Interferon-gamma induces a decrease in the intracellular calcium pump in a human salivary gland cell line

Sean Meehan, Ava J. Wu, Elaine C. Kang, Takayuki Sakai, and Indu S. Ambudkar

Secretory Physiology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interferon-gamma (IFN-gamma ) ± tumor necrosis factor-alpha (TNF-alpha ) induces antiproliferation and intracellular Ca2+ store depletion in a human submandibular ductal cell line (HSG), which can be reversed on cytokine removal [A. J. Wu, G. C. Chen, B. J. Baum, and I. S. Ambudkar. Am. J. Physiol. 270 (Cell Physiol. 39): C514-C521, 1996]. Here we have examined a possible mechanism for the IFN-gamma -induced intracellular Ca2+ store depletion. There was a time-dependent decrease in thapsigargin-dependent internal Ca2+ release after exposure of the cells to the cytokines. The intracellular Ca2+ pump [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] protein in lysates and membranes of cells treated with IFN-gamma  ± TNF-alpha , but not with TNF-alpha alone, showed a similar time-dependent decrease (examined using a SERCA2 antibody). Removal of the cytokines, which resulted in recovery of cell growth and refill of internal Ca2+ stores, also increased the level of SERCA protein. The decrease in SERCA is not a result of decreased cell proliferation, since thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone, or serum-free growth conditions induced antiproliferative effects on HSG cells without any corresponding decrease in SERCA. We suggest that the IFN-gamma -induced decrease in the level of SERCA accounts for the depleted state of internal Ca2+ stores in cytokine-treated HSG cells. These data suggest a novel mechanism for the inhibition of HSG cell growth by IFN-gamma .

sarco(endo)plasmic reticulum calcium adenosinetriphosphatase protein; intracellular calcium ion store; salivary cell line; cell proliferation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

gamma -INTERFERON (IFN-gamma ) is an immunoregulatory cytokine that exerts well-documented effects on the growth and differentiation of cells of the immune system (2, 15). It is now recognized that IFN-gamma also decreases the proliferative ability of several types of nonimmune cells (21, 22). More recently, IFN-gamma treatment has been reported to induce alterations in the physiological characteristics of epithelial cells. For example, IFN-gamma induces expression of the immunomodulatory cell surface proteins, HLA-DR and intracellular adhesion molecule-1 (ICAM-1), and loss of epithelial barrier function in colonic epithelial cells (4, 6). In addition, a number of ion transport functions are altered in these cells. The exact mechanism(s) involved in these pleiotropic effects of IFN-gamma on immune and nonimmune cell types is not yet clearly understood.

The cytokines, IFN-gamma and tumor necrosis factor-alpha (TNF-alpha ), have been suggested to be involved in salivary gland pathogenesis of Sjogren's syndrome, an autoimmune disease associated with a dramatic decrease in salivary gland function (i.e., fluid secretion) and chronic inflammation of major and minor salivary glands. Loss of salivary acinar tissue, extensive lymphocytic infiltrates, elevated levels of IFN-gamma and TNF-alpha , and expression of HLA-DR and ICAM-1 have been reported in the diseased glands (7, 20). We have previously reported that IFN-gamma  ± TNF-alpha treatment of a human salivary gland ductal cell line (HSG) induces a strong antiproliferative effect and decreases the progression of cells through the cell cycle (25, 26). Although the IFN-gamma -induced effect on HSG cell growth is enhanced in the presence of TNF-alpha , TNF-alpha alone does not alter HSG cell growth. IFN-gamma -treated HSG cells express the cell surface proteins HLA-DR and ICAM-1, indicating an alteration in the physiological status of these cells, which would make them more "immune responsive" (26). Furthermore, we had also reported that IFN-gamma  ± TNF-alpha treatment of HSG cells induces depletion of intracellular Ca2+ stores, which is reversed when the cytokines are removed from the growth medium (24). It has been previously suggested that internal Ca2+ stores have a role in the maintenance of cell proliferation and protein synthesis (11, 14). Loss of cell proliferation has been associated with the depletion of internal Ca2+ stores in smooth muscle cells treated with inhibitors of the intracellular Ca2+ pump [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)], thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) (8, 19, 23). A similar association between the internal Ca2+ store status and cell proliferation has been seen in several other cell types after treatment with antimycotic agents, e.g., miconazole, (3). We have also previously shown that treatment of HSG cells with thapsigargin, which results in a persistent depletion of internal Ca2+ stores, exerts a strong antiproliferative effect (24). On the basis of these previous findings, we have suggested that the depletion of internal Ca2+ stores in IFN-gamma  ± TNF-alpha -treated cells may have a role in the cytokine-induced inhibition of cell growth.

