Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria.

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.


INTRODUCTION
ceramide, even physiological Ca 2+ responses of mitochondria to InsP 3 -generating agonists are sufficient to induce apoptosis, possibly via Ca 2+ -dependent opening of the permeability transition pore (20). The Ca 2+ content of the ER also affects the cell sensitivity to apoptotic stimuli. A decreased [Ca 2+ ] ER was observed in cells overexpressing the antiapoptotic protein Bcl-2 (21,22), and a variety of conditions that decreased [Ca 2+ ] ER has been shown to protect cells from ceramide-induced cell death (23). The opposite effect was observed in cells overexpressing the Ca 2+ -ATPases (SERCA2b) or the ER-resident Ca 2+ -binding chaperone calreticulin, which increased the Ca 2+ content of the ER (23)(24)(25). Conversely, cells lacking the calreticulin had a decreased ER Ca 2+ content and were more resistant to apoptotic stimuli (26). Calreticulin-deficient cells, however, had normal [Ca 2+ ] ER levels, suggesting that the ability of calreticulin to modulate the cell sensitivity to apoptotic stimuli might be linked to changes in the total Ca 2+ content of the ER rather than to changes in [Ca 2+ ] ER .
Calreticulin is a 46-kDa Ca 2+ -binding chaperone that interacts in a Ca 2+ -dependent fashion with several ER resident proteins, with unfolded glycoproteins, and with Ca 2+ transporters at the ER membrane (27,28). Calreticulin is composed of three structural and functional domains: a highly conserved N-terminal domain, involved in chaperone function and in the interactions with other ER chaperones; a proline-rich P-domain, which shares significant amino acid sequence identity with calnexin, calmegin, and CALNUC, and is involved in the chaperone function of calreticulin; and a C-terminal domain that binds Ca 2+ ions with low affinity and high capacity (29). The Ca 2+ -binding C-domain has been postulated to be the "Ca 2+ -sensor" that regulates calreticulin interactions with other proteins (25,29).
Because of the central role of the ER in Ca 2+ signaling, both the chaperoning functions of calreticulin as well as its interactions with ER Ca 2+ transporters can interfere with Ca 2+ signals. For example, calreticulin inhibits repetitive Ca 2+ waves by interacting selectively with distinct isoforms of SERCA2 (30,31). On the other hand, conflicting results have been reported regarding the role of calreticulin in the modulation of store-operated Ca 2+ influx calreticulin , P+C-domain and calnexin by western blotting with anti-calreticulin, anti-HA and anti-calnexin antibodies. Three cell lines with the highest inducible expression of calreticulin , P+C-domain and calnexin were selected for this study.
Cell Culture -HEK-293 or Tet-On cell lines were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and were maintained in a humidified incubator at 37°C in the presence of 5% CO 2 / 95% air. Cells (~200,000) were plated on 25 mm glass coverslips. HEK-293 at 60% of confluency, cells were transiently transfected with cDNAs encoding the yellow cameleons probes. Cells were imaged 3 to 5 days after transfection.
Stable HEK-293 transfectants were grown in the presence of geneticin (100 µg/ml) for 3 weeks and ~20 clones were expanded for each condition and tested for expression of the probes. 2 µg Dox/ml was added into the culture medium to induce expression of calreticulin, its P+C-domain, or calnexin in Tet-On cell lines.
Immunoblotting and Immunocytochemistry -Western blot analysis with the use of goat anti-calreticulin, anti-HA and rabbit anti-calnexin antibodies was carried out as described (25). For indirect immunofluorescence of calreticulin expressing HEK Tet-On cells were plated on coverslips pre-treated with polylysine and cultured in the presence or absence of 2 µg of Dox /ml for 72 h. Cells were washed 3 times with PBS, fixed with 3.7% paraformaldehyde for 20 min and permeabilized with 0.3% Triton X-100 for 20 min.
Calreticulin was detected by incubation with a goat anti-calreticulin antibody followed by staining with a rabbit anti-goat antibody conjugated to Texas-Red (Jackson Immunoresearch).
