Article |
Address correspondence to Erica Werner, Dept. of Cell Biology, Emory University, Whitehead Biomedical Research Bldg., 615 Michael St., Atlanta, GA 30322. Tel.: (404) 727-6277. Fax: (404) 727-6456. E-mail: ericaw{at}cellbio.emory.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: integrin; actin; Rac; ROS; mitochondria
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In most of these pathways, the mechanisms for ROS production and their molecular targets remain largely unknown. At least three distinct mechanisms have been identified: upon neutrophil activation, superoxide is produced in the phagosome by a membrane oxidase to kill the endocytosed pathogen. In nonphagocytic cells, a different mechanism is used in association with activation of EGF (Bae et al., 1997) and PDGF receptors (Sundaresan et al., 1995). In these systems, ROS are produced by a distinct oxidase at the plasma membrane, inhibiting phosphatases and potentiating tyrosine kinases signaling (Lee et al., 1998; Barrett et al., 1999).
Mitochondria are the third and most important source for ROS in every cell type, where 15% of the transported electrons is diverted to the formation of superoxide instead of water (Boveris et al., 1972). Although the molecular mechanisms involved in the mitochondrial switch to ROS production are not well understood, multiple pathways may converge to modify mitochondrial function. Moreover, recent studies also indicate that the production of ROS may be accompanied by changes in mitochondrial metabolism (Nemoto et al., 2000; Nicholls and Budd, 2000). The rate of mitochondrial superoxide production is modulated during TNF signaling (Schulze-Osthoff et al., 1993; Sanchez-Alcazar et al., 2000), hypoxia (Vanden Hoek et al., 1997; Chandel et al., 1998), and apoptosis (Cai and Jones, 1998).
The small GTPase Rac has emerged as a common mediator of ROS production in diverse signaling pathways that lead to mitogenesis, gene expression, and stress responses (Irani et al., 1997; Page et al., 1999; Ozaki et al., 2000; Suzukawa et al., 2000). The molecular mechanisms involved have been identified through the use of mutations in Rac that modify its interaction with effector proteins. The best understood mechanism is promotion of ROS production in neutrophils where Rac2 binds to a NAD(P)H oxidase through the insertion domain and residue 26 in the effector domain to mediate assembly and function of the burst oxidase at the plasma membrane (Freeman et al., 1996). In most other cell types, the oxidase remains unidentified. In Ras-induced NIH 3T3 cell transformation, Rac1 is necessary and sufficient to induce ROS production by a mechanism dependent on the insertion domain (residues 124135) (Irani et al., 1997; Joneson and Bar-Sagi, 1998). However, there are other pathways where Rac promotes ROS production by a distinct mechanism. During PDGF-induced cyclin D expression, Rac-mediated ROS production depends on the residues 33 and 37 of the effector domain (Joyce et al., 1999; Page et al., 1999). Rac mediates ROS production during noncytotoxic TNF signaling by an indirect mechanism involving phospholipase A2 (Woo et al., 2000). These observations suggest that Rac may act at different steps to control ROS production.
In rabbit synovial fibroblasts (RSFs), clustering of integrins by anti-5 integrin antibodies leads to altered adhesion followed by the reorganization of the actin cytoskeleton and the production of ROS. As a result, the transcription factor NF
B is activated, inducing the expression of IL-1 and matrix metalloproteinase (MMP)-1/collagenase-1 (CL-1) (Kheradmand et al., 1998). In the present study, we have elucidated the pathway by which integrin-mediated signaling induces ROS production.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To gain insights into the mechanism used by GTPases to trigger an increase in ROS production and induce CL-1 expression, we used point mutations that selectively disrupt effector domain functions of the constitutively active mutants RacV12 and RacL61 (Westwick et al., 1997). When we cotransfected RSFs with a panel of different Rac effector domain point mutants together with a CL-1 promoter reporter construct, we observed that only the mutant L37 was completely unable to induce expression of a CL-1 (Fig. 2 A) or NFB (Fig. 2 B) promoterreporter constructs. We compared the RacV12 H26 mutant in the switch 1 effector domain with the RacV12 N130 mutant in the insertion domain, both of which can activate other Rac effectors, such as PAK, but cannot activate the neutrophil oxidase in vitro (Freeman et al., 1996). In contrast to the L37 mutant, neither mutant was impaired in the induction of the expression of the NF
B promoter, a step in the pathway more proximal to the integrin signal than CL-1 expression (Fig. 2 B), or of the CL-1 promoter (unpublished data). These results suggest that Rac activates a signaling pathway by an effector molecule that requires binding through residue 37 to induce NF
B activation and CL-1 expression, thus pointing to a novel mechanism for induction of ROS.
|
Mitochondria are the source of integrin-mediated ROS production
An important source of ROS is the nonenzymatic generation of superoxide as a by-product of mitochondrial metabolism. In our assay, the H2O2 signal was inhibited when the cells were preincubated with a superoxide scavenger, Tiron, and abolished by the simultaneous addition of catalase to the reaction mixture (Fig. 3 A), corroborating that the detected signal is effectively H2O2 and suggesting that the H2O2 is originated by superoxide. The H2O2 signal induced by anti-5 mAb was abrogated by inhibition of the function of mitochondrial respiratory chain complex 1 by 1 µM rotenone, complex 3 by 25 µM antimycin A, or complex 4 by 500 µM KCN (Fig. 3 A). These results suggest that the mitochondrial respiratory chain is responsible for the rise in ROS induced by anti-
5 mAb.