The Ca2+ content of internal Ca2+ stores is regulated by a number of Ca2+ transporters that transport Ca2+ either in or out of the endoplasmic reticulum (ER) and by proteins that retain Ca2+ in the ER lumen (1). SERCA are located in the ER membrane and mediate the active transport of Ca2+ from the cytosol into the lumen of the ER. The isoform SERCA2b appears to be ubiquitously present in nonexcitable cells, whereas SERCA3 is less widely distributed. The other isoforms, SERCA1 and SERCA2a, are more specifically distributed in excitable cells (27). The present study was aimed at understanding the mechanism by which internal Ca2+ store depletion is induced after treatment of HSG cells with IFN-gamma and TNF-alpha . To this end, we have examined the status of internal Ca2+ stores and the level of SERCA protein in cytokine-treated cells. The data demonstrate that cytokine-induced internal Ca2+ store depletion is associated with a decrease in the SERCA protein level. We suggest that the IFN-gamma -induced decrease in SERCA protein accounts for the depleted state of the internal Ca2+ stores in cytokine-treated cells and contributes to the antiproliferative effect exerted by the cytokine on HSG cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. The HSG cell line was a gift of Dr. Mitsunabo Sato, Tokushima University, Shikoku, Japan (18). HSG cells were cultured in Eagle's minimal essential medium (Biofluids, Rockville, MD) supplemented with 10% bovine calf serum (Biofluids), L-glutamine (2 mM), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C with 5% CO2, as described previously (24-26). Cells (5 × 105) were plated onto tissue culture plates (Falcon, NJ). After 24 h, cytokines or other agents, as specified, and fresh media were added at the following concentrations: 1,000 U/ml IFN-gamma (Genzyme), 20 U/ml TNF-alpha (Genzyme), and 200 nM thapsigargin or 4 µM BHQ (both from Calbiochem). For the serum-free condition, the cell medium was replaced with serum-free medium containing 0.1% fatty acid-free bovine serum albumin, and the cells were grown for the number of days specified. The concentration of cytokines used in this study was based on our previous studies (24-26). Media cytokines (or other agents) were changed every 3 days for 7-9 days. For the "reversal" condition, cells were grown in cytokine-containing media for 6 days as described above. Thereafter, the media were removed and replaced with media without cytokines. After 4 days of "recovery" in cytokine-free media, cells displayed normal proliferative activity and morphology.

Cell proliferation assay. Assays were performed as described previously (24-26). Briefly, 104 cells were plated onto 12-well tissue culture plates and treated with cytokines or other conditions, as described in the text, for 7 days. Cell proliferation was calculated from optical density measurements at 410 nm with a Gilford spectrophotometer (Gilford, Oberlin, OH) to detect bromodeoxyuridine incorporated into cellular DNA (Cell Proliferation Assay; Amersham Life Science, Arlington Heights, IL). Cell number and viability were determined using the 0.4% trypan blue exclusion technique (24-26). All assays were done in triplicate, and experiments were repeated at least three times.

Cytosolic Ca2+ concentration measurements. Cells were detached from the plates with Versene (Biofluids) and then were washed twice with Hanks' balanced salt solution (HBSS) buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) containing 0.01% bovine serum albumin. Cells were incubated in HBSS at 30°C for 15 min. Fura 2-acetoxymethyl ester (2 µM; Molecular Probes, Eugene, OR) was then added to the medium, and the cells were further incubated for 45 min at 30°C (10, 24). Cells were then washed two times with HBSS and maintained at 30°C until use. Immediately before use, aliquots of cells were removed, spun gently (500 g for 30 s), washed, and resuspended in the required incubation medium at 37°C. Fluorescence was measured using an SLM 8000-DMX 100 spectrofluorometer. Cells were gently stirred in a cuvette maintained at 37°C. The emission wavelength was set at 510 nm, and excitation wavelengths were 340 and 380 nm. Cytosolic Ca2+ concentration ([Ca2+]i) was calculated from the 340/380 nm fluorescence ratio. Each experiment was repeated at least three times.

