Diaphot immunofluorescence microscope and Nikon Coolpix 995 digital camera.
Measurement of complement-dependent cytotoxicity. Complement-mediated cytolysis was determined by measuring release of lactate dehydrogenase (LDH), similarly to the method described previously (8). Specific release of LDH was calculated as [NS-HIS]/[100-HIS], where NS represents the percentage of total LDH released into cell supernatants in incubations with NS, and HIS is the percentage of total LDH released into cell supernatants in incubations with HIS.
Statistics. Data are presented as means ± SE. The t-statistic was used to determine significant differences between two groups. One-way ANOVA was used to determine significant differences among groups. Where significant differences were found, individual comparisons were made between groups using the t-statistic and adjusting the critical value according to the Bonferroni method.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Complement-induced transactivation of EGF-R is not dependent on the actin cytoskeleton. The present study confirms that incubation of antibody-sensitized GEC with NS as the source of complement induces EGF-R phosphorylation, consistent with transactivation (Fig. 2A). This transactivation of EGF-R was also induced by C8-deficient serum reconstituted with purified C8, but not with C8-deficient serum alone, confirming that transactivation is dependent on assembly of C5b-9 (Fig. 2A). Moreover, EGF-R transactivation is a prerequisite for downstream signals in GEC, including activation of cPLA2 and ERK (9). To determine whether EGF-R transactivation is dependent on an intact actin cytoskeleton, GEC were preincubated with cytochalasin D or latrunculin B. Pretreatment did not impair the ability of complement to induce EGF-R phosphorylation (Fig. 2A). To verify that phosphorylated EGF-R was actually activated, we assessed whether EGF-R was able to bind its substrate, PLC-1. Lysates of complement-treated and untreated GEC were absorbed with a GST fusion protein that contains the SH2 and SH3 domains of PLC-
1. GST-PLC-
1-SH2-SH2-SH3 bound to EGF-R only in lysates of complement-treated GEC, and the bound EGF-R was tyrosine phosphorylated (Fig. 2B). Previously, we also showed that complement induces tyrosine phosphorylation of PLC-
1 (6). Together, the results imply that there is activation of PLC-
1. A result analogous to PLC-
1 was obtained using a GST-Grb-2 fusion protein in an earlier study (9).
|
The above experiments demonstrate that complement induces EGF-R transactivation in cultured GEC, but it is important to determine whether analogous changes occur in C5b-9-mediated GEC injury in vivo. To address this question, we assessed EGF-R tyrosine phosphorylation and protein expression in the PHN model of membranous nephropathy, where GEC injury and proteinuria are due to C5b-9 assembly. Glomeruli were isolated from control rats and from rats with PHN on day 14, a time point when these rats show marked proteinuria (8). Glomerular EGF-R phosphorylation was enhanced about twofold in rats with PHN, compared with control, and there were no differences in EGF-R protein expression (Fig. 3). In earlier studies, we demonstrated that signals downstream of EGF-R, including cPLA2, are also activated in PHN (10). Together, the results imply that cultured GEC reflect pathophysiological changes in vivo.
