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Epidermal Growth Factor Activates Store-Operated Calcium Channels in Human Glomerular Mesangial Cells

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska.

Correspondence to Dr. Steven C. Sansom, Department of Physiology and Biophysics, University of Nebraska Medical Center, 984575 Nebraska Medical Center, Omaha, NE 68198-4575. Phone: 402-559-2919; Fax: 402-559-4438. E-mail: ssansom{at}unmc.edu

	Abstract

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Abstract. A cellular influx of Ca²⁺ is critical for initiating and maintaining growth in a variety of cell types. Experiments were performed to determine whether epidermal growth factor (EGF), which is known to initiate a proliferative response in mesangial cells, could regulate by intracellular signal transduction the store-operated Ca²⁺ channels (SOC) of human mesangial cells (HMC) in culture. The cell-attached patch configuration was used to monitor the activity of SOC, with 90 mM Ba²⁺ in the pipette and physiologic saline solution in the bath. Under control conditions, the mean NP_o value was 1.06 at a holding potential of -80 mV. When 100 nM EGF was added to the bath, SOC were activated by 53%. The EGF-evoked response was dose-dependent, with a half-maximal activation concentration of 4.8 nM. An inhibitor of tyrosine kinase, i.e., tyrphostin A23 (100 µM), completely abolished EGF-evoked channel activation. EGF combined with the inactive control compound tyrphostin A1 (100 µM) elicited significant (85%) activation of SOC. Calphostin C, an inhibitor of protein kinase C (PKC), did not affect the baseline activity of SOC but abolished the EGF-evoked enhancement of SOC activity. The PKC activator phorbol-12-myristate-13-acetate (PMA) significantly activated SOC. However, the effects of PMA were duplicative rather than additive or potentiating with maximal concentrations (100 nM) of EGF, suggesting that PMA and EGF activate SOC through a common PKC pathway. In addition, downregulation of PKC via incubation of HMC with PMA for 1 to 20 h depressed both basal activity and EGF-induced activation of SOC. It is concluded that EGF stimulates SOC in HMC through an intracellular signaling mechanism involving tyrosine kinase and PKC.

	Introduction

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Glomerular mesangial cells are normally contractile cells strategically oriented among the glomerular capillary network to regulate filtration surface area. Several ion-selective channels are thought to be involved in the mesangial contractile process, including large-conductance, Ca²⁺-activated K⁺ channels (1) and voltage-dependent, L-type Ca²⁺ channels (2).

Although mesangial cells are normally contractile, in certain pathophysiologic states, such as diabetes mellitus, glomerular mesangial cells proliferate as an autocrine response to secreted cytokines and growth factors (3). Several growth factors, including epidermal growth factor (EGF), are involved in diabetic proliferation of mesangial cells (4). EGF stimulates proliferation of nonexcitable cells (5,6,7,8) and initiates both proliferation (9,10,11) and contraction (12) of certain excitable cells, such as the smooth muscle of rat thoracic aorta (12). Studies suggest that proliferative responses to growth factors are dependent on the cellular influx of Ca²⁺ through Ca²⁺-selective channels. In A 431 carcinoma cells, EGF activated a 10-pS Ca²⁺ channel in excised or cell-attached patches (13). In cultured rat mesangial cells, Ma et al. (14) observed that platelet-derived growth factor (PDGF), when added to the pipette solution in patch-clamp experiments, dramatically stimulated a "receptor-operated" Ca²⁺ channel (ROC).

Recently, a small Ca²⁺-selective channel of approximately 2 pS was detected in human mesangial cells (HMC) using patch-clamp techniques (15). The properties of this channel were consistent with those defined for store-operated Ca²⁺ channels (SOC), which represent the predominant Ca²⁺ entry pathway in nonexcitable cells (16,17,18). The SOC are governed by the Ca²⁺ content of agonist-sensitive intracellular stores. However, the SOC are not only involved in replenishing intracellular stores of Ca²⁺. In nonexcitable cells, an influx of Ca²⁺ via SOC regulates several diverse processes, such as apoptosis, exocytosis, enzyme control, gene regulation, and cell proliferation. Identification of SOC in vascular smooth muscle (19) and HMC (20,21) has led to the hypothesis that SOC are involved in stimulating and maintaining growth of these excitable cells.