Crude membrane preparation. Cells, treated as specified in the legends to Figs. 1-5, were detached from the plates by incubation with Versene as described above. Cells were washed with ice-cold buffer containing 50 mM tris(hydroxymethyl)aminomethane (Tris)-HEPES, pH 7.4, and 0.1 mM phenylmethylsulfonyl fluoride. Cells were homogenized in the same medium using a Polytron homogenizer and then were centrifuged at low speed [3,000 revolutions/min (rpm)]. The pellet was rehomogenized as described above and recentrifuged at 3,000 rpm. The supernatants were combined and centrifuged at 40,000 rpm. The resulting pellet was washed once with buffer, suspended in a minimal volume of buffer, divided into aliquots, and stored at -80°C after quick-freezing. As needed, aliquots were thawed on ice and then used. Protein was estimated using the Bio-Rad protein assay kit.

Gel electrophoresis and Western blot procedures. Control HSG cells or cells treated with the agents, as described, were lysed with buffer containing 300 mM NaCl, 50 mM Tris-HCl (pH 7.6), 0.5% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The lysates were centrifuged to remove cellular debris, and the supernatants were stored at -80°C (it should be noted that all reactivity to the antibody was found in the supernatant, which is referred to as cell lysate below). Cell lysates (50 µg protein) or crude membranes (3-5 µg protein) were treated with SDS-containing sample buffer for 30 min at 37°C and electrophoresed on 8% acrylamide gels (12). To allow comparison between the various samples, equal amounts of protein were loaded on gels. The protein was transferred electrophoretically to polyvinylidene difluoride filters and immunoblotted with a monoclonal antibody (1:1,000 dilution) to the SERCA pump (obtained from Affinity Bioreagents, Golden, CO), which has been reported to react with the SERCA2 isoform of the protein. The proteins reacting with the antibody were detected by using the enhanced chemiluminescence (ECL) assay (Amersham). Autoradiographs were scanned on a Pharmacia Biotech imaging system, and the band volume corresponding to SERCA was calculated using an Image Master program.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Time-dependent effect of IFN-gamma and TNF-alpha on the thapsigargin-sensitive internal Ca2+ store in HSG cells. We had previously reported that internal Ca2+ stores are depleted in HSG cells treated for 7 days with IFN-gamma  ± TNF-alpha , at which time maximal effects on cell number are also observed (24, 26). To examine when internal Ca2+ store depletion occurs during exposure of cells to the cytokines, we have examined thapsigargin-stimulated internal Ca2+ release in control cells and cells treated with the cytokines for a various numbers of days. In these experiments, cells were allowed to attach to the plate for 24 h before the cytokines were added to the growth medium. Control cells were treated with vehicle (dimethyl sulfoxide) and grown in the absence of cytokines for the same time period. The status of the internal Ca2+ stores was assessed by measuring the elevation of [Ca2+]i stimulated by thapsigargin in cells suspended in medium containing 5 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to prevent Ca2+ influx. Under these conditions, addition of thapsigargin (2 µM) to HSG cells induces a sharp rise in [Ca2+]i, which is transient and returns to resting [Ca2+]i within 100-150 s (data not shown here; see Refs. 10 and 24). This increase in [Ca2+]i is due to Ca2+ release from internal stores, and the magnitude of the increase is directly related to the Ca2+ content of the store. The data in Fig. 1 show that thapsigargin induces a 100 nM increase in [Ca2+]i in control cells (above the resting level of ~80 nM). A similar increase in [Ca2+]i is seen in cells treated with cytokines for 1 or 2 days. On day 4 after treatment, the thapsigargin-stimulated [Ca2+]i increase in cytokine-treated cells is decreased by ~50% compared with that in control cells, whereas resting [Ca2+]i in both sets of cells are similar and not altered. A similar 50% reduction in the thapsigargin-stimulated [Ca2+]i elevation is also observed on day 7 of treatment. The thapsigargin-stimulated internal Ca2+ release in control cells on days 1 through 7 are similar even though the cells are at different densities (i.e., on day 1 they are <25% as confluent as on day 7). Thus it appears that the difference in thapsigargin-stimulated Ca2+ release observed between control and cytokine-treated cells is not due to a difference in cell densities of two sets of cells. These results are consistent with our previous report (24) and suggest that the thapsigargin-sensitive Ca2+ stores are depleted (~50%) when HSG cells are exposed to IFN-gamma ± TNF-alpha . Furthermore, the results demonstrate that store depletion is achieved between days 2 and 4 of treatment. It should be noted that some other effects of IFN-gamma were observed earlier. For example, activation of JAK2 and STAT-1 is seen within 10 min (unpublished data), whereas expression of HLA-DR and ICAM-1 is seen by 3 days (24, 26). Thus there does not appear to be a temporal correlation between these different effects induced by IFN-gamma on HSG cells.