|
Because the diversion of electrons to form superoxide can lead to a dissipation of membrane potential () (Zoratti and Szabo, 1995; Madesh and Hajnoczky, 2001), we examined whether integrin cross-linking induces changes in mitochondrial membrane potential. We analyzed the distribution of the dual emission potentiometric probe JC-1. When high negative potential drives the dye concentration above a threshold, the green fluorescent monomers (low membrane potential) form red fluorescent aggregates (high membrane potential). After 15 min of dye loading at 37°C, most of the mitochondria in control RSFs were bright orange (Fig. 4 A, a), indicative of highly energized mitochondria. When the membrane potential was dissipated with the protonophore FCCP, all of the mitochondria stained green (Fig. 4 A, f), corroborating that JC-1 accumulation is driven by membrane potential. After 2 h of anti-
5 mAb treatment, a population of cells had only green-stained mitochondria (Fig. 4 A, c). Frequently, the green staining mitochondria were present in cells that had rounded in response to the anti-
5 mAb treatment. The number of cells with green-stained mitochondria increased with time from 7 ± 1% (SEM) at 0 h to 21 ± 4% after 2 h and to 43% after 8 h of anti-
5 mAb addition. However, by 24 h the mitochondria in all cells were highly polarized again (Fig. 4 A, e), and no cells with only green mitochondria were detected, indicating that this is a reversible change. Cytochalasin D, which also induces ROS production (Kheradmand et al., 1998), induced mitochondrial depolarization after 2 h of treatment (Fig. 4 A, b). These results further support the biochemical evidence for cell shapedependent control of mitochondrial function.
|
Bcl-2 blocks integrin-mediated signaling for CL-1 expression
Cell rounding and detachment can induce apoptosis in some cell types (Ruoslahti and Reed, 1994). During the induction of apoptosis, mitochondria are engaged to produce ROS and undergo membrane depolarization, membrane permeability transition (MPT), release of specific proteins from the intermembrane space (cytochrome C, procaspases 2 and 9, Apaf-1, and AIF), and caspase activation through a mechanism that is not clear (Kroemer and Reed, 2000). Members of the Bcl-2 family can inhibit ROS production (Hockenbery et al., 1993; Kane et al., 1993), membrane potential loss (Gottlieb et al., 2000), MPT (Marzo et al., 1998), cytochrome C release (Yang et al., 1997), and caspase activation (Strasser et al., 2000). We hypothesized that in response to triggering integrin-mediated signaling in RSFs the mitochondrial signal transduction is engaged by a mechanism common to apoptosis, yet is resolved into induction of gene expression instead of cell death.
Neither control cells nor cells treated for 4 h with anti-5 mAb showed cytochrome C release from mitochondria to the cytosol by Western blot analysis (Fig. 5 A) or by immunofluorescence (unpublished data). The addition of selective inhibitors of caspases 1 or 3 or the general caspase inhibitor Z-VAD fluoromethyl ketone did not inhibit anti-
5 mAbinduced CL-1 production (Fig. 5 B), although they effectively inhibited staurosporine-induced apoptosis (unpublished data). Anti-
5 mAb induced equivalent amounts of CL-1 in control cells or cells treated with an inhibitor of MPT, cyclosporin A (Fig. 5 C). In contrast, we found that Bcl-2, which blocks several mitochondrial-dependent processes during apoptosis, inhibited integrin ligation-induced signal transduction. The transient overexpression of human Bcl-2 in RSFs inhibited RacV12-induced NBT precipitation as a measure of mitochondrial metabolic/redox function and transcriptional activation of both NF
B and CL-1 promoters (Fig. 5 D). Importantly, the anti-
5 mAb treatment did not induce apoptosis at 24 h cells cultured without serum as analyzed by the TUNEL method (unpublished data) or by growth rates upon serum restitution as measured by dimethylthiazolyldiphenyltetrazolium bromide (MTT) metabolism (Fig. 5 E). After 24 h of anti-
5 mAb treatment, all of the cells expressing CL-1 exhibited a normal nuclear morphology by DAPI staining, confirming that CL-1 induction is not associated with cell death (Fig. 5, F and G). Together, these results further support a role for mitochondria in integrin-dependent signal transduction by a mechanism that can be regulated by Bcl-2 but that is different from apoptosis.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrin control of mitochondrial function
Four independent lines of evidence advocate a role for mitochondria in integrin-induced signal transduction. The first line of evidence for mitochondrial involvement in signal transduction is anti-5 mAbinduced mitochondrial depolarization in cells committed to CL-1 gene expression.