It is now generally accepted that the binding of EGF to its receptor results in enhanced receptor-receptor affinity, thereby causing receptor dimerization. In turn, this initiates the activation of EGF receptor tyrosine kinase, which is essential for further signal transduction (5). The initial step of the intracellular signaling pathway involves the phosphorylation of a group of substrates, among which are phospholipase C- and various protein kinases. Activation of phospholipase C- results in the generation of two intracellular second messengers, i.e., diacylglycerol and inositol-1,4,5-trisphosphate (IP₃). Diacylglycerol activates protein kinase C (PKC), which induces a variety of cellular effects. PKC participation in cell growth was previously demonstrated. For example, downregulation of PKC inhibits insulin-like growth factor-1-induced proliferation of vascular smooth muscle cells (22). Moreover, PKC plays a pivotal role in the cellular proliferation of mononuclear cells (23). We therefore tested the hypothesis that EGF stimulation consists of an intracellular signaling cascade involving tyrosine kinase and PKC, in which Ca²⁺ can enter the cell via plasma membrane SOC.

	Materials and Methods

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HMC Cultures
The procedure for culturing HMC was described previously (2). Briefly, HMC were cultured in Dulbecco's modified Eagle's medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10 mM Hepes, 2.0 mM glutamine, 0.66 U/ml insulin, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20% fetal bovine serum. The pH value of the medium was adjusted to 7.2 to 7.4. After reaching confluence, cells were studied within 56 h after passage onto 22 x 22 x 1-mm cover glasses (Fisher Scientific, Pittsburgh, PA). Only subpassages of <11 generations were used in this study.

Patch-Clamp Recordings
Glass pipettes (plain; Fisher Scientific) were prepared with a patch pipette puller and a pipette polisher (PP-830 and MF-830, respectively; Narishige, Tokyo, Japan). Single-channel patch-clamp configurations were established on the membranes of HMC cultured on glass coverslips.

Single-channel analysis was performed using standard patch-clamp techniques (2,24,25). The patch pipette, which was partially filled with solution, was in contact with an Ag-AgCl wire on a polycarbonate holder connected to the headstage of a patch clamp (PC-501A; Warner Instrument Corp., Hamden, CT). The pipettes were lowered onto the cell membrane, and suction was applied to obtain a high-resistance (>10-G) seal. All experiments were conducted at room temperature (22 to 23°C). Data were digitized for single-channel analysis using an analog-to-digital interface (Axon Instruments, Foster City, CA) and were recorded by a computer system. The low-pass filter was set at 500 Hz.

Single-Channel and Statistical Analyses
The unitary current (i), defined as zero for the closed state (C), was determined as the mean of the best-fit Gaussian distribution of the amplitude histograms. Channels were considered to be in an open state (O) when the total current (I) was >(n - 1/2)I and <(n + 1/2)I, also meant for this term?` where n is the maximal number of current levels observed. The open probability (P_o) was defined as the time spent in an open state divided by the total time of the analyzed record. When multiple channels occupied a patch, the channel activity was calculated as NP_o = nP_n, where P_n is the probability of finding n channels open. Therefore, NP_o could be calculated without making assumptions regarding the total number of channels in a patch or the open probability for individual single channels. The Axoscope acquisition program and pClamp program, version 6.02 (Axon Instruments, Foster City, CA), were used to record and analyze currents.

All NP_o values were calculated from 10 s of single-channel recording and are reported as mean ± SEM. Whenever possible, experiments were conducted in a paired manner, with each patch acting as its own control. In these cases, the average change in NP_o for a group of experiments (i.e., before versus after an experimental manipulation) was analyzed using a paired t test. One-way ANOVA, followed by Student-Newman-Keuls test, was used for multiple comparisons. Significance was established as P < 0.05. Statistical analyses were performed using SigmaStat software (Jandel Scientific, San Rafael, CA).