|
An intact actin cytoskeleton is required for complement-induced stimulation of cPLA2 catalytic activity and release of AA. In keeping with previous results, incubation of antibody-sensitized GEC with complement (NS) induced an increase in free [3H]AA (Fig. 4A). Furthermore, incubation with C8-deficient serum reconstituted with purified C8 increased free [3H]AA (5.47 ± 0.47% of total radioactivity) compared with unreconstituted C8-deficient serum (1.57 ± 0.42% of total radioactivity), indicating that release of AA is due to assembly of C5b-9 (8). The complement-induced release of AA is dependent on the activation of cPLA2 (23), and the mechanism involves Ca2+-dependent association of cPLA2 with membranes of GEC organelles (18, 23), as well as an increase in cPLA2 catalytic activity due to phosphorylation via a PKC-dependent pathway (6, 18, 23). Complement-induced [3H]AA release was inhibited by 45% with cytochalasin D and by
80% with latrunculin B (Fig. 4, A and B). Disruption of the cytoskeleton may have reduced [3H]AA release either by interfering with the association of cPLA2 with membranes or by inhibiting the catalytic activity of cPLA2. To distinguish between these possibilities, we first employed an in vitro cPLA2 activity assay that monitors release of [14C]AA from [14C]phosphatidylethanolamine (23). In keeping with prior results, it was demonstrated that cPLA2 activity was stably increased in lysates of GEC that had been incubated with antibody and complement, compared with control (Fig. 4C). Pretreatment of GEC with cytochalasin D reduced basal PLA2 activity and abolished the complement-stimulated increase. The inhibitory effect of cytochalasin D on PLA2 activity appeared to be greater than its effect on AA release in intact cells, but it should be noted that the two assays measure complementary, although not identical, parameters (e.g., the in vitro assay does not reflect the role of Ca2+ in AA release). The cPLA2 activity assay was also employed to address the effect of latrunculin B (protocol as in Fig. 4C). In the absence of latrunculin B pretreatment, complement stimulated an increase in cPLA2 activity in GEC extracts that was 2.58 ± 0.47-fold of control (P < 0.015). After pretreatment with 1 µM latrunculin B, the complement-stimulated increase in cPLA2 activity was trivial (0.07 ± 0.15-fold of control; 3 experiments). Thus the effect of latrunculin B was in keeping with that of cytochalasin D.
|
Unlike cytochalasin D or latrunculin B, which depolymerize the actin cytoskeleton, jasplakinolide is a cell-permeant compound that stabilizes F-actin and organizes actin filaments at the cell periphery, near the plasma membrane (29). (Because jasplakinolide competes with phalloidin for the F-actin binding site, it is not possible to examine rhodamine-phalloidin staining in cells after jasplakinolide treatment.) We predicted that preincubation of GEC with jasplakinolide may enhance the complement-induced increase in free [3H]AA, but contrary to expectations, jasplakinolide inhibited the release of free [3H]AA by complement (Fig. 4D) and did not reverse the inhibitory effect of latrunculin B (Fig. 4E). Phosphorylation of the ezrin-radixin-moesin family of proteins is required for cross-linking of actin to the plasma membrane (41). GEC were treated with calyculin A to induce phosphorylation-dependent association of these proteins with the plasma membrane. Calyculin A is a serine/threonine protein phosphatase inhibitor that inhibits phosphatases 1 and 2, and treatment of many cell lines with calyculin A results in a condensation of actin filaments at the plasma membrane (1, 29). Preincubation of GEC with calyculin A inhibited the complement-induced increase in free [3H]AA (Fig. 4F). Calyculin A also partially inhibited the complement-induced stimulation of cPLA2 activity, measured by release of [14C]AA from exogenously added phospholipid substrate in vitro (complement: 152 ± 15% of control, complement+calyculin A: 130 ± 20% of control, P < 0.005, 5 experiments).
Disruption of the actin cytoskeleton affects pathways upstream of cPLA2. The next series of experiments assessed whether the actions of the compounds that affect the cytoskeleton were directed specifically at cPLA2 or at upstream mediators, i.e., production of [3H]DAG and/or activation of PKC. In GEC, complement increases inositol trisphosphate and DAG ([3H]DAG and DAG mass) (4, 7), and the complement-induced activation of cPLA2 is dependent on the activation of the DAG-PKC pathway, but is independent of ERK (6). In keeping with previous results, incubation of GEC with complement increased [3H]DAG and PKC activity (Fig. 5, A and B), and depletion of PKC reduced the complement-mediated increase in free [3H]AA by 75% (Fig. 4A). Latrunculin B blocked both the complement-induced increases in [3H]DAG and PKC activity almost completely (Fig. 5, A and B). Cytochalasin D reduced the complement-induced increases in [3H]DAG and PKC activity by
35%, although the increase in [3H]DAG remained statistically significant (Fig. 5, A and B). Thus changes in [3H]DAG correlate closely with changes in PKC activity, and depolymerization of the actin cytoskeleton reduces [3H]DAG production (and PKC activation) by blocking PLC-mediated phospholipid hydrolysis. Although jasplakinolide and calyculin A blocked the complement-dependent increase in free [3H]AA, these compounds had no effect on changes in [3H]DAG (Fig. 5A) or PKC activity (not shown).