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Fig. 1.   Time course of internal Ca2+ store depletion in cytokine-treated human submandibular ductal cell line (HSG) cells. Cells were grown for number of days indicated in presence of interferon-gamma (IFN-gamma ) and tumor necrosis factor-alpha (TNF-alpha ), as described in MATERIALS AND METHODS. For cytosolic Ca2+ concentration ([Ca2+]i) measurements, cells were detached from the plates and loaded with fura 2. Two-milliliter aliquots of cells were washed in Hanks' balanced salt solution medium and maintained in a cuvette at 37°C, with gentle stirring. EGTA (5 mM) was added to the medium followed 30 s later by 2 µM thapsigargin. Thapsigargin induced the typical transient [Ca2+]i elevation as previously described (34). Changes in [Ca2+]i values (peak minus resting) determined from 4-5 experiments are shown as means ± SE for control cells (open bars) and cytokine-treated cells (solid bars). Resting [Ca2+]i in control and cytokine cells were not significantly different from each other at any time point. Thapsigargin-stimulated [Ca2+]i increases on days 4 and 7 are significantly different (P < 0.01, Student's t-test) from all other values. All other values are not significantly different from each other.

SERCA levels in cytokine-treated HSG cell lysates and isolated membranes. To determine a possible mechanism to account for the depletion of internal Ca2+ stores, we examined the levels of the SERCA protein in control and cytokine-treated cells by using a monoclonal antibody against the SERCA2 isoform (28). Western blots of total cell lysates run on 8% polyacrylamide gels are shown in Fig. 2A. A protein of molecular mass of ~100 kDa reacts with the antibody in the samples from control cells, which is consistent with the reported molecular mass of SERCA2 (28). Because SERCA2a is not likely to be present in HSG cells, we suggest that the protein detected by the antibody is probably SERCA2b. However, a contribution of SERCA3 to this reactivity cannot be presently ruled out, since the antibody we have used has been recently reported to also recognize the SERCA3 subtype of the Ca2+ pump (13). Thus further studies using a SERCA3-specific antibody are needed to investigate the presence of SERCA3 in HSG cells. In the results described below, we have referred to the protein(s) that reacts with this antibody as SERCA. The level of SERCA does not appear to be altered in lysates of HSG cells grown for various days in a normal growth medium (i.e., in control cells). However, the level is significantly decreased to 85 ± 4% of control (P < 0.01, by paired Student's t-test, n = 6) determined by densitometric scanning of Western blots (6 gels, 3 different experiments) of lysates of HSG cells treated with IFN-gamma  ± TNF-alpha for 4 days and further decreased to 49 ± 3.5% of controls (P < 0.001, paired Student's t-test, n = 6) in cells treated for 7 days. The level of SERCA in cytokine-treated cells on various days has been expressed relative to the respective control (i.e., untreated cells on same day of culture, as shown in Figs. 1 and 2A). In Fig. 2A, the blots were subjected to the ECL reaction for a longer period of time (5-10 min, until the band on day 7 was clearly detected) to allow comparison of the SERCA level in the different samples. Shortening the time of the ECL reaction allowed for better resolution of the decrease in SERCA on day 4; e.g., with 2-5 min of exposure, a 40-50% decrease was noted. However, in this case, the band on day 7 was not clearly detected. Thus there appears to be a greater decrease in SERCA on day 7 compared with day 4. Although it is not possible from the present data to directly quantitate the SERCA levels, the temporal pattern of the decrease in SERCA is similar to the cytokine-induced decrease in the internal Ca2+ store(s) content in HSG cells. Furthermore, when the cytokines are removed from the medium and cells are allowed to recover for 7 days, the SERCA level is increased to levels comparable with that in control cells. The recovery of SERCA on cytokine removal is greater in Fig. 2A than in 2B, since the cells were grown for a longer time in regular medium (7 days vs. 4 days). Thus the cytokine-induced decrease in the SERCA protein level appears to be a reversible process. These results are consistent with our previous report (24), showing recovery of internal Ca2+ stores under the same conditions. Furthermore, in cells treated with TNF-alpha alone, there is no decrease in SERCA, whereas, in those treated with IFN-gamma , the level of SERCA is similar to that in IFN-gamma ± TNF-alpha -treated cells (Fig. 2B). These results are similar to the effects of these cytokines on HSG cell growth; TNF-alpha alone does not alter the proliferative activity of HSG cells, whereas IFN-gamma alone reduces proliferation (25, 26).