[Ca 2+ ] measurements -Cells plated on 25 mm coverslips were superfused at 37°C in a thermostatic chamber (Harvard Apparatus, Holliston, MA) equipped with gravity feed inlets and vacuum outlet for solution changes. Dual-emission ratio imaging of [Ca 2+ ] with cameleons probes was performed as previously described (11). Cameleon fluorescence from by guest on July 9, 2020 http://www.jbc.org/ Downloaded from cells was imaged on a Axiovert S100 TV using a 100X, where R max and R min are the ratios obtained respectively in the absence of Ca 2+ and at saturating Ca 2+ . K' d is the apparent dissociation constant and n is the Hill coefficient of the Ca 2+ calibrations curves obtained in situ for each cameleon.
For better 3D rendering widefield or confocal image stacks were deconvoluted after acquisition on a Silicon Graphics Octane workstation using the Huygens 2 software and shadow projections were constructed using the Imaris software (Bitplane AG, Zurich, Switzerland) by guest on July 9, 2020 http://www.jbc.org/ Downloaded from

RESULTS
To generate cells inducible for calreticulin, we stably transfected HEK-293 cells with a rabbit calreticulin cDNA construct driven by the tetracyclin promoter (Tet-ON). The activation of calreticulin gene transcription by doxycyclin (Dox), added to the culture medium, was confirmed by immunoblotting with a goat polyclonal CRT antibody (Fig. 1A).
Quantification of the immunoblot indicated that the cellular calreticulin content increased by 2.5 fold within 24h, and remained at this level for up to 5 days in culture. The induction was specific for calreticulin, as addition of Dox had no effect on the expression of other ER luminal chaperones such as ERp57 or Bip (not shown). An immunostaining with a calreticulin-specific antibody confirmed that protein expression was much stronger in Doxinduced cells, and still displayed the reticular pattern typical of the ER (Fig. 1B, left). No immunoreactivity was observed in the cytosol or at the plasma membrane, confirming that, after induction, calreticulin remained localized within the ER lumen. The ER structure was not noticably altered, since Dox induction did not affect the intracellular distribution of the ER-targeted Ca 2+ indicator YC4 ER (Fig. 1B, right). This indicated that the increase in calreticulin did not interfere with the import, ER retention, or folding efficiency of the GFPbased indicator. Moreover, the Ca 2+ affinity of both the ER-targeted probe YC4 ER and of the cytosolic probe YC2, measured in situ in cells permeabilized with ionomycin or digitonin, were not affected by the increased expression of calreticulin ( Fig. 1C). Thus, Dox induction increased the amount of calreticulin within the ER lumen in a controlled manner, without interfering with the targeting specificity or Ca 2+ dependency of the cameleon Ca 2+ indicators. 2+ ] homeostasis. To assess whether the sustained increase in calreticulin levels interfered with ER Ca 2+ homeostasis, we measured the changes in the free Ca 2+ concentration within the ER lumen, [Ca 2+ ] ER , using the low-affinity ER-targeted ratiometric "cameleon" indicator YC4 ER (KD=290 µM, ref (11)). YC4 ER measurements revealed that the induction of calreticulin markedly increased the resting [Ca 2+ ] ER levels ( Fig. 2), the basal [Ca 2+ ] ER values averaging 306±31 µM in the absence and 595±53 µM 72h after Dox-dependent induction of calreticulin expression . The increase could by guest on July 9, 2020 http://www.jbc.org/ Downloaded from not be attributed to a specific ER region, as higher [Ca 2+ ] ER levels were observed throughout the ER network in the ratio images ( Fig. 2A). Thus, the 2.5 fold increase in calreticulin levels caused, after 3 days of induction, a doubling in the free Ca 2+ concentration within the ER lumen.