|
To further delineate the sites of action of cytoskeleton-altering drugs, we studied the effects of PMA on the release of [3H]AA. In these experiments, we employed an experimental model developed earlier, in which GEC are first incubated with PMA (to activate PKC), and then [Ca2+]i is clamped by permeabilizing GEC with buffers containing specific concentrations of Ca2+, as PMA does not independently increase [Ca2+]i in GEC (6). Permeabilization of untreated GEC with buffer containing 1 mM free Ca2+ induced an upward trend in free [3H]AA, compared with 0.1 µM free Ca2+ (resting Ca2+; Fig. 6). A greater increase in free [3H]AA was induced by treatment of GEC with PMA, plus permeabilization with buffer containing 1 mM Ca2+ (Fig. 6). Pretreatment of GEC with latrunculin B had no significant inhibitory effect on the PMA-induced increase in free [3H]AA (Fig. 6). Similarly, cytochalasin D had no effect on [3H]AA release by PMA (4-7 experiments; data not shown). In contrast to cytochalasin D and latrunculin B, pretreatment of GEC with jasplakinolide or calyculin A inhibited the Ca2+- and PMA-stimulated release of [3H]AA by 100% (5-6 experiments; data not shown). Therefore, the inhibitory effects of latrunculin B and cytochalasin D on complement-induced activation of cPLA2 are at due, least in part, to inhibition of steps upstream of PKC, involving inhibition of PLC (Fig. 5), whereas jasplakinolide and calyculin A most likely inhibit the action of PKC or PKC effectors on cPLA2 activity.
|
Disruption of the actin cytoskeleton does not affect the membrane association of cPLA2. The above experiments indicate that disruption of the cytoskeleton leads to inhibition of complement-stimulated cPLA2 catalytic activity. In the next series of experiments, we examined whether cytoskeleton-disrupting drugs affected the association of cPLA2 with membranes, which is an essential step for the release of AA. In an earlier study, we demonstrated that in resting GEC, a portion of cPLA2 was associated with the membrane (microsomal) fraction and that, in complement-stimulated GEC, the majority of phospholipid hydrolysis occurred at the ER (18). However, translocation of cPLA2 from the cytosol to the membrane compartment was not detected with a physiological agonist, such as C5b-9, but only after incubation of GEC with the Ca2+ ionophore ionomycin (which induces a greater increase in [Ca2+]i compared with C5b-9). In untreated GEC, cPLA2 was found in both cytosolic and microsomal fractions, whereas the ER protein calnexin (18) was exclusively microsomal (Fig. 7A). Cytochalasin D and latrunculin B did not affect the amount of cPLA2 recovered in the microsomal fraction (Fig. 7A). In a second set of experiments, GEC were untreated, or treated with ionomycin (to facilitate cPLA2 translocation) or ionomycin plus calyculin A. Ionomycin induced a small increase in microsomal cPLA2, compared with untreated cells, but by analogy to cytochalasin D and latrunculin B, calyculin A did not affect the microsomal association of cPLA2 (Fig. 7B).
|
The effects of latrunculin B and cytochalasin D on cPLA2 were also studied using immunofluorescence microscopy (Fig. 8A). In GEC, cPLA2 staining was predominantly cytosolic, and in some cells (particularly after treatment with the Ca2+ ionophore A-23187), there was perinuclear enhancement, in keeping with localization at the membrane of the ER or nuclear envelope (18). The perinuclear enhancement was not affected by pretreatment with latrunculin B (Fig. 8A). Similarly, cytochalasin D did not affect cPLA2 staining (results not shown). GEC were also stained with antibody to calnexin, to localize the ER (Fig. 8B). The distribution of calnexin in resting cells was mainly perinuclear, with some extension of calnexin from the nucleus toward the cell periphery. Calnexin staining was not affected by A-23187. Treatment with latrunculin B (Fig. 8B) or cytochalasin D (not shown) did not have any significant effect on the perinuclear staining pattern of calnexin, but there was slightly less peripheral extension of the staining from the perinuclear regions. These results indicate that latrunculin B or cytochalasin D, while altering the actin cytoskeleton (Fig. 1), did not alter the perinuclear localization of the ER and cPLA2 (Fig. 8), and, together with the biochemical data (Fig. 7, A and B), suggests that ER-cPLA2 interaction remains intact despite cytoskeletal disruption.
|
Finally, we assessed whether cPLA2 was associated with the actin cytoskeleton. In these experiments, GEC were incubated with or without ionomycin+PMA and were then treated with buffer containing 1% Triton X-100. Almost all of the cPLA2 was recovered in the Triton-soluble fraction (Fig. 7C), and only trivial amounts were present in the Triton-insoluble (cytoskeleton) fraction, suggesting no significant association of cPLA2 with the cytoskeleton.