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Fig. 2.   IFN-gamma -induced decrease in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) protein levels of HSG cells. Cells were treated with IFN-gamma and TNF-alpha for various days, as described for Fig. 1. On days noted (A), cells were exposed to lysis buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as described in MATERIALS AND METHODS. Control cell lysates exhibit a protein with a molecular mass of ~98 kDa that reacts with SERCA2 antibody (control blots not treated with primary antibody showed no reactivity and are not shown). Levels of this protein in control cells (c) and IFN-gamma  + TNF-alpha -treated cells (t) on various days (1, 2, 4, and 7) after treatment are shown. Last lane shows reactivity in samples from "reversed cells" (r) (i.e., grown with cytokines for 7 days and then in normal medium for 7 days). B: Western blot of cell lysates (50 µg protein/sample) from control cells (C) 7 days after plating, cells treated with IFN-gamma  + TNF-alpha (I+T) for 7 days, cells treated with IFN-gamma (I) for 7 days, cells treated with TNF-alpha (T) for 7 days, and cells treated with both cytokines for 7 days and then grown in control medium for 4 days (R). Results are representative of 3 similar experiments. C: levels of SERCA in membranes isolated from cytokine-treated cells. Cells were treated for 7 days with IFN-gamma  ± TNF-alpha , as described previously. A crude membrane (40,000 g pellet, see MATERIALS AND METHODS) was prepared from control cells (C) and cells treated with IFN-gamma (I) or IFN-gamma and TNF-alpha (I+T) for 7 days. SDS-PAGE and Western blotting with anti-SERCA antibody were performed as described above with 3 µg protein/lane. Results are representative of 3 similar experiments.

To further examine this decrease in SERCA levels in cytokine-treated HSG cells, we prepared a crude membrane fraction (40,000 g pellet) from control, IFN-gamma -treated, and IFN-gamma  ± TNF-alpha -treated (for 7 days) cells. Figure 2C shows the reactivity of an ~100 kDa protein in these membranes to the SERCA antibody. The level of this protein (SERCA) is markedly lower in membranes prepared from IFN-gamma  ± TNF-alpha -treated cells compared with that in membranes from untreated cells. Membranes from cells treated with IFN-gamma alone show a similar decrease in SERCA. Silver staining of comparable gels further demonstrates that the general protein profile of membranes from cytokine-treated and control cells is similar (data not shown).