Second, we found that integrin cross-linking induces a ROS increase that requires the function of complexes 1, 3, or 4 of the respiratory chain. Glycolysis maintains the levels of ATP upon inhibition of the respiratory chain in primary fibroblasts (McKay et al., 1983). Thus inhibition of respiration does not hinder the mitochondrial pathway by causing ATP depletion. In confirmation, blocking mitochondrial respiration with rotenone alone was not sufficient to cause mitochondrial depolarization due to reverse activity of the ATP synthase. In isolated mitochondria, complexes 1 and 3 participate directly in ROS production. NADPH or succinate induces superoxide production under conditions of slow respiration (state 4, not conductive to ATP synthesis) where the reduced state of the ubisemiquinone favors the direct transfer of electrons to oxygen (Turrens et al., 1985). Antimycin A enhances but rotenone and cyanide inhibit this mechanism. Although integrin-induced ROS production was sensitive to rotenone and cyanide, we could not detect enhanced ROS production induced by antimycin A. This deviation can be explained by differences in the mitochondrial metabolic state or in antimycin A sensitivity of intact cells. Alternatively, the site for superoxide production could be at cytochrome B or downstream of the antimycin A block. Such a mechanism has been invoked for ROS production induced by release of cytochrome C to the cytosol during apoptosis (Cai and Jones, 1998; Sanchez-Alcazar et al., 2000). However, this mechanism seems unlikely because we did not detect cytochrome C release, indicating the existence of a different mechanism.
The third line of evidence for mitochondrial participation in integrin-mediated signal transduction comes from the increase in anti-5 mAbinduced NBT reduction into insoluble formazan. Even though tetrazolium salts can be reduced by several dehydrogenases, the results are consistent with NBT reduction in mitochondria. We found that NBT reduction occurs at the same time of the detected rise in H2O2 production (unpublished data) and distributes into an intracellular and vesicular compartment. RacV12, which induced H2O2 production and signal transduction, was sufficient to induce a similar staining pattern. Additionally, formazan deposition was abolished by interfering with mitochondrial function using rotenone and inhibited by the effector domain mutant L37 or by Bcl-2 when induced by RacV12.
A fourth line of evidence constitutes Bcl-2 inhibition of RacV12 signaling. Although Bcl-2 localizes to mitochondria, nucleus, and ER, its inhibition of NBT reduction manifests a role in the control of mitochondrial homeostasis. Accordingly with this view, accumulating evidence indicates that rather than antagonizing specific steps of apoptosis Bcl-2 regulates mitochondrial metabolism, including ADP/ATP exchange (Vander Heiden et al., 1999), permeability to metabolic anions (Vander Heiden et al., 2000), and preventing membrane potential dissipation by increasing proton efflux (Shimizu et al., 1998). These mechanisms predict the participation of Bcl-2 in nonapoptotic processes. Interestingly, overexpression of Bcl-xL in pancreatic islet ß cells inhibits not only apoptosis but also mitochondrial response to glucose (Zhou et al., 2000). Thus, the simplest interpretation for our results is that Bcl2 interferes with signal transduction by counteracting Rac-induced changes in mitochondrial metabolism.
Rac mediates ROS production through a novel mechanism
We show that integrins engage mitochondria through the activation of Rac, which constitutes a novel mechanism for this GTPase to mediate ROS production. We have shown previously that integrin ligation induces ROS production by a Rac-dependent mechanism (Kheradmand et al., 1998). Now we show that this GTPase is activated downstream of integrin cross-linking before ROS levels increase at 2 h. These nonoverlapping kinetics suggest a novel indirect mechanism for Rac-dependent ROS induction, involving one or more intermediate steps after the GTPase activation. This mechanism is further supported by our observation that RhoA, although unable to activate the neutrophil oxidase (Abo et al., 1991), also induces H2O2 and CL-1 production in RSFs. This is not as a result of cross-talk between the GTPases because integrin-mediated signaling is selectively inhibited by interfering with the function of one of the GTPases, i.e., only RacN17 inhibits anti-5 mAb signaling (Kheradmand et al., 1998) and only RhoA N19 interferes with integrin-mediated phagocytosis signaling (Werner et al., 2001). Our findings with the effector domain mutants of Rac further support an indirect mechanism distinct from the activation of a membrane oxidase.
Our results indicate that Rho GTPases modulate mitochondrial function through an indirect mechanism, which could involve the control of the anti/proapoptotic function of the Bcl-2 family members and/or the organization of the cytoskeleton. Integrins can engage multiple mechanisms that could sustain cell survival by controlling the function of different members of the Bcl-2 family. Phosphatidylinositol 3-kinase, which can be activated by Rac (Bokoch et al., 1996), activates PKB/Akt kinase and modulates the apoptotic function of Bad (del Peso et al., 1997). However, in our system inhibitors of phosphatidylinositol 3-kinase did not affect anti-5 mAbinduced signaling (unpublished data), suggesting that signaling proceeds by a different mechanism. Integrins can also restrict Bax localization during epithelial cell adhesion to extracellular matrix (Gilmore et al., 2000). An additional mechanism implied in the literature, but not yet demonstrated, is the control of the function or localization of members of the Bcl-2 family by integrins through interaction with members of the 14-3-3 scaffolding protein family. These proteins have multiple interaction partners including ß1 integrins during cell spreading (Han et al., 2001) and p190RhoGEF (Zhai et al., 2001). On the other end of the cascade, the interaction of 14-3-3 with Bad prevents its proapoptotic function (Datta et al., 2000).