Solutions and Chemicals
The extracellular bath solution contained 140 mM KCl, 10 mM Hepes, and 1 mM CaCl₂. The pipette solution contained 90 mM BaCl₂ and 10 mM Hepes. The pH of all solutions was adjusted to 7.4. EGF, tyrphostin A1 and A23, calphostin C, and phorbol-12-myristate-13-acetate (PMA) were purchased from Calbiochem (La Jolla, CA). Lanthanum chloride was obtained from Sigma.

	Results

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Effects of EGF on SOC
As demonstrated in a previous study, with 90 mM BaCl₂ in the pipette solution, the SOC is identified in cell-attached patches as a highly abundant but low (approximately 2-pS)-conductance channel with voltage-independent gating properties (26). This SOC is also identified as being nearly completely inhibited by the addition of 2 µM La³⁺ to the pipette solution (26). In cell-attached patches, SOC is abundantly expressed and active under basal unstimulated conditions. Figure 1A shows representative tracings for SOC under control conditions (NP_o = 0.75) (Figure 1A, top tracing), with 2 µM La³⁺ in the pipette (NP_o = 0.15) (Figure 1A, middle tracing), and 30 s after addition of 100 nM EGF to the bathing solution (NP_o = 1.35) (Figure 1A, bottom tracing). For each condition, the holding potential was -80 mV. The time course for the effects of EGF is shown in Figure 1B. After a 10-s exposure to 100 nM EGF, the response of SOC was significantly increased, from a NP_o of 1.07 ± 0.05 to 1.28 ± 0.10. The NP_o remained significantly elevated (1.44 ± 0.07) 3 min after addition of EGF. As demonstrated in Figure 1C, the single-channel conductance was slightly but significantly increased after EGF administration; however, the reversal potential was not significantly different.

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effects of addition of epidermal growth factor (EGF) to the extrapipette (bath) solution on store-operated Ca2+ channels (SOC), monitored in the cell-attached configuration. (A) Representative tracings of SOC under control conditions (top), with 2 µM La3+ in the pipette (middle), and 10 s after addition of 100 nM EGF to the bathing solution (bottom), at a holding potential of -80 mV. The pipette solution contained 90 mM BaCl2. The bathing solution contained 140 mM KCl. Arrows indicate the closed state. Dashed lines indicate the different current levels. Inward currents are downward. (B) Plot of NPo versus time of exposure to 100 nM EGF (holding potential, -80 mV). *, P < 0.05, compared with the value at time 0 (before EGF addition). (C) Plot of current (I) versus holding potential (-VP) before and after addition of 100 nM EGF to the bathing solution. The calculated single-channel conductances were 2.1 ± 0.4 pS under control conditions and 2.8 ± 0.3 pS after EGF addition. The reversal potentials (extrapolated) were 67.5 ± 17.9 and 50.0 ± 3.5 mV before and after EGF addition, respectively.

The dose-response relationship for the EGF-evoked activation of SOC is presented in Figure 2. The percentage increase in NP_o reached statistical significance at EGF concentrations as low as 5 nM and was maximal at concentrations between 10 and 100 nM. The half-maximal activation concentration for EGF was 4.8 nM.

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Plot of EGF concentration (logarithmic scale) versus response of SOC, as determined by measuring the percentage increase in NPo. The half-maximal activation concentration of EGF was 4.8 nM. *, P < 0.05, compared with the corresponding baseline value.