Effect of calreticulin induction on ER [Ca
The doubling in resting [Ca 2+ ] ER could reflect either an increased Ca 2+ pumping activity, or a decrease in the passive Ca 2+ permeability, or "leak", of the ER. To distinguish between these possibilities, we studied the effect of the SERCA inhibitor thapsigargin (Tg) on calreticulin-dependent changes in free ER Ca 2+ . Tg induced a slow decrease in [Ca 2+ ] ER in both control and calreticulin-induced cells (Fig. 2B) 2C). The recovery rates were 1.9-fold higher in calreticulin overexpressers than in control, non-induced cells, at any given [Ca 2+ ] ER (Fig.2C inset). Because this assay measures the net flow of Ca 2+ from the external space to the ER, this indicates that both the influx of Ca 2+ across the plasma membrane and the ER Ca 2+ pumping activity were increased in cells expressing high levels of calreticulin. In the absence of agonist stimulation, the increased rates of ER refilling were not balanced by a parallel increase in the endogenous ER Ca 2+ permeability, resulting in higher [Ca 2+ ] ER levels at rest. However, induction of calreticulin expression markedly increased the agonist-induced ER Ca 2+ permeability, and therefore, upon stimulation, more Ca 2+ was released from the ER lumen.

Effect of CRT induction on cytosolic Ca 2+ signals. To assess how these changes in ER
luminal Ca 2+ homeostasis influenced Ca 2+ signals in the cytosol, we monitored changes in cytoplasmic Ca 2+ , [Ca 2+ ] cyt , with the cytosolic YC2 probe (K D =1.24µM). Ca 2+ release from ER stores was measured in the absence of external Ca 2+ , and Ca 2+ influx was subsequently measured by re-adding Ca 2+ to the external medium. Figure 3 shows that both CCh and Tg elicited a much larger increase in [Ca 2+ ] cyt in calreticulin overexpressing cells, indicating that substantially more Ca 2+ was released from the intracellular Ca 2+ stores. Compared to previous studies using fura-2 (32), the differences between control and calreticulin overexpresser cells were striking, reflecting the better adequacy of the YC2 probe to quantify [Ca 2+ ] cyt changes in the micromolar range. Subsequent addition of Ca 2+ to assess the activity of store-operated Ca 2+ channels at the plasma membrane revealed that, as previously reported (32), Ca 2+ influx was severely blunted in Tg-stimulated calreticulin overexpressing cells (Fig. 3 responses at different times following the induction of protein expression. Figure 4A shows that the resting [Ca 2+ ] ER levels were increased 24 hours after Dox-dependent induction of calreticulin expression, and remained elevated thereafter. In contrast, Ca 2+ influx, taken as the peak [Ca 2+ ] cyt upon Ca 2+ re-addition to Tg-treated cells, was inhibited only 3 days after the induction with Dox (Fig. 4B, circles). The amount of releasable Ca 2+ followed a similar delayed time-course: the peak of Tg-induced [Ca 2+ ] cyt release was only marginally increased 24 hours post-induction, and became significantly increased only 2 or 3 days after Doxinduction of calreticulin expression (Fig. 4B,  chelator, as expected from the capacitative mechanism (Fig. 5B). This indicates that SOC by guest on July 9, 2020 http://www.jbc.org/ Downloaded from channels were fully functional in calreticulin overexpresser cells when [Ca 2+ ] ER was artificially clamped to the level found in non-induced cells. Therefore, the high expression of calreticulin had no effect per se on the activity of SOC channels, which is determined primarily by the [Ca 2+ ] ER level in the ER lumen.
Effect of calreticulin induction on mitochondrial Ca 2+ signals. In addition to communicating with the plasma membrane, the ER is also involved in a cross-talk with mitochondria, which are strategically located close to the sites of Ca 2+ release, and can capture part of the Ca 2+  (Fig. 6B). This suggested that mitochondria were still able to take up the Ca 2+ released by the ER, but that Ca 2+ extrusion from mitochondria was facilitated. As a result, the average [Ca 2+ ] mit level measured during CCh application was significantly reduced in calreticulin induced cells (Fig. 6B). This finding was unexpected, as calreticulin induction increased both the amount of releasable Ca 2+ , the driving force for ER-to-cytosol Ca 2+ release, and the InsP 3induced Ca 2+ permeability of the ER (Figs. 2 and 3). The shorter duration of the [Ca 2+ ] mit signal suggested that the ability of mitochondria to retain Ca 2+ loads was impaired.