Alternatively, this indirect mechanism could involve the actin cytoskeleton. Rac induction of ROS production through a signal conveyed by reorganization of the actin cytoskeleton is consistent with the ability of cytochalasin D (Kheradmand et al., 1998) and activated RhoA to induce H2O2 and CL-1 production. A tantalizing candidate to couple mitochondrial function to actin cytoskeleton reorganization is gelsolin, a member of a multigene family of proteins with actin filament severing and capping activity. In preliminary studies, we have found that microinjection of gelsolin protein into RSFs induces shape change, motility, and expression of CL-1 (unpublished data). Gelsolin is a downstream target of Rac in fibroblasts (Azuma et al., 1998) and neutrophils (Arcaro, 1998). Gelsolin inhibits caspase activation (Azuma et al., 2000) and modifies mitochondrial membrane potential by modulating VDAC activity (Kusano et al., 2000), thus inhibiting apoptosis (Koya et al., 2000). Gelsolin may also be involved in signal transduction, since null fibroblasts have a blunted proinflammatory response (Witke et al., 1995). Interestingly, in RSFs, integrin-mediated activation of Rho and Rac and ROS production induces the expression of proinflammatory cytokines through activation of NFB (Kheradmand et al., 1998; Werner et al., 2001).
Together, we favor a model for the participation of mitochondria in integrin signaling in a nonapoptotic cascade that leads to gene expression and cell differentiation.
Inhibition of integrin (this paper) and RacV12 (Kheradmand et al., 1998) signaling by antioxidants indicates that mitochondria are engaged to generate superoxide as an intermediary in the signal transduction pathway. We also found that in cells committed to signaling mitochondria undergo membrane depolarization. Our results are insufficient to demonstrate a direct relationship between both mitochondrial changes. However, the time course of both events (at the time we observe a sharp decrease in H2O2 production, we still detect an increase in the number of cells with depolarized mitochondria) indicates that superoxide is produced first, and then membrane potential is lowered. This order of events would be consistent with the known mitochondrial superoxide production dependency from membrane potential (Korshunov et al., 1997) and the regulation of VDAC function by superoxide (Zoratti and Szabo, 1995; Madesh and Hajnoczky, 2001). Further experiments are needed to demonstrate the soundness of this model.
Few other examples exist showing that mitochondria participate in signal transduction by capturing Ca+2 or producing ROS (for review see Duchen, 1999). In most cases, ROS production and membrane potential loss have been associated with apoptosis. However, induction of apoptosis and gene expression appear to share a common pathway of signaling through mitochondria where the outcome is dictated by survival signals. It is instructive to compare the integrin-triggered pathway with TNF-induced signal transduction. TNF
-induced apoptosis has been studied mainly in cells treated with cycloheximide or actinomycin D where the absence of survival pathways provided by the activation of gene expression shifts the outcome of signaling to cell death. However, in most normal cell types TNF
induces gene expression through the activation of NF
B, AP-1, JNK, and MAPKK by a mechanism dependent on mitochondrial respiratory chain function (Schulze-Osthoff et al., 1993; Goossens et al., 1995), a pathway that is abrogated by stable manganese superoxide dismutase overexpression (Manna et al., 1998). Rac is also part of these dual pathways. Its activity is necessary both for TNF
-mediated apoptosis (Embade et al., 2000) and gene expression (Esteve et al., 1998). Apoptosis induced by altered adhesion and FAK function can be compensated by Rac activation (Almeida et al., 2000). The participation of Rac and mitochondria in these pathways with dual outcomes suggests that an additional signal is required. This interpretation is consistent with observations that Rho and Rac can mediate apoptosis in the absence of survival signals from serum (Esteve et al., 1998; Embade et al., 2000) or mediate cell proliferation and transformation in the presence of these signals.
By largely unexplored mechanisms, mitochondrial function must be coupled to other cellular functions to accommodate changes in metabolism requirements. Mitochondria could be recruited to respond to new metabolic requirements by controlling ATP levels or lipid metabolism or, as it is in this case, to promote oxidative signaling. More interestingly, mitochondria may coordinate parallel signal transduction pathways triggered by the altered cytoarchitecture that takes place during changes in tissue organization, cell locomotion, inflammatory responses, wound healing, or tumor progression. It is interesting to note that many tumor cells have altered mitochondrial function with increased glycolytic metabolism, a process known as the Warburg effect (Racker, 1983). Altered adhesion and activation of Rac and Rho GTPase isoforms are also features of many tumor cells. Whether these processes are functionally coupled remains to be determined.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells
RSFs were isolated as described previously (Aggeler et al., 1984) and cultured between passages 3 and 10 in DME H-21 supplemented with 10% FBS (Hyclone) and 2 mM glutamine in 5% CO2 at 37°C. For the experiments, RSFs from confluent cultures were plated in DME supplemented with 0.2% lactalbumin hydrolysate (LH) on dishes coated previously for 2 h with 20 µg/ml of human fibronectin in PBS and then blocked for 30 min with 0.2% LH-DME. After 1 h to allow cell spreading, 10 µg/ml of protein Gaffinity-purified anti-5 mAb (BIIG2, a gift from Dr. C. Damsky, University of California, San Francisco, CA) was added. As controls, purified rat immunoglobulins or unrelated antibodies of the same species and isotype were used.
