©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Rac-dependent and -independent Pathways Mediate Growth Factor-induced Ca Influx (*)

(Received for publication, January 4, 1996)

Maikel P. Peppelenbosch(§) (1) Leon G. J. Tertoolen(§) (2) Alida M. M. de Vries-Smits (1) Rong-Guo Qiu (3) Laura M'Rabet (1) Marc H. Symons (3) Siegfried W. de Laat (2) Johannes L. Bos (1)(¶)

From the  (1)Laboratory for Physiological Chemistry, Utrecht University, Universiteitsweg 100, NL-3584 CG Utrecht, The Netherlands, (2)Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands, and (3)Onyx Pharmaceuticals, Richmond, California 94806

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We report that expressing interfering mutants of the small Ras-related GTPase Rac, using either recombinant vaccinia virus or stable DNA transfection, eliminates epidermal growth factor-induced Ca signaling, without affecting Ca mobilization or influx from G protein-coupled receptors. Platelet-derived growth factor-dependent Ca influx, however, is only partly sensitive to dominant negative Rac proteins. Thus, whereas epidermal growth factor-induced Ca influx is completely mediated by Rac proteins, platelet-derived growth factor-induced Ca influx involves Rac-dependent and -independent signaling pathways.


INTRODUCTION

Epidermal growth factor (EGF) (^1)and its receptor are important regulators of cell growth and differentiation. Binding of EGF to its receptor results in receptor dimerization and subsequent activation of the EGF receptor tyrosine kinase, which mediates further signal transduction(1, 2, 3, 4) . Among the biochemical changes initiated by the EGF receptor is a transient increase in [Ca](5) , which is essential for EGF-induced proliferation(6, 7) . This Ca transient has been linked to EGF-stimulated hydrolysis of polyphosphoinositides by phospholipase C, as the resulting production of inositol 1,4,5-trisphosphate causes release of Ca from intracellular stores(8, 9) . Ca release, however, constitutes only a minor part of the overall EGF-induced elevation in [Ca], since the major part originates from the activation of voltage-independent, Ca-specific ion channels, resulting in a Ca influx through the plasma membrane(5, 10, 11, 12, 13) .

The molecular mechanism, responsible for the induction of this Ca influx, is unclear. Several types of Ca-permeable channels are activated in response to EGF, including a 10-pS Ca-selective conductance(12, 14) . Stimulation of the latter channel requires the liberation of AA from cellular membranes and its subsequent conversion to leukotrienes(15, 16) . The importance of activation of the 10-pS Ca channel, with respect to the EGF-induced Ca transient, is at present unclear. Apart from EGF, other growth factors open plasma membrane-localized calcium channels as well. PDGF, for instance, is a potent inducer of Ca influx in Rat-1 fibroblasts(17) . The underlying Ca channels, as well as the signaling mechanisms leading to their activation, have remained unexplored.

A candidate molecule to function in the signaling upstream of growth factor-induced Ca influx is the Rac protein. Rac is a member of the Rho family of Ras-related GTPases (for a review, see (18) ). Recently, it has been shown that Rac has an essential function in the cellular transformation induced by oncogenic Ras(19) , although the downstream elements of the Rac signaling cascade leading to transformation are not clear. Rac proteins are implicated in growth factor-induced cytoskeletal remodeling. For instance, Rac mediates polymerization of actin at the plasma membrane (so-called ruffling (20) ). Rac also activates the Rho protein, an event necessary for growth factor-dependent stress fiber formation(21) . Interestingly, apart from Rac, AA release and subsequent leukotriene production are implicated in growth factor-induced actin remodeling as well(22) , suggesting a relationship between Rac and AA metabolism. Indeed, we recently established that growth factor-induced AA release requires functional Rac protein and that Rac-dependent Rho activation is dependent on the metabolism of AA to leukotrienes(23) . This connection between Rac and leukotriene production and the requirement for leukotriene synthesis in the EGF-dependent activation of the 10-pS Ca channel therefore raise the possibility that Ca influx is one of the effectors of the Rac signaling cascade.