Role of Tyrosine Kinase Activity in Channel Activation by EGF
Like other growth factors, EGF binds to its receptor to activate the catalytic activity of tyrosine kinase, resulting in tyrosine autophosphorylation followed by a cascade of intracellular signal transduction events (5). To determine whether this initial tyrosine kinase step contributed to EGF-induced activation of SOC, the EGF-evoked response was examined in the presence of tyrphostin A23, a specific inhibitor of tyrosine kinase. Figure 3 is a bar graph summarizing the effects of EGF (100 nM) alone and in combination with either the active inhibitor tyrphostin A23 (100 µM) or the nonactive inhibitor tyrphostin A1 (100 µM). As shown, tyrphostin A23 completely inhibited the effects of EGF; however, in combination with tyrphostin A1 (which was used as a negative control for tyrphostin A23), EGF evoked a significant increase in channel activity.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Bar graph illustrating the effects of the tyrosine kinase inhibitor tyrphostin A23 and the inactive control compound tyrphostin A1 on the response (NPo) of SOC to 100 nM EGF. *, P < 0.05, compared with the control value (before treatment).

Role of PKC in EGF-Induced Activation of SOC
It was postulated that PKC is an important mediator of the EGF-evoked activation of SOC. This hypothesis was tested by examining the effects of specific activators and blockers of the PKC system on EGF-evoked responses. These results are summarized in Figure 4. As shown, EGF alone, PMA (100 µM) alone, and the combination of EGF plus PMA produced similar levels (40 to 55%) of SOC activation. These results are consistent with the idea that PMA and EGF both activate SOC through the PKC transduction pathway. Calphostin C (1 µM), a selective inhibitor of PKC, completely abolished EGF-induced channel activation; however, calphostin C did not affect the basal activity of SOC.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Role of protein kinase C (PKC) in EGF-evoked activation of SOC. The bar graph illustrates the effects of the PKC inhibitor calphostin C and the PKC activator phorbol-12-myristate-13-acetate (PMA) on the basal SOC activity and the EGF-evoked activation of SOC. EGF (100 nM) alone activated SOC by 50%. Calphostin C alone did not affect SOC but inhibited the EGF-evoked response. PMA and PMA plus EGF activated SOC by 40 and 45%, respectively. *, P < 0.05, compared with the corresponding baseline value. , P < 0.05, compared with EGF.

In other systems, phorbol esters such as PMA stimulate PKC within the first 15 min; however, after 1 h, PKC is down-regulated because of an increase in degradation (27). Therefore, to further establish the involvement of PKC, the activity of SOC was determined after exposure of the HMC to PMA for 1 h. As demonstrated in Figure 5, incubation of HMC with 100 µM PMA for 1 to 20 h significantly inhibited not only the baseline activity of SOC but also the EGF-induced activation. This inhibition was not time-dependent after 1 h, because no difference could be observed among different time periods (Figure 5).

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Bar graph illustrating downregulation of PKC activity by long-term (1- to 20-h) incubation of mesangial cells with PMA. Basal and EGF-evoked activation (NPo) of SOC was significantly decreased after 1 to 2 h and remained decreased after 10 to 20 h. *, P < 0.05, 100 nM EGF versus the corresponding baseline value. , P < 0.05, baseline value at each time point of PMA treatment versus the baseline value at time 0 (without PMA treatment).

	Discussion

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Role of SOC
The results of this study demonstrated that EGF, when added to the bathing solution of HMC in culture, could activate a low-conductance Ca²⁺ channel with properties consistent with those of the SOC. The EGF-evoked activation of SOC was abolished by inhibitors of tyrosine kinase and PKC. Moreover, the effects of short-term application of PMA on SOC were duplicated but not potentiated by maximal concentrations of EGF. Therefore, it is proposed that EGF activates SOC of mesangial cells via an intracellular signal transduction pathway involving tyrosine kinase and PKC.

Several observations suggest that the SOC plays a pivotal role in cell proliferation and differentiation. For example, a form of T cell immunodeficiency with impaired lymphocyte proliferation is marked by an absence of SOC-mediated Ca²⁺ influx and the failure to express an agonist-stimulated increase in the cytoplasmic Ca²⁺ concentration (28). A study using fluorescence microscopy found that capacitative Ca²⁺ entry in proliferating vascular smooth muscle cells was fivefold greater than that in growth-arrested cells (19).