H2O2 assay
Confluent RSFs were plated on fibronectin-coated plates for at least 4 h before the experiments. Anti-5 mAb was added at 20-min intervals, and at the end the cells were washed with PBS and incubated with Hank's balanced salt solution without phenol red, 1 mM Hepes, 100 µM homovanillic acid, and 5 U/ml HRP type VI for 1 h at 37°C (Baggiolini et al., 1986). The samples were alkalinized with 0.1 M glycine-NaOH, pH 10, and the formation of the fluorescent product was measured in a Fluorolog 3 Instruments SA fluorimeter at excitation of 321 nm and emission at 421 nm. No reading was detected when peroxidase or substrate was omitted. Standards were prepared from a 30% wt/vol H2O2 stock solution. The limit of detection was 0.05 µM, whereas the basal activity detected was 0.1 µM. The amount of H2O2 produced was calculated by adding the three readings where anti-
5 mAbinduced H2O2 production peaked. The respiratory chain inhibitors were added at the first time of anti-
5 mAb addition (2.2 h). After the PBS wash, the residual amount of inhibitors did not interfere with the reading of a standard amount of H2O2 added (unpublished data). The readings were corrected for cell number.
Rac activation assay
To measure Rac activation, confluent cultures were cultured overnight in DME-LH. RSFs (5 x 106) were plated on fibronectin-coated plates for 4 h and then incubated with 10 µg/ml of anti-5 mAb for different periods. Cells were lysed in 500 µl of lysis buffer and 450 µl incubated with PAK1-RBD agarose following the manufacturer recommendations (Upstate Biotechnology). The total Rac present in the lysate was measured in 50 µl before incubation with PAK1-RBD and activated Rac bound to PAK-RBD were detected by Western blot.
JNK kinase assay
Transfected Cos 7 cells were lysed in 20 mM Tris, pH 7.6, 250 mM NaCl, 3 mM EDTA, 20 mM ß glycerophosphate, 0.5% (vol/vol) NP-40, 1 mM DTT, 1 mM sodium orthovanadate, and protease inhibitors. JNK was immunoprecipitated for 2 h with anti-JNK antibody (Cell Signaling) and washed two times with kinase buffer. The immune complex-associated kinase activity was measured in kinase buffer (25 mM Hepes, pH 7.5, 20 mM ß glycerophosphate, 2 mM DTT, 20 mM MgCl2, 0.1 mM sodium orthovanadate) incubated with 1 µg GST-c-jun and 5 µCi [-35S]ATP for 30 min at 30°C. After gel electrophoresis, c-jun phosphorylation was revealed by autoradiography (Sudo and Karin, 2000).
NBT staining
RSFs were incubated for 1 h at 37°C with a filtered solution of 0.3 mg/ml of NBT in Hank's balanced salt solution without phenol red during the second hour of anti-5 mAb treatment. These conditions were established to optimize signal over the background differences in staining. The cells were washed once with PBS and fixed with 0.4% paraformaldehyde for light microscopy. To quantify NBT precipitation, cells were washed twice with 70% methanol and fixed for 5 min in 100% methanol. After the wells are allowed to air dry, the formazan is solubilized with 120 µl 2M KOH and 140 µl DMSO. The OD was read in an ELISA plate reader at 590 nm (Rook et al., 1985).
JC-1 staining
RSFs were plated on glass coverslips coated with fibronectin and were stained with 10 µg/ml of JC-1 in 0.2% LH DME for 15 min at 37°C (Smiley et al., 1991). JC-1 is a hydrophobic carbocyanine compound with delocalized positive charge that allows its cellular distribution driven by negative potential; when the concentration reaches a threshold, it forms aggregates with a shift in the absorbed and emitted fluorescence. Therefore, when the membrane potential is < -100 mV the mitochondria appear green, but under conditions of high membrane potential (< -140 mV) high amounts of JC-1 aggregate and form a red fluorescent complex (Smiley et al., 1991). Cells were photographed within 5 min under epifluorescence using a FITC/TR dual band pass filter in a Leica DMR microscope.
For the cell isolation experiments, RSFs were treated for 4 h without or with 10 µg/ml of anti-5 mAb, stained with JC-1 for 15 min at 37°C, and then released with trypsin-EDTA. Soybean trypsin inhibitor was added, and the cells were sorted in a Becton Dickinson FACSVantage SE cell sorter gated to separate cells by green and orange fluorescence. 5 x 105 cells collected from each condition were plated on fibronectin-coated glass coverslips and then incubated for an additional 24 h. Secreted CL-1 in the supernatant was measured by slot blot. The number of cells recovered after 24 h was confirmed by counting nuclei after staining with DAPI.
Cytochrome C release
RSFs were incubated for 4 h without or with 10 µg/ml of ant-5 mAb and homogenized as described (Carthy et al., 1999). Cytochrome C was detected by Western blot analysis of protein in the high speed pellet (100 µg of protein) for mitochondria and supernatant (50 µg) for cytosol.
MTT assay
To quantify viable cells, 20 µl of 5 mg/ml MTT in PBS was added to 100 µl of culture medium and incubated with RSFs at 37°C for 1 h. Metabolized MTT was solubilized with 300 µl of DMSO, and color development was measured at 570 nm. Each point shown is the average of triplicates with a standard deviation too small to be represented in the graph.