In the present study, we have investigated the possible relationship between the Rac signaling and the growth factor-dependent Ca transient. The results show that EGF-induced Ca signaling is mediated by Rac. PDGF-induced Ca influx, however, is only partly dependent on functional Rac protein. These results identify Ca influx as a Rac-specific effector and define this Rac-mediated Ca influx as the major pathway by which EGF activates Ca influx.


EXPERIMENTAL PROCEDURES

Measurements of [Ca](i)

Cells were grown according to routine procedures. For [Ca](i) determinations, cells were grown on glass coverslips and put on 0.5% fetal calf serum containing medium for 16 h. Cells were loaded with 5 µM indo-1-AM (Molecular Probes) for 45 min at 33 °C in Hepes-buffered, 10 mM glucose-supplemented saline, washed, and measured with a Perkin-Elmer 3000 fluorescence spectrometer in the same medium at 33 °C. Excitation wavelength was 355 nm (slit, 5 nm), and emission wavelength was 405 nM (slit, 10 nm). Calibration of the fluorescent signal was performed as described earlier(5) .

Construction and Expression of Recombinant Vaccinia Viruses

For expression of mutated Rac proteins a Rac N17 cDNA and a Rac wild type cDNA were introduced in a viral growth factor minus strain of vaccinia virus by homologous recombination as described earlier(24, 25) . The authenticity of the constructs was confirmed by sequence analysis. Viruses were grown in RK-13 cells. HeLa cells were infected with 10-20 plaque-forming units/cell for 60 min, after which the medium was replaced with 0.5% fetal calf serum-containing medium. Experiments were performed 16-20 h later.

Expression of Rac and Rho Proteins in Rat-1 Fibroblasts

Rat-1 cells expressing mutant Rac and Rho proteins are described elsewhere(19) . In brief, a plasmid expressing a tetracyclin-controlled transactivator was transfected into Rat-1 cells in the presence of a G418 resistance marker, and subsequently a construct expressing Rac and Rho mutants controlled by a hybrid element containing a tet operator and a cytomegalovirus minimal promoter was transfected in the presence of a puromycin resistance marker. The presence of the Rac and the Rho protein was established by Western blotting. The transfected Rat-1 cells were grown in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal calf serum, 0.4 mg/ml G418, 1 µg/ml puromycin, and 2 µg/ml tetracyclin. Tetracyclin was withdrawn 48 h before experimentation, which resulted in a 2-3-fold increase in expression of the transfected genes. 16 h prior to experimentation, the fetal calf serum concentration was reduced to 0.5%.


RESULTS

Vaccinia Virus-mediated Expression of Dominant Negative Rac Proteins Inhibits EGF-induced Ca Signaling

For assessing the role of Rac in the EGF-induced Ca transient, we introduced a dominant negative mutant of Rac, Rac N17 (21, 26, 27) , into HeLa cells. We employed a vaccinia virus expression system, which we have shown earlier to be a successful tool for studying the role of Ras and Rac in signal transduction(23, 24, 25) . The Ca signaling in uninfected HeLa cells, HeLa cells infected with vv-wt, and cells infected with vv-Rac N17 was investigated. Uninfected and vv-wt-infected HeLa cells showed, upon addition of EGF (50 ng/ml), a Ca response having a slow onset (the effect is first noted 10-20 s after application), an amplitude of approximately 100 nM (Fig. 1; Table 1), and a return to basal levels within 3 min. Influx of Ca from the extracellular medium underlies this transient, as it is not observed in the presence of EGTA or La (not shown). Also HeLa cells infected with a vaccinia virus expressing non-mutant Rac proteins were not at all inhibited with respect to the EGF-induced Ca response but showed enhanced Ca signaling when challenged with EGF (Table 1). In contrast, vv-Rac N17-infected HeLa cells did not show any increase in [Ca](i) upon the application of EGF (Fig. 1; Table 1). Therefore, the EGF-dependent Ca transient in HeLa cells requires functional Rac protein.