Effects of calreticulin on mitochondria morphology and membrane potential. The abnormal mitochondrial response of calreticulin cells suggested that calreticulin induction might cause by guest on July 9, 2020 http://www.jbc.org/ Downloaded from structural or functional damages to mitochondria. Because mitochondria are tightly coupled to Ca 2+ release sites at the ER membrane (6,37), subtle change in the architecture of the mitochondrial network might be sufficient to cause dramatic effects on [Ca 2+ ] mit signals. On the other hand, changes in mitochondrial membrane potential, ∆ψ m , which determines the driving force for Ca 2+ , also directly impact on [Ca 2+ ] mit . To distinguish between these two possibilities, we measured ∆ψ m and assessed the morphology of mitochondria as well as their interactions with the ER. To assess the morphology of mitochondria without relying on the extent of their negative membrane potential, we used the genetically targeted indicator DsRed mit . Figure 7A shows that the staining pattern of DsRed mit was not markedly altered in calreticulin-induced cells. Upon Dox induction, mitochondria retained their "worm-like" appearance and did not appear swollen or condensed (Fig. 7A). Although a variety of mitochondria morphologies were observed both in control and Dox-induced cells, no systematic alterations could be observed in association with the induction of calreticulin expression. More importantly, the overlap between the mitochondrial and the ER signal was similar in control and calreticulin cells, as assessed by co-labeling cells with YC4 ER and Mitotracker Red (Fig. 7B). In both conditions, mitochondria appeared embedded into the ER, suggesting that the induction of calreticulin did not disrupt the interactions between the ER and mitochondria. Thus, although the resolution of the confocal microscope did not allow us to resolve the contact points between the ER and mitochondria, the structural integrity as well as the relationship between the two organelles appeared to be preserved. We next measured the mitochondrial membrane potential, ∆ψ m , using the rhodaminebased dye TMRM, which accumulates into polarized mitochondria. The ∆ψ m -driven accumulation of TMRM into mitochondria was quantified as the ratio of the mitochondrial over cytosolic fluorescence intensity (38). The TMRM ratio was significantly lower in Doxinduced cells (Fig. 7C, left panel), indicating that ∆ψ m was reduced by long term overexpression of calreticulin. The decrease in ∆ψ m was not due to TMRM photoactivation and subsequent local generation of reactive oxygen species (ROS) (39), as determined by time-lapse imaging. The TMRM ratio was already lower in Dox-induced cells illuminated for the first time, and did not change subsequently over the 20 minutes recording period (data not shown). The decrease in ∆ψ m was confirmed by measurements with JC-1, a potentiometric dye that forms red-emitting aggregates at negative ∆ψ m (38). As shown in Fig. 7C, the proportion of red-emitting JC-1 aggregates was markedly reduced in Dox-induced cells (Fig.   7C, right panel). Thus, the abnormal [Ca 2+ ] mit response of calreticulin-overexpressing cells correlated with a decreased mitochondrial membrane potential, with no visible alteration in the mitochondrial architecture.  Fig. 8C, D). In addition the reduction of TMRM fluorescence was also measured in P+C induced cells (Fig. 8E). This suggested that the "Ca 2+ -sensing" and Ca 2+ storage C-domain of calreticulin was responsible for the deleterious effects. Despite repeated attempts we were unable to generate cells overexpressing either the N-or C-domain alone. However, it is unlikely that the chaperone P-domains of calreticulin plays a role because Dox-inducible expression of ER chaperone calnexin, which contains a similar P-domain did not reproduce the effect on [Ca 2+ ] ER (Fig.8B). In summary, these data suggest that the low-affinity, high capacity Ca 2+ -binding C-domain, rather that the chaperone interacting regions of calreticulin, mediate the effects on [Ca 2+ ] ER leading to modulation of SOC and mitochondrial Ca 2+ homeostasis.