Western and slot blot
CL-1 expression was measured by slot blot analysis of serial dilutions of the treated cell culture supernatants as described previously or by Western blot analysis of the cell culture supernatants using a mixture of six monoclonal antirabbit CL-1 mAb (oligoclonal) and an HRP-conjugated antimouse secondary antibody (Tremble et al., 1994). HRP binding was evidenced by enhanced luminescence reaction, and the bands were quantified by densitometry using Image Quant software for analysis. The reference value to calculate fold of induction was the basal expression of CL-1 of cells plated on fibronectin without anti-5 mAb.
Immunofluorescence
The cells were plated on fibronectin-coated glass coverslips. The monolayer was washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 for 3 min, and blocked for 1 h with 15% FBS in PBS. Primary antibodies were diluted in 15% FBS and incubated for 1 h at 37°C. After three washes with PBS 0.1% Triton X-100, the samples were incubated with Alexa-labeled secondary antibodies (Molecular Probes) for 45 min and embedded in Vectashield with DAPI (Vector Laboratories). Samples were analyzed and photographed using a Leica DMR microscope.
Plasmids and transfections
Dr. Marc Symons (Picower Institute for Medical Research, Manhasset, NY) provided the expression plasmids with RhoA V14 (Qiu et al., 1995), and Dr. Gaston Hayes (Onyx Pharmaceuticals, Richmond, CA) provided the panel of Rac mutants (Westwick et al., 1997). The NFB reporter plasmid corresponds to the ELAM promoter from 730 to +52 coupled to luciferase and was provided by Dr. Ulrike Schindler (Tularik Inc., South San Francisco, CA) (Schindler and Baichwal, 1994). To measure CL-1 induction, a minimal CL-1 promoter (-517 to 63) coupled to luciferase was used (Kheradmand et al., 1998). In control cells, empty vector or pEGFP1C (CLONTECH Laboratories, Inc.) was transfected in equal amounts. Dr. Stanley Korsmeyer (Dana Farber Cancer Institute, Harvard Medical School, Boston, MA) provided the expression vector with RSV-human Bcl-2 (Seto et al., 1988). The histone 2BGFP construct is from Dr. Jennifer Lippincott-Schwartz (National Institutes of Health, Bethesda, MD).
The point mutants of RacV12 in the residues 26 and 130 were prepared using the QuickChange site Mutagenesis Kit (Stratagene). RacV12 in pcDNA3 was used as a template with the oligo 5'-CTGATCAGTTACACAACCCATGCATTTCCTGGAG-3' to generate the H26 mutant and the oligo 5'-CGAGAAACTGAACGAGAAGAAGCTGACTCC-3' to generate the N130 change. The mutations were verified by sequencing.
Cells were transfected using adenovirus as described previously (Forsayeth and Garcia, 1994) (Kheradmand et al., 1998). Briefly, the cells were subcultured the day before. 3 x 106 cells were transfected in a 3.5-cm dish with 2.5 ml of the following mixture: adenovirus stock diluted 1:20 in serum and antibiotic-free DME, 2 µg/ml of plasmid, and 80 µg/ml DEAE-dextran. The cells and the mixture were incubated for 2 h at 37°C and then washed for 1 min with 10% DMSO in PBS and incubated overnight with DME 10% FBS. The medium was then changed to DME-0.1% LH and 24 h later. Equal numbers of cells were plated on fibronectin-coated dishes. The cells were analyzed after 24 h by measuring luciferase activity in whole cell lysates using a commercial kit (Promega). The readings were corrected by protein concentration (Bradford) or ß-galactosidase activity (Tropix).
Data analysis
The experimental data are represented as means ± SEM. Statistical comparisons were performed using the two-tailed Student's t test. Values of P < 0.05 were considered to be statistically significant.
![]() |
Footnotes |
---|
* Abbreviations used in this paper: CL-1, collagenase-1; LH, lactalbumin hydrolysate; mAb, monoclonal antibody; MMP, matrix metalloproteinase; MPT, membrane permeability transition; MTT, dimethylthiazolyldiphenyltetrazolium bromide; NBT, nitroblue tetrazolium; ROS, reactive oxygen species; RSF, rabbit synovial fibroblast.
![]() |
Acknowledgments |
---|
This work was supported by funds from the National Institutes of Health (AR20684 and CA72006) and the Ruth and Milton Steinbach Fund and a PEW International Fellowship to E. Werner.
Submitted: 8 November 2001
Revised: 12 April 2002
Accepted: 30 May 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aggeler, J., S.M. Frisch, and Z. Werb. 1984. Collagenase is a major gene product of induced rabbit synovial fibroblasts. J. Cell Biol. 98:16561661.[Abstract]
Almeida, E.A., D. Ilic, Q. Han, C.R. Hauck, F. Jin, H. Kawakatsu, D.D. Schlaepfer, and C.H. Damsky. 2000. Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase. J. Cell Biol. 149:741754.
Arcaro, A. 1998. The small GTP-binding protein Rac promotes the dissociation of gelsolin from actin filaments in neutrophils. J. Biol. Chem. 273:805813.
Azuma, T., W. Witke, T.P. Stossel, J.H. Hartwig, and D.J. Kwiatkowski. 1998. Gelsolin is a downstream effector of rac for fibroblast motility. EMBO J. 17:13621370.
Azuma, T., K. Koths, L. Flanagan, and D. Kwiatkowski. 2000. Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression. J. Biol. Chem. 275:37613766.