Figure 1: Inhibition of the EGF-induced Ca transient by Rac N17. Effects are shown of EGF and histamine (HA) on the intracellular Ca concentration in indo-2-AM-loaded HeLa cells (Hela), in wild type vaccinia virus-infected HeLa cells (Hela vv-WT), and in HeLa cells infected with a vaccinia virus expressing Rac N17 (Hela vv-Rac N17). Also the effects of EGF on ERK2 phosphorylation (as assayed by altered gel mobility; for experimental procedures, see (17) ) in vv-wt-infected cells and vv-Rac N17-infected HeLa cells are shown.





The specificity of the inhibitory effect of Rac N17 on EGF-induced Ca signaling was demonstrated in experiments in which the effect of vv-Rac N17 infection on the EGF-dependent phosphorylation of ERK2 was analyzed. This phosphorylation of ERK2 is mediated by a complex signal transduction cascade (reviewed in e.g.(28) ). vv-Rac N17 infection did not inhibit EGF-dependent ERK2 phosphorylation in comparison with uninfected or vv-wt-infected cells (Fig. 1). We concluded that Rac proteins act specifically in the signal transduction pathway leading to EGF-induced Ca signaling.

Histamine-induced Ca Signals Are Not Dependent on Functional Rac Proteins

Apart from EGF, histamine also can induce a Ca transient in HeLa cells. Addition of 1 µM histamine to HeLa cells gives rise to a H(1) receptor-mediated biphasic increase in [Ca](i). The first phase is immediate upon application of histamine, is EGTA-insensitive, and is mediated by inositol 1,4,5-trisphosphate production. The second phase consists of an EGTA- and La-sensitive Ca influx through the plasma membrane(29, 30) . Importantly, vv-Rac N17 infection inhibited neither histamine-induced Ca release nor histamine-dependent Ca influx, when compared with uninfected or vv-wt-infected cells (Fig. 1). Therefore, Rac N17 is not a general inhibitor of cellular Ca signaling.

Expression of Rac N17 by Stable DNA Transfection Inhibits the EGF-dependent Ca Transient in Rat-1 Fibroblasts

Independent support for a role of Rac in EGF-induced Ca influx was obtained from experiments in which we tested Ca signaling in Rat-1 fibroblasts in which mutant Rac proteins were introduced by stable DNA transfection(19) . In a Rac N17 expressing clone, Rac function was clearly inhibited, as EGF did not produce ruffling in this clone. Significantly, addition of EGF (50 ng/ml) to these cells did not yield any elevation of [Ca](i), whereas endothelin (1 µM) and ATP (50 µM) elicited a substantial increase in [Ca](i) (Fig. 2; Table 1). In contrast, application of EGF to untransfected Rat-1 cells or Rat-1 cells transfected with an empty transfection vector gave rise to a transient increase in [Ca](i) with a maximal amplitude of approximately 75 nM (Fig. 2; Table 1). EGF-induced receptor autophosphorylation (not shown) or ERK2 activation (Fig. 2) was not affected by the expression of the Rac N17 protein. Together, these results provide strong support for the notion that Rac proteins are implicated in the signal transduction cascade leading to EGF-induced Ca influx.


Figure 2: Effects of EGF and ATP on [Ca] in Rat-1 fibroblasts (Rat1), Rat-1 fibroblasts expressing an empty transfection vector (Rat1 Vector Control), Rat-1 fibroblasts expressing Rac N17 (Rat1 Rac N17), and Rat-1 fibroblasts expressing Rac V12 (Rat1 Rac Val12). Also the effects of EGF on ERK2 gel mobility in vector control, Rac N17-expressing, and Rac V12-expressing Rat-1 fibroblasts are shown.