Internal Ca2+ store content and SERCA levels in HSG cells grown in the presence of thapsigargin, BHQ, or serum-free conditions. To determine whether the decrease in SERCA in cytokine-treated HSG cells is due to more general effects of antiproliferation, e.g., decreased protein synthesis, we examined the status of internal Ca2+ stores and SERCA levels of cells grown 1) in the presence of thapsigargin or BHQ, two strong SERCA inhibitors, or 2) in a serum-free medium. Figure 3 shows that all three conditions induce strong antiproliferative effects on HSG cells. Compared with control cells, the cell number after 7 days is 10, 15, and 20%, respectively, in the +thapsigargin, +BHQ, and serum-free growth conditions. These data are consistent with an earlier report (24) and further demonstrate that BHQ, a SERCA inhibitor like thapsigargin, also induces antiproliferative effects on HSG cells. Both thapsigargin and BHQ also induce total depletion of the thapsigargin-sensitive internal Ca2+ stores (data not shown). Importantly, lysates of cells grown for 7 days in medium containing BHQ or thapsigargin do not show any decrease in SERCA protein levels (data not shown).


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Fig. 3.   Effect of SERCA inhibitors, cytokines, and serum-free conditions on HSG cell growth. Cell number was determined by trypan blue exclusion. Bromodeoxyuridine incorporation also gave similar results (not shown). All conditions for cell culture were as described in MATERIALS AND METHODS. Cells were grown for 7 days in 1) control medium (Con), 2) medium with 4 µM 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), 3) medium with 200 nM thapsigargin (Tg), 4) medium without fetal bovine serum (- serum), and 5) medium with IFN-gamma  + TNF-alpha (I+T). Data are means ± SE from 3-5 experiments. All values are significantly different from control condition (P < 0.005, by Student's t-test).

Fetal bovine serum exerts a strong mitogenic effect on HSG cells, and, in the absence of serum in the culture medium, HSG cell growth is greatly reduced (Fig. 3). However, the thapsigargin-induced Ca2+ release in these cells is similar to those in control cells that are maintained in a serum-containing medium (Fig. 4), demonstrating the intactness of the internal Ca2+ store(s). Consistent with this observation, we have observed that the SERCA level in these cells is not decreased. These data demonstrate that the decrease in SERCA in IFN-gamma -treated cells is not a result of a decrease in cell proliferation, protein synthesis, or decrease in intracellular Ca2+ stores.


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Fig. 4.   Effect of SERCA inhibitors and serum-free growth conditions on SERCA protein and intracellular Ca2+ stores in HSG cells. Thapsigargin-stimulated [Ca2+]i elevation was measured as described for Fig. 1, in control cells (open bar) and in cells grown in a serum-free condition (solid bar). Values were not statistically different from each other. [Ca2+]i increase was undetectable in cells grown in presence of thapsigargin or BHQ and is not shown.

Effect of BHQ on the recovery of HSG cell growth induced by removal of the cytokines from the culture medium. Figure 5 shows the effect of TNF-alpha and IFN-gamma on HSG cell growth. As reported by us previously (24-26), treatment of HSG cells with these cytokines induces a persistent decrease (>95%) of cell growth (compare curves 1 and 2). When the cytokines are removed from the growth medium on day 6, the cells resume normal growth after day 10 (curve 3). Notably, at this time, the SERCA level shows a recovery (see Fig. 2B), and the internal Ca2+ store(s) is also partially refilled (24). To examine whether functional SERCA is required for the reversal of cell growth in cytokine-treated cells, we treated the cells with 4 µM BHQ when the cytokine was removed on day 6. Under these conditions, the recovery of cell growth is completely inhibited and cell number remains >95% lower than that of controls (curve 4 ). However, when BHQ is removed from the medium on day 12, increased cell growth is seen after day 17 (curve 5 ). These data are consistent with the effects of SERCA inhibitors, e.g., BHQ and thapsigargin, on cell growth reported by us and others (8, 19, 23, 24). The data suggest that functional SERCA is required 1) for normal growth of HSG cells and 2) for recovery of growth of HSG cells after IFN-gamma treatment.