Effect of L63RhoA expression on the cytoskeleton and AA release. Rho GTPases are known to stabilize actin filaments and induce stress fiber formation in various cells (2). In GEC, stable expression of a constitutively active RhoA mutant (L63RhoA) (Fig. 9A) resulted in increased actin polymerization, as reflected by the appearance of stress fibers superimposed on the cortical distribution of F-actin (Fig. 1E). In addition, while stress fibers disappeared after incubation with latrunculin B, the L63RhoA-transfected GEC showed a relative resistance to depolymerization of cortical actin by latrunculin B, compared with Neo GEC (Fig. 1F). Complement-induced increases in [3H]AA and [3H]DAG were attenuated significantly in the L63RhoA-transfected cells compared with Neo GEC (Fig. 9B). The association of cPLA2 with the microsomal membrane fraction did not appear to be significantly different between the GEC that overexpress L63RhoA and Neo GEC (Fig. 9C), suggesting that L63RhoA acted via attenuation of the complement-mediated stimulation of cPLA2 catalytic activity. We were not able to verify directly that L63RhoA attenuated cPLA2 activity, because the GEC that overexpress L63RhoA express endogenous cPLA2 (i.e., these cells do not overexpress cPLA2), and changes in endogenous cPLA2 activity were too small to be quantitated reliably in the in vitro PLA2 assay.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stabilization of actin polymerization by jasplakinolide reduced the complement-induced increase in [3H]AA release (Fig. 4). The same effect was observed with calyculin A, which condenses actin filaments at the cell periphery near the plasma membrane. Moreover, calyculin A partially inhibited the complement-induced increase in cPLA2 activity (we were not able to directly test the effect of jasplakinolide on cPLA2 activity for practical reasons). Jasplakinolide and calyculin A blocked the PMA-induced release of [3H]AA (in the presence of increased [Ca2+]i) and did not affect changes in [3H]DAG. Therefore, these two drugs most likely interfered with the stimulation of cPLA2 catalytic activity downstream of PKC. Stable expression of L63RhoA attenuated complement-induced increases in [3H]AA and [3H]DAG (Fig. 9). This effect was associated with enhanced actin polymerization (Fig. 1E). In addition, L63RhoA may have acted via a mechanism analogous to calyculin A, because constitutively active RhoA was reported to induce phosphorylation of ezrin-radixin-moesin proteins (2). Together, the results demonstrate that both depolymerization and stabilization of the actin cytoskeleton can reduce AA release and suggest that AA release is dependent on cytoskeletal remodeling (see below).
Our results indicate that the cytoskeleton-disrupting drugs most likely affected signaling events in the vicinity of the plasma membrane. Cytochalasin D and latrunculin B interfered with PLC-1, which is believed to function mainly at the plasma membrane, where substrate, i.e., phosphatidylinositol 4,5-bisphosphate (PIP2) is most abundant (44). Integrin engagement by extracellular matrix results in accumulation of talin, focal adhesion kinase, vinculin, and
-actinin around the integrin cytoplasmic domain. Actin filaments interact with
-actinin and talin to form a supporting structure that organizes focal contacts. F-actin assembly may be dependent on Rho GTPases, Rho kinases, phosphatidylinositol 4-phosphate 5-kinase, as well as ezrin-radixin-moesin proteins (2). An equilibrium exists between PIP2, F-actin, actin-binding proteins (profilin, gelsolin, cofilin), and Rho GTPases, such that changes in actin polymerization could be associated with changes in PIP2 (15, 26). Alternatively, it has been reported that activation of PLC-
1 by EGF may depend on association with the actin cytoskeleton (46). Production of DAG after PIP2 hydrolysis leads to the activation of PKC. On stimulation of cells, various isoforms of PKC translocate to the plasma membrane (as well as other subcellular sites) (12, 17). The regulation of actin polymerization and its potential role in PKC activation (e.g., Ca2+ and lipid dependence, anchoring proteins) (28) will require further study. Additional studies will also be required to define the relevant PKC isoforms in GEC (14). It should also be noted that in GEC there was no direct association of cPLA2 with the actin cytoskeleton, although cPLA2 has been reported to interact with the intermediate filament protein vimentin (21).