Effects of Rac V12 Expression

If Rac activation is sufficient to produce Ca influx, constitutive activation of Rac would be expected to maximally activate the signaling pathway leading to Ca influx and therefore abolish the ability of EGF to increase [Ca](i). In contrast, if Rac acts as an essential cofactor in this signaling pathway, constitutive Rac activation would be expected to lead to enhanced Ca signaling in response to EGF. To distinguish between these two possibilities, a mutant Rac V12 protein was introduced into Rat-1 fibroblasts by DNA transfection(19) . Two resulting clones expressing the activated Rac protein show constitutive ruffling and were tested for EGF-dependent Ca signaling. The highly transient nature of the growth factor-produced Ca signal implies the presence of efficient feedback mechanisms. We were therefore not surprised not to observe elevated basal levels of [Ca](i), especially as high Ca levels are not compatible with long term cell survival. Importantly, neither of the clones displayed any increase in [Ca](i) when challenged with EGF, although responses to endothelin and ATP were normal (Fig. 2; Table 1). Also EGF-induced receptor autophosphorylation and ERK2 activation were not affected by the expression of the mutated protein (Fig. 2). These findings imply that Rac V12 is not synergizing with some other signal in the activation of Ca channels but that Rac mediates a single pathway from the EGF receptor to the activation of Ca channels.

PDGF-induced Ca Influx Involves Rac-dependent and -independent Signaling Mechanisms

In order to test whether the role of Rac in the induction of Ca influx is limited to the EGF receptor signal transduction, we investigated PDGF-induced Ca signaling in wild type Rat-1 fibroblasts, Rat-1 fibroblasts expressing Rac N17, and Rat-1 cells containing an empty transfection vector. In wild type Rat-1 cells and Rat-1 fibroblasts and cells transfected with an empty transfection vector, PDGF produced an increase in [Ca](i) of approximately 250 nM (Fig. 3; Table 2). This response is dependent on Ca influx, as it is completely inhibited by application of EGTA (not shown). Importantly, Ca signaling in response to PDGF in the Rac N17-expressing clone was significantly reduced but not completely eliminated (Fig. 3; Table 2). In Rac N17-expressing cells, PDGF-induced activation of Rac signal transduction was completely inhibited, as PDGF did no longer increase leukotriene production in these cells (Table 2). Therefore, PDGF-dependent Ca influx involves Rac-dependent and -independent signal transduction pathways.


Figure 3: PDGF-induced Ca transients in Rat-1 fibroblasts and Rat-1 fibroblasts expressing Rac N17.





Ca Signaling Is Not Abolished by Rho V14 or Rho N19 Expression

The observed inhibition of the growth factor-induced Ca transient by expressing interfering mutants of the Rac protein raises questions as to the specificity of these effects. We therefore have tested the consequences of expressing mutants of Rho, as Rho and Rac are highly homologous proteins. Clones of Rat-1 fibroblasts stably expressing either activated Rho (Rho V14) or dominant negative Rho (Rho N19) were isolated. The Rho V14-expressing cell line had a very high number of stress fibers in comparison with the parental line, whereas the Rho N19 cell line had reduced stress fibers (not shown). Significantly, in both cell lines EGF induced Ca transients. For the Rho V14 cell line, this calcium transient was similar as the parental Rat-1 cell line (Fig. 4; Table 1and Table 2). The Rho N19 cell line showed a reduced calcium influx compared with the the parental cell line, probably due to the fact that the Rho N19 cell line showed reduced viability upon serum starvation. We concluded that expression of either constitutively active or dominant negative Rho-like proteins per se does not inhibit growth factor-induced Ca influx.


Figure 4: Effects of EGF and ATP in Rho V14-expressing Rat-1 fibroblasts.