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Fig. 5.   Effect of BHQ on recovery of HSG cell growth induced by cytokine removal. HSG cell growth was determined as described in MATERIALS AND METHODS for a period of 23 days after plating. Growth conditions were as follows: 1) control growth medium was used for entire experiment; 2) growth medium containing IFN-gamma and TNF-alpha was used during entire experiment; 3) cytokine-containing medium was replaced by control medium on day 6; 4) cytokine-containing medium was replaced by 4 µM BHQ-containing medium on day 6; 5) cytokine-containing medium was replaced with BHQ-containing medium on day 6 and then BHQ-containing medium was replaced with control medium on day 12. Values are means ± SE from 3 experiments, each done in duplicate.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have demonstrated here that treatment of HSG cells with IFN-gamma and TNF-alpha induces a time-dependent, persistent depletion of internal Ca2+ stores. Furthermore, there is a concurrent time-dependent decrease in the level of the intracellular Ca2+ pump protein (SERCA). IFN-gamma , which by itself induces a substantial reduction in HSG cell growth and internal Ca2+ pool depletion, reduces the level of SERCA to the same extent as that in cells treated with IFN-gamma  ± TNF-alpha . TNF-alpha , which by itself does not decrease HSG cell proliferation (24-26), does not alter SERCA levels. Thus the ability of the cytokines to decrease SERCA appears to be related to their ability to reduce cell proliferation. This decrease in SERCA does not appear to be a result of a general decrease in protein synthesis, reduced cell proliferation, or depletion of intracellular Ca2+ stores. We have shown that HSG cells in a growth-arrested state, due to removal of serum from the growth medium or due to inclusion of Ca2+ pump inhibitors (thapsigargin or BHQ), in the growth medium do not exhibit decreases in SERCA levels. Finally, the levels of SERCA recover to control levels on removal of IFN-gamma . Therefore, we suggest that the decrease in SERCA is specifically induced in response to the treatment of HSG cells with IFN-gamma .

The activity of SERCA is involved in the accumulation of Ca2+ into the ER and the refill of internal Ca2+ stores, and it is well documented that inhibition of SERCA activity induces depletion of internal Ca2+ stores (1, 8, 10). Thus we suggest that the IFN-gamma -induced decrease in SERCA protein accounts for the decreased Ca2+ content of the internal Ca2+ stores detected in IFN-gamma -treated cells. Another possible mechanism that can be proposed to account for the internal Ca2+ store depletion is IFN-gamma -stimulated internal Ca2+ release, mediated via an increase in inositol trisphosphate generation, as has been previously reported in renal carcinoma cells (9).

However, we can rule out this possibility since we have previously demonstrated that IFN-gamma does not induce any acute (within 10 min) release of Ca2+ from internal Ca2+ store(s) or increase in inositol trisphosphate in HSG cells (24). Thus our data are more consistent with our first suggestion. Although this is the first report describing an effect of IFN-gamma on the SERCA protein level, it has been previously reported (4, 6) that IFN-gamma reduces the levels of the Na+-Cl--K+ cotransporter and the cystic fibrosis transmembrane conductance regulator in the colon-derived epithelial cell lines (T84 and HT-29, respectively). The mechanism(s) involved in the modulation of gene expression by IFN-gamma is presently not completely understood. Both upregulation and downregulation of various genes have been reported (17). Possible effects at the promoter level of the genes as well as on regulation of transcription factors have been described. In addition, IFN-gamma has also been suggested to induce posttranscriptional modifications, such as alterations in mRNA stability (4).

Previous studies with smooth muscle and neuroblastoma cells have demonstrated that loss of cell proliferation can be correlated with depletion of internal Ca2+ stores, whereas synthesis of new Ca2+ pump protein in the store membrane precedes reentry of cells into the cell cycle (19, 23). Our present and previous data show that depletion of internal Ca2+ store(s), decrease of SERCA protein, loss of cell proliferation, and arrest of cell cycle are temporally correlated in IFN-gamma -treated HSG cells (24, 26). We have also shown here that addition of BHQ to cells before removal of the cytokines prevents recovery of cell proliferation. Thus the functional integrity of SERCA appears to be critical for the normal growth and cycling of HSG cells. Furthermore, the SERCA activity also appears to be critical for the recovery of HSG cell growth after removal of the cytokines. Our studies suggest that SERCA inhibitors and IFN-gamma essentially induce antiproliferative effects by a similar mechanism. In both cases, the function of the SERCA protein is impaired, and the net result is a decrease in Ca2+ uptake into internal Ca2+ stores and internal Ca2+ store depletion. It should be noted that the cytokine treatment induces >95% inhibition of cell growth but a maximum depletion of ~50% of the thapsigargin-sensitive Ca2+ store. This suggests that total Ca2+ store depletion is not required for the inhibition of cell growth and that only part of the thapsigargin-sensitive Ca2+ store is important for the normal proliferative activity of HSG cells. Furthermore, likely compartmentalization of intracellular Ca2+ stores and a relatively heterogeneous distribution of SERCA in these store membranes could account for the discrepancy between the extent of decrease in SERCA and content of thapsigargin-sensitive internal Ca2+ store(s). However, further studies are required to clarify why the SERCA level continues to decrease until day 7 while there is no further detectable depletion of the thapsigargin-sensitive Ca2+ store after day 4.