DISCUSSION
In this study we report that differential expression of calreticulin in the lumen of ER affects the Ca 2+ homeostasis of distinct cellular compartments. Altered Ca 2+ signals were observed in the ER, in the cytosol, at the plasma membrane, and in the mitochondria. The most predominant effects of increased expression of calreticulin occurred at the level of ER, where the protein resides. Consistent with all previous studies (23,32,34,40), we found that calreticulin overexpression increased the total amount of Ca 2+ stored in the ER, an effect that occurred within days after the induction of protein expression. In addition, we found that the increased expression of calreticulin has a significant effect on the free intraluminal ER Ca 2+ .
The free Ca 2+ concentration within the ER lumen, [Ca 2+ ] ER , nearly doubled within 24h of induction of calreticulin expression and remained at these elevated levels for several days. This is in contrast to earlier report where in oocytes [Ca 2+ ] ER levels were either not affected (34) or slightly decreased (31) when calreticulin was overexpressed. Although different expression systems were used, these diverging effects of calreticulin relate to cellular systems expressing the same SERCA isoform. In this study, increased [Ca 2+ ] ER levels and Ca 2+ pumping activity were observed in calreticulin overexpressing HEK-293 cells, which contain predominantly the SERCA 2b isoform (Fig. 2) (24). In the present study, calreticulin levels were increased by 2.5 fold, Ca 2+ pumping activity by 1.9 fold, and [Ca 2+ ] ER by 1.8 fold. This excellent correlation reflected the imbalance between the increased Ca 2+ pumping activity and the endogenous Ca 2+ permeability of the ER, which was unaffected by calreticulin.
The increased Ca 2+ pumping activity, however, was not mediated by SERCA isoforms, as inferred from the effects of thapsigargin. Thapsigargin, added at concentrations that fully inhibit SERCA, unmasked a nearly identical passive ER Ca 2+ permeability in control and calreticulin overexpressers (Fig. 2B). Because at steady state the Ca 2+ pumping activity is equal to the ER Ca 2+ leak, this indicates that, under resting conditions, the activity of SERCA was not altered in the calreticulin overexpressers. Thus, thapsigargin-insensitive Ca 2+ pumps mediate the increased ER refilling observed during Ca 2+ re-addition to Ca 2+ -depleted cells (Fig. 2C). A likely candidate is the Pmr1 family of Ca 2+ transport ATP ases, which has recently been shown to be expressed and functional in mammalian cell lines (41). The thapsigargin-insensitive Pmr1 pump is localized mainly to the Golgi complex, but a substantial fraction is present and functional in the ER. The Pmr1 store had a reduced Ca 2+ leak and weak InsP 3 responses, and COS-7 cells overexpressing the Pmr1 pump had delayed Ca 2+ influx (42). It is tempting to speculate that calreticulin, by interacting with the Golgitargeted Pmr1 pump, might promote its retention in the ER, thereby accounting for the increased Ca 2+ pumping activity observed in calreticulin overexpressers. In any case, the existing evidence strongly suggests that calreticulin interacts differentially with distinct Ca 2+ pump isoforms and modulate the rates of Ca 2+ uptake into the ER, thereby directly altering [Ca 2+ ] ER . The physiological relevance of these interactions is not clear, but a decreased [Ca 2+ ] ER has been shown to activate the transcription of the calreticulin gene (43). Therefore, an increase in calreticulin level in the ER would rapidly restore normal [Ca 2+ ] ER levels, thereby abrogating its transcriptional activation. Consistent with such a feed-back mechanism, the [Ca 2+ ] ER increase was the first perturbation observed upon the induction of calreticulin.
In addition to increasing the total and free Ca 2+ of the ER, calreticulin also increased the rates of agonist-induced Ca 2+ release. Increased release was observed over a wide range of [Ca 2+ ] ER , indicating that it did not simply reflect the increased driving force for Ca 2+ , but increased fluxes though InsP 3 -gated channels. This was unexpected, because it was reported recently that the rates of ATP-induced Ca 2+ release were decreased in cells with increased [Ca 2+ ] ER due to overexpression of SERCA (24). This effect was attributed to the Ca 2+dependent inhibition of InsP 3 gated channels. Because in our calreticulin-induced cells the InsP 3 channels were also exposed to higher amounts of Ca 2+ ions, both on the ER and on the cytosolic side, the increased release might reflect a direct action of calreticulin on InsP 3 -gated Ca 2+ channels.