DISCUSSION

In the present study, we have investigated a possible relationship between the growth factor-induced Ca transient and the small GTPase Rac. We showed that expressing interfering mutants of the Rac protein, using either a vaccinia virus expression system or stable DNA transfection, abolished Ca signaling in response to EGF but did not affect the Ca transients induced by histamine, endothelin, and ATP. PDGF-induced Ca influx, however, was only partly inhibited by Rac N17 expression, showing that the PDGF receptor can activate Ca channels via Rac-dependent and -independent signaling pathways. We concluded that Ca influx is a Rac-specific effector mechanism and that this Rac-dependent Ca influx is the major signaling pathway mediating the EGF-induced increase in [Ca](i).

Our results are the first example for a role of a small GTPase in the control of intracellular Ca levels. Ca is an important second messenger in the cell, and our finding that Rac mediates growth factor-induced calcium influx stresses the importance of this protein in cellular processes. In addition to calcium influx, Rac mediates growth factor-induced release of AA (23) and is implicated in the control of the JNK/SAPK pathway (31, 32) as well as in the stimulation of the mammalian STE20 homologue PAK65(33) . In particular the finding that Rac mediates AA metabolism is interesting, since in patch clamp analysis we have shown that the opening of a 10-pS calcium channel by EGF can be blocked by inhibitors of AA metabolism and can be partially restored by leukotrienes(16) . Therefore, Rac-mediated AA release and leukotriene production may account for the effects of Rac N17 expression on Ca signaling. Alternatively, Rac-dependent Ca influx may act upstream of AA release, as activation of the cytosolic form of phospholipase A(2) is Ca-dependent(23) .

Expression of Rac N17 caused a 50-70% decrease in the PDGF-induced Ca transient in Rat-1 fibroblasts. Therefore, a function of Rac in the production of Ca influx is not limited to the EGF receptor. The PDGF receptor, however, can apparently partly bypass the requirement for Rac for elevating [Ca](i), since expression of Rac N17 was not able to completely inhibit this response. In agreement, in cells expressing PDGF receptors lacking the kinase insert domain, PDGF does not activate Rac (34) but still induces Ca signaling. (^2)Therefore, whereas the EGF-induced increase in [Ca](i) is mostly mediated by Rac, the PDGF receptor employs Rac N17-sensitive and -insensitive signal transduction pathways to produce Ca signaling. Interestingly, in cell-attached patch clamp experiments, PDGF, but not EGF, activates a 6-pS Ca-permeable ion channel, (^3)which may mediate this Rac N17-insensitive Ca influx.

The functioning of Rac in signal transduction is most clear with respect to the growth factor-induced cytoskeletal reorganization. Rac mediates the formation of lamellipodia and membrane ruffles(21) . Furthermore, as an upstream regulator of Rho, it also regulates stress fiber formation(20) . The biochemical pathways, leading to cytoskeletal reorganization, are not clear. Importantly, the activity of many actin-controlling proteins is highly dependent on [Ca](i). Increases in [Ca](i) directly activate actin filament-severing proteins(35) . Preventing Ca signaling, by intracellular Ca chelation with quin-2, inhibits lamellipodia formation upon activation of human blood platelets(36) . This suggests that at least some of the cytoskeletal effects of Rac require an increase in [Ca](i). Therefore, Rac-mediated Ca influx may serve as one of the biochemical signals by which Rac proteins effect cytoskeletal organization.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors made equal contributions to this work.

To whom correspondence should be addressed. Tel.: 31-30-538989/8977; Fax: 31-30-539035.

(^1)
The abbreviations used are: EGF, epidermal growth factor; pS, picosiemens; AA, arachidonic acid; ERK2, extracellular signal-regulated kinase 2; PDGF, platelet-derived growth factor; vv-wt, wild type vaccinia virus; vv-Rac N17, recombinant vaccinia virus expressing Rac N17; [Ca], intracellular Ca concentration.

(^2)
L. G. J. Tertoolen, unpublished observations.

(^3)
M. P. Peppelenbosch, unpublished observations.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.