We have observed in the present study that Ca2+ store depletion is induced between days 2 and 4 and that a significant decrease in SERCA is also induced within this same time period. In our previous report (25), we have shown that there is an increase in cell number up to day 3 of cytokine treatment, after which the cell number remains unchanged until day 6. This is associated with cell cycle arrest at G0/G1. We have recently reported that HSG cells undergo cell death when treated with IFN-gamma  ± TNF-alpha , but not TNF-alpha alone, for >4 days (25). It has been suggested that Ca2+ may be a key factor in the regulation of cell proliferation (14) and other functions such as protein synthesis (5), expression of transcription factors (14, 29), and PKC-dependent activation of mitogen-activated protein kinase (28). Several recent studies have suggested that the depletion of internal Ca2+ stores(s) is also involved in mediating the process of apoptosis and cell death in several different cell types (14). Thus IFN-gamma -induced internal Ca2+ store depletion could also have a role in the cell death observed in HSG cells exposed to IFN-gamma and TNF-alpha .

The depleted state of the internal Ca2+ store(s) in salivary cells exposed to IFN-gamma and TNF-alpha can also provide a possible explanation for the two major effects of Sjogren's syndrome on salivary gland cells, i.e., loss of acinar tissue and reduced fluid secretion. Depletion of the internal Ca2+ store(s) could induce a reduction in cell growth or an enhancement in cell death/apoptosis, both of which would lead to reduced acinar tissue. A reduction in fluid secretion would then result, due to the loss of acinar tissue, which is the sole site of water movement in salivary glands. Decreased internal Ca2+ store(s) could also have more direct effects on the level of fluid secretion, since intracellular Ca2+ mobilization events are required for initiating and maintaining fluid secretion in salivary glands (1, 16). Further studies are required to determine whether there are alterations in the status of intracellular Ca2+ store(s) in salivary glands during the pathogenesis of Sjogren's syndrome or other inflammatory conditions and whether these alterations are significant in the functional damage of the glands induced by the disease.

In summary, we have demonstrated that IFN-gamma -induced depletion of the internal Ca2+ store(s) in HSG cells is temporally associated with the cytokine-induced decrease in the level of the SERCA protein. Although further studies are required to determine the mechanism by which IFN-gamma decreases the level of SERCA in salivary gland epithelial cells, on the basis of the correlation observed in the status of internal Ca2+ store(s), the level of SERCA, and the proliferation of HSG cells after cytokine treatment, we suggest that the IFN-gamma -induced decrease in functional SERCA results in the depletion of internal Ca2+ stores. The depletion of the internal Ca2+ store may have a role in the antiproliferative effects and/or apoptosis induced by this cytokine. Thus our present findings suggest a novel mechanism by which IFN-gamma induces antiproliferative effects on salivary gland cells, i.e., by depletion of intracellular Ca2+ stores mediated via a decrease in the level of SERCA. It will be interesting to see whether this cytokine induces similar effects on the SERCA and intracellular Ca2+ stores in other cell types.

    ACKNOWLEDGEMENTS

We thank Dr. Bruce J. Baum for support during the course of this work. We also thank all our collegues for their help and cooperation.

    FOOTNOTES

Address for reprint requests: I. S. Ambudkar, Rm. 1N-113, Bldg. 10, National Institutes of Health, Bethesda, MD 20892.

Received 14 March 1997; accepted in final form 21 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C2030-C2036




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