Bae, Y.S., S.W. Kang, M.S. Seo, I.C. Baines, E. Tekle, P.B. Chock, and S.G. Rhee. 1997. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J. Biol. Chem. 272:217221.
Barrett, W.C., J.P. DeGnore, Y.F. Keng, Z.Y. Zhang, M.B. Yim, and P.B. Chock. 1999. Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B. J. Biol. Chem. 274:3454334546.
Benard, V., B.P. Bohl, and G.M. Bokoch. 1999. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274:1319813204.
Boveris, A., N. Oshino, and B. Chance. 1972. The cellular production of hydrogen peroxide. Biochem. J. 128:617630.[Medline]
Cai, J., and D.P. Jones. 1998. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J. Biol. Chem. 273:1140111404.
Caron, E., and A. Hall. 1998. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science. 282:17171721.
Chandel, N.S., E. Maltepe, E. Goldwasser, C.E. Mathieu, M.C. Simon, and P.T. Schumacker. 1998. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl. Acad. Sci. USA. 95:1171511720.
del Peso, L., M. Gonzalez-Garcia, C. Page, R. Herrera, and G. Nunez. 1997. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 278:687689.
Droge, W. 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82:4795.
Duchen, M.R. 1999. Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J. Physiol. 516:117.
Embade, N., P.F. Valeron, S. Aznar, E. Lopez-Collazo, and J.C. Lacal. 2000. Apoptosis induced by Rac GTPase correlates with induction of FasL and ceramides production. Mol. Biol. Cell. 11:43474358.
Flohe, L., and F. Otting. 1984. Superoxide dismutase assays. Methods Enzymol. 105:93104.[Medline]
Forsayeth, J.R., and P.D. Garcia. 1994. Adenovirus-mediated transfection of cultured cells. Biotechniques. 17:354356, 357358.
Freeman, J.L., A. Abo, and J.D. Lambeth. 1996. Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65. J. Biol. Chem. 271:1979419801.
Gilmore, A.P., A.D. Metcalfe, L.H. Romer, and C.H. Streuli. 2000. Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J. Cell Biol. 149:431446.
Goossens, V., J. Grooten, K. De Vos, and W. Fiers. 1995. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc. Natl. Acad. Sci. USA. 92:81158119.[Abstract]
Gottlieb, E., M.G. Vander Heiden, and C.B. Thompson. 2000. Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis. Mol. Cell. Biol. 20:56805689.
Hockenbery, D.M., Z.N. Oltvai, X.M. Yin, C.L. Milliman, and S.J. Korsmeyer. 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 75:241251.[Medline]
Irani, K., Y. Xia, J.L. Zweier, S.J. Sollott, C.J. Der, E.R. Fearon, M. Sundaresan, T. Finkel, and P.J. Goldschmidt-Clermont. 1997. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science. 275:16491652.
Joneson, T., and D. Bar-Sagi. 1998. A Rac1 effector site controlling mitogenesis through superoxide production. J. Biol. Chem. 273:1799117994.
Joneson, T., M. McDonough, D. Bar-Sagi, and L. Van Aelst. 1996. RAC regulation of actin polymerization and proliferation by a pathway distinct from Jun kinase. Science. 274:13741376 (erratum published 276:185).
Joyce, D., B. Bouzahzah, M. Fu, C. Albanese, M. D'Amico, J. Steer, J.U. Klein, R.J. Lee, J.E. Segall, J.K. Westwick, et al. 1999. Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway. J. Biol. Chem. 274:2524525249.
Kheradmand, F., E. Werner, P. Tremble, M. Symons, and Z. Werb. 1998. Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science. 280:898902.
Koya, R.C., H. Fujita, S. Shimizu, M. Ohtsu, M. Takimoto, Y. Tsujimoto, and N. Kuzumaki. 2000. Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release. J. Biol. Chem. 275:1534315349.
Kusano, H., S. Shimizu, R.C. Koya, H. Fujita, S. Kamada, N. Kuzumaki, and Y. Tsujimoto. 2000. Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC. Oncogene. 19:48074814.[CrossRef][Medline]
Lee, A.C., B.E. Fenster, H. Ito, K. Takeda, N.S. Bae, T. Hirai, Z.X. Yu, V.J. Ferrans, B.H. Howard, and T. Finkel. 1999. Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. J. Biol. Chem. 274:79367940.
Lee, S.R., K.S. Kwon, S.R. Kim, and S.G. Rhee. 1998. Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J. Biol. Chem. 273:1536615372.
Madesh, M., and G. Hajnoczky. 2001. VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release. J. Cell Biol. 155:10031015.
Manna, S.K., H.J. Zhang, T. Yan, L.W. Oberley, and B.B. Aggarwal. 1998. Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappaB and activated protein-1. J. Biol. Chem. 273:1324513254.
Marzo, I., C. Brenner, N. Zamzami, S.A. Susin, G. Beutner, D. Brdiczka, R. Remy, Z.H. Xie, J.C. Reed, and G. Kroemer. 1998. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J. Exp. Med. 187:12611271.
Nemoto, S., K. Takeda, Z.X. Yu, V.J. Ferrans, and T. Finkel. 2000. Role for mitochondrial oxidants as regulators of cellular metabolism. Mol. Cell Biol. 20:73117318.