Our previous studies showed that association of cPLA2 with subcellular membranes occurs in resting and stimulated GEC and is essential for AA release (18). The ER is the principal site of phospholipid hydrolysis by cPLA2 and the most important source of free AA. Treatment of GEC with cytochalasin D, latrunculin B, or calyculin A did not alter the amount of cPLA2 associated with microsomal membranes (which include ER), suggesting that an intact actin cytoskeleton is not essential for the association of cPLA2 with the membrane compartment (Fig. 7). Furthermore, the ER (as visualized by calnexin staining) appeared to be unaffected (or affected to only a minor extent) by cytochalasin D and latrunculin B (Fig. 8). Similarly, treatment of hepatocytes with cytochalasin D induced only minor changes in ER ultrastructure (43).
Our study, which has revealed an important role for the actin cytoskeleton in complement signaling, is in keeping with studies in other systems, which showed that the actin cytoskeleton is important in cell cycle progression, including expression of immediate early genes and cyclins (40). In response to insulin treatment, actin filament disassembly blocked activation of Ras, ERK, and p38 mitogen-activated protein kinase, but not insulin receptor autophosphorylation, phoshatidylinositol 3-kinase, or S6 kinase (16, 40). Moreover, binding of Shc to the insulin receptor was not affected, but binding of Grb2 to Shc was disrupted (40). The authors were not able to determine whether there was direct association of Shc or Grb2 with the cytoskeleton. Serum response factor regulates transcription of many serum-inducible genes and is activated by LIM kinase-1. Activation is blocked by latrunculin B (34). In rat mesangial cells, disruption of the actin cytoskeleton with latrunculin B upregulated interleukin-1-induced expression of inducible nitric oxide synthase, whereas jasplakinolide suppressed the enhancement by latrunculin B. Also, latrunculin B decreased serum response factor activity, and serum response factor played a negative regulatory role in the expression of inducible nitric oxide synthase (47). It would seem that jasplakinolide (which facilitates actin polymerization) should produce an effect opposite to that of cytochalasin D or latrunculin B (which depolymerize the cytoskeleton). However, all of these drugs were inhibitory to AA release in the present study, and parallel effects of jasplakinolide and latrunculin B or cytochalasin D have been reported in several other systems. For example, jasplakinolide and latrunculin B both inhibited insulin-stimulated glucose uptake in adipocytes (16), lipopolysaccharide-mediated production of reactive oxygen species in monocytes (30), and accumulation of phosphatidylinositol 3,4,5-trisphosphate in response to a chemotactic stimulus in neutrophils (42), whereas jasplakinolide and cytochalasin D both induced apoptosis in airway epithelial cells (45).
The protocol employed in the present study did not result in complement-induced changes in F-actin, although we observed that F-actin decreases with more prolonged complement exposure (37). In another study (39), sublethal GEC injury by complement was associated with loss of actin stress fibers and focal contacts, but not integrins. There was a reduction in tyrosine phosphorylation of paxillin but no change in content of focal contact proteins. The complement-induced disassembly of the actin cytoskeleton may have been due to ATP depletion, or loss of other cytosolic components, and recovery from injury was seen in 18 h (39). In vivo, GEC (podocytes) contain F-actin as a thin layer at the base of the foot processes, and abnormalities in actin-associated proteins may lead to a disruption in GEC architecture (24). Podocyte foot process effacement and focal detachment from the glomerular basement membrane are prominent in C5b-9-mediated glomerular injury. This raises the possibility of disassembly of focal adhesion complexes or actin filaments, which support foot processes. Further studies will be required to determine how the cytoskeleton modulates the pathophysiology of complement-dependent GEC injury in vivo.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|