Activation of SOC by EGF
Previous studies using fluorescence microscopy revealed a store-operated Ca²⁺ entry pathway in HMC in culture (20,21). The electrophysiologic equivalent of this pathway was recently identified in patch-clamp experiments, which demonstrated a low-conductance (2 pS with barium as the charge carrier), voltage-independent channel that was sensitive to block by La³⁺, compared with Cd²⁺, and was selective for Ca²⁺, compared with Ba²⁺ (15). These properties are identical to the biophysical and pharmacologic characteristics previously described for SOC in various nonexcitable cells (17,18,29).

Using the cell-attached patch-clamp technique, it was found that SOC were activated by EGF applied in the extrapipette (bathing) solution of the cultured HMC. This response to EGF was observed within 10 s after application but was sustained for as long as the EGF was present in the bathing solution (usually approximately 180 s). The response to EGF was dose-dependent, with a half-maximal activation concentration near 5 nM.

Many previous studies have explored the long-term regulation of ion channels by growth factors (5,30,31). Typically, these studies have identified growth factor-related increases in the de novo synthesis of ion channels. However, a few recent studies have reported short-term effects of growth factors on Ca²⁺ entry pathways, as demonstrated in this study. Nerve growth factor potentiated voltage-gated calcium currents in motor neurons of the freshwater snail Lymnaea (32). Nerve growth factor and neurotrophin-3 activated calcium-dependent potassium channels in neurons isolated from embryonic mouse brain (33). In rat mesangial cells, PDGF induced immediate activation of a low-conductance, voltage-independent, calcium channel, which was defined as a ROC (14). Therefore, growth factors may stimulate Ca²⁺ entry in the long term by promoting synthesis of new channels and in the short term by modulating the open probability of channels already present in the membrane.

As mentioned previously, PDGF activated a ROC in cultured rat mesangial cells (14). In contrast to the findings presented here, this Ca²⁺ channel was activated only when PDGF was placed in the pipette. No response could be detected when PDGF was added to the extrapipette (bath) solution. However, it is difficult to compare these studies because different species were used (rat versus human), different growth factors were used (PDGF versus EGF), and a different Ca²⁺ channel may have been activated. Although the ROC had a single-channel conductance close to that observed in this study (approximately 1 pS with Mn²⁺ versus 2 pS with Ba²⁺ as the charge carrier), the Ca²⁺ selectivity of the ROC was very low, in comparison with the SOC. It is therefore possible that the PDGF-activated Ca²⁺ channel is distinct from the SOC in this study.

It was unexpected to find that EGF increased the single-channel conductance of SOC. It is probable, however, that the presence of EGF caused more complete openings of SOC, resulting in higher average current measurements in the EGF-activated SOC, compared with the basal state.

Involvement of Tyrosine Kinase and PKC
Tyrphostin A23 is a relatively specific blocker of the EGF receptor tyrosine kinase, with an IC₅₀ of 40 µM (34). The inactive analog tyrphostin A1 (35) was used to rule out nonspecific effects of tyrphostin on the SOC. The finding that 100 µM tyrphostin A23 (but not tyrphostin A1) inhibited the EGF-evoked activation of SOC supports the idea that EGF interacts with a tyrosine kinase receptor at the plasma cell membrane (12,14,36,37). EGF binds to its receptor, resulting in enhanced receptor-receptor affinity and thus receptor dimerization. This initiates activation of the EGF receptor tyrosine kinase, which is essential for phosphorylation of the tyrosine residues of phospholipase C (5,12,38). As a result, activated phospholipase C produces the second messengers 1,2-diacylglyerol and IP₃. 1,2-Diacylglyerol activates PKC, which phosphorylates intracellular signaling molecules and membrane channel proteins (39).