Because of the increased ER Ca 2+ load and the increased driving force for Ca 2+ , more Ca 2+ was released into the cytosol during stimulation with agonists and/or thapsigargin, and store-operated Ca 2+ influx was reduced when measured with the Ca 2+ re-addition protocol (Fig. 3).
However, analysis of the cytosolic and ER responses at different times after induction indicated that calreticulin levels had no direct effects on store-operated Ca 2+ influx.
Decreased SOC activity was only observed in cells induced to express CRT for 3 days, and correlated with an increase in total stored Ca 2+ , rather than with the resting [Ca 2+ ] ER levels ( Fig. 4). In previous studies, decreased Ca 2+ influx was observed in stable calreticulin overexpressers (32), but not in cells transiently transfected with calreticulin (33). Our observations reconcile these apparently discrepant findings, and caution against the Ca 2+ readdition protocol to assess store-operated Ca 2+ influx, because 1) the degree of store depletion cannot be readily estimated from the cytosolic Ca 2+ responses, and 2) the concomitant activity of SERCA greatly affects the dynamics of the [Ca 2+ ] cyt signal, precluding accurate estimates of the influx component.
The effects of calreticulin extended beyond the ER and affected another organelle, the mitochondria. However, the larger release of Ca 2+ from the ER was not associated with an equally larger Ca 2+ accumulation in mitochondria, but with a reduced signal as [Ca 2+ ] mit rapidly returned to basal levels despite the presence of InsP 3 -generating agonists (Fig. 6). The abnormal [Ca 2+ ] mit response did not reflect structural damage, because the shapes and numbers of mitochondria as well as their relationship to the ER appeared normal by confocal microscopy, but was associated with a mitochondrial depolarization (Fig. 7). The depolarization, by reducing the driving force for Ca 2+ , is expected to reduce mitochondrial Ca 2+ uptake and might thus account for the blunted [Ca 2+ ] mit response. In addition, the activity of the mitochondrial Ca 2+ uniporter might be further inhibited by the high Ca 2+ concentrations found at the ER/mitochondria microdomain. Prolonged exposures to high Ca 2+ concentrations might desensitize the uniporter, as exposures to low Ca 2+ concentrations are needed to reset the uniporter into rapid uptake mode, its most efficient mode of Ca 2+ uptake (44).
Furthermore, the mitochondria Ca 2+ uptake sites have been shown to be already close to saturation during physiological stimulations (6,37) concentrations.
These perturbations of Ca 2+ homeostasis are unlikely due to the chaperone function of calreticulin, as impaired ER and mitochondrial Ca 2+ responses were observed in cells induced to express a truncated calreticulin lacking the chaperone N-domain of the protein (Fig. 8).
Most importantly, the overexpression of calnexin, an ER chaperone similar to calreticulin and containing a chaperoning P-domain, did not affect cytosolic or ER Ca 2+ homeostasis. This indicates that the effects do require neither the N-nor the P-domain, but are mediated by the unique C-domain of calreticulin. Thus, alterations in "Ca 2+ -sensing", rather that in chaperone activity, are responsible for the increased Ca 2+ pumping and release activity, which lead to higher Ca 2+ turnover between the ER and mitochondria. These findings have important physiological implications because different levels of calreticulin are expressed in different tissue (28). Furthermore, expression of the protein is up-regulated under the conditions of stress and starvation (28). In the immune system, the CRT gene is activated in stimulated cytotoxic T-cells (45) where it may play a role in a Ca 2+ -dependent signaling and/or cytotoxic T-cell killing. In many cancer cells, including prostate cancer, calreticulin expression is increased or up-regulated by different steroids (46,47). Expression of calreticulin is also differentially regulated during development (48          by guest on July 9, 2020