Nicholls, D.G., and S.L. Budd. 2000. Mitochondria and neuronal survival. Physiol. Rev. 80:315360.
Ozaki, M., S.S. Deshpande, P. Angkeow, S. Suzuki, and K. Irani. 2000. Rac1 regulates stress-induced, redox-dependent heat shock factor activation. J. Biol. Chem. 275: 3537735383.
Page, K., J. Li, J.A. Hodge, P.T. Liu, T.L. Vanden Hoek, L.B. Becker, R.G. Pestell, M.R. Rosner, and M.B. Hershenson. 1999. Characterization of a Rac1 signaling pathway to cyclin D(1) expression in airway smooth muscle cells. J. Biol. Chem. 274:2206522071.
Qiu, R.G., J. Chen, D. Kirn, F. McCormick, and M. Symons. 1995. An essential role for Rac in Ras transformation. Nature. 374:457459.[CrossRef][Medline]
Rook, G.A., J. Steele, S. Umar, and H.M. Dockrell. 1985. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J. Immunol. Methods. 82:161167.[CrossRef][Medline]
Sanchez-Alcazar, J.A., E. Schneider, M.A. Martinez, P. Carmona, I. Hernandez-Munoz, E. Siles, P. De La Torre, J. Ruiz-Cabello, I. Garcia, and J.A. Solis-Herruzo. 2000. Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells. J. Biol. Chem. 275:1335313361.
Schindler, U., and V.R. Baichwal. 1994. Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol. Cell. Biol. 14:58205831.[Abstract]
Schulze-Osthoff, K., R. Beyaert, V. Vandevoorde, G. Haegeman, and W. Fiers. 1993. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J. 12:30953104.[Abstract]
Seto, M., U. Jaeger, R.D. Hockett, W. Graninger, S. Bennett, P. Goldman, and S.J. Korsmeyer. 1988. Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. EMBO J. 7:123131.[Abstract]
Shimizu, S., Y. Eguchi, W. Kamiike, Y. Funahashi, A. Mignon, V. Lacronique, H. Matsuda, and Y. Tsujimoto. 1998. Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc. Natl. Acad. Sci. USA. 95:14551459.
Smiley, S.T., M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T.W. Smith, G.D. Steele, Jr., and L.B. Chen. 1991. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA. 88:36713675.[Abstract]
Smith, J., E. Ladi, M. Mayer-Proschel, and M. Noble. 2000. Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell. Proc. Natl. Acad. Sci. USA. 97:1003210037.
Sudo, T., and M. Karin. 2000. Assays for JNK and p38 mitogen-activated protein kinases. Methods Enzymol. 322:388392.[CrossRef][Medline]
Sundaresan, M., Z.X. Yu, V.J. Ferrans, K. Irani, and T. Finkel. 1995. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 270:296299.[Abstract]
Suzukawa, K., K. Miura, J. Mitsushita, J. Resau, K. Hirose, R. Crystal, and T. Kamata. 2000. Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species. J. Biol. Chem. 275:1317513178.
Thannickal, V.J., and B.L. Fanburg. 2000. Reactive oxygen species in cell signaling. Am. J. Physiol. Lung Cell. Mol. Physiol. 279:L1005L1028.
Tremble, P., R. Chiquet-Ehrismann, and Z. Werb. 1994. The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts. Mol. Biol. Cell. 5:439453.[Abstract]
Vanden Hoek, T.L., Z. Shao, C. Li, P.T. Schumacker, and L.B. Becker. 1997. Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J. Mol. Cell. Cardiol. 29:24412450.[CrossRef][Medline]
Vander Heiden, M.G., N.S. Chandel, X.X. Li, P.T. Schumacker, M. Colombini, and C.B. Thompson. 2000. Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc. Natl. Acad. Sci. USA. 97:46664671.
Werner, E., F. Kheradmand, R. Isberg, and Z. Werb. 2001. Phagocytosis mediated by Yersinia invasin induces collagenase-1 expression in rabbit synovial fibroblasts through a pro-inflammatory cascade. J. Cell Sci. 114:33333343.
Westwick, J.K., Q.T. Lambert, G.J. Clark, M. Symons, L. Van Aelst, R.G. Pestell, and C.J. Der. 1997. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways. Mol. Cell. Biol. 17:13241335.[Abstract]
Woo, C.H., Y.W. Eom, M.H. Yoo, H.J. You, H.J. Han, W.K. Song, Y.J. Yoo, J.S. Chun, and J.H. Kim. 2000. Tumor necrosis factor- generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J. Biol. Chem. 275:3235732362.
Yang, J., X. Liu, K. Bhalla, C.N. Kim, A.M. Ibrado, J. Cai, T.I. Peng, D.P. Jones, and X. Wang. 1997. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 275:11291132.
Zhai, J., H. Lin, M. Shamim, W.W. Schlaepfer, and R. Canete-Soler. 2001. Identification of a novel interaction of 14-3-3 with p190RhoGEF. J. Biol. Chem. 276:4131841324.
Zhou, Y.P., J.C. Pena, M.W. Roe, A. Mittal, M. Levisetti, A.C. Baldwin, W. Pugh, D. Ostrega, N. Ahmed, V.P. Bindokas, et al. 2000. Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion. Am. J. Physiol. Endocrinol. Metab. 278:E340E351.