The conclusion that the effects of EGF are mediated by PKC is based on several experimental findings in this study. First, inhibition of PKC with calphostin C dramatically depressed EGF-induced increases in the open probability of SOC. Second, PMA significantly activated SOC, by 50%; this increase was comparable to the response to EGF. Finally, after a 60-min incubation, PMA nearly completely suppressed the EGF activation of SOC. This finding is consistent with the known ability of PMA to down-regulate PKC in the long term. These results indicate that the PKC system plays a critical role in mediating the acute EGF-evoked activation of SOC.

Other studies have revealed PKC-mediated Ca²⁺ entry via SOC in other cell types. In Xenopus oocytes, acute stimulation of PKC with a phorbol ester initially enhanced thapsigargin-evoked Ca²⁺ entry (via SOC), as indicated by the endogenous Ca²⁺-dependent Cl^— current (40). In NR8383 alveolar macrophages, stimulation of PKC corresponded to activation of Ca²⁺ influx (41). In whole-cell patch-clamp experiments with RBL-2H3 cells, Parekh and Penner (42) demonstrated that inward Ca²⁺ current through SOC was accelerated by PMA and prevented by inhibitors of PKC.

This study is seemingly in conflict with at least two studies that have demonstrated that PKC mediates SOC inhibition. In HMC, Menè et al. (21) observed that PMA inhibited angiotensin II-stimulated Ca²⁺ influx through SOC. In RBL-2H3 cells, Kuchtey and Fewtrell (43) observed that PMA reduced the intracellular Ca²⁺ response to antigen-evoked depletion of intracellular Ca²⁺ stores. However, in both of those studies, Ca²⁺ stores were released after addition of either angiotensin II or an antigen. Therefore, PMA could have been inhibiting the IP₃-sensitive Ca²⁺ release channels instead of SOC. Because we observed a very minimal increase in the intracellular Ca²⁺ concentration after addition of EGF (R. Ma and S. C. Sansom, unpublished observations), it is not likely that EGF activates SOC by releasing Ca²⁺ stores. Therefore, in the EGF signaling pathway, the IP₃-sensitive Ca²⁺ release channels are bypassed with PKC, leading to the direct activation of SOC.

Although the mechanism for SOC activation by PKC was not determined in this study, it is possible that PKC either activates SOC directly or activates an associated regulator of SOC. In support of the former possibility was a recent finding that the transient receptor potential of Drosophila melanogaster, which has been proposed to be the genetic equivalent of SOC (44,45), was regulated by PKC (46).

It is worth noting that PKC inhibition by calphostin C did not significantly affect the basal open probability of SOC. This finding suggests that the PKC system is not involved in spontaneous SOC activity, as we observed in cultured HMC using the cell-attached configuration. However, degradation of PKC after incubation with PMA for at least 1 h. significantly reduced the Ca²⁺ entry current, indicating a significant contribution by PKC to the basal activity of SOC. These disparate results may be explained by the complexity of the PKC system. It is known that the PKC family contains at least 11 members, including PKC-, -ß, -, -, -, -, and - (47,48,49,50). Studies indicate that the different PKC isoforms have different functions and subcellular distributions. Therefore, different PKC isoforms may be regulated by different agonists.

In conclusion, this study reports that EGF can acutely activate a low-conductance Ca²⁺ channel with properties identical to those of SOC observed in a previous study by this laboratory. The EGF-induced response is dependent on tyrosine kinase activation, and PKC is a key component of this intracellular signaling pathway. It has been suggested that calcium entry through SOC is involved in cell growth, proliferation, and differentiation. Moreover, EGF-evoked activation of SOC may be important in some disease states, such as diabetic glomerulosclerosis, which often results in abnormal proliferation of glomerular mesangial cells. Continued investigation of the modulation of SOC activity by growth factors via short-term and long-term mechanisms would facilitate understanding of the pathologic development of this renal disease.

	Acknowledgments

 
This work was supported by National Institutes of Health Grant DK49561 (to Dr. Sansom). Dr. Ma was supported by a fellowship grant from the American Heart Association (Heartland Affiliate). We are grateful to Hanna Abboud, of the University of Texas Health Science Center at San Antonio, for providing us with cultures of HMC.

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