Cell-surface calreticulin has been characterized as an important factor in inducing anti-tumor immune responses (7
). Anti-cancer regimens that induce an anti-tumor immune response are likely to be more effective than non-immunogenic regimens with similar cancer cell cytotoxicity (4
). Identifying conditions that induce cell-surface calreticulin, and understanding mechanisms and functional consequences of cell-surface calreticulin expression could help design more effective anti-tumor therapies. Our findings indicate that thapsigargin induces transient, pre-apoptotic, cell-surface calreticulin exposure in some cell types ( and ), that thapsigargin treatment of target cells increases pro-inflammatory cytokine production by BMDC in a manner independent of target cell-derived calreticulin (), that extracellular calreticulin either in a cell-surface bound ( and S2D
) or soluble form (, S2C
) is unable to stimulate production of IL-6, IL-1β, TNF-α, IL-23 or IL-12p70, and that thapsigargin treatment of cells impacts their phagocytic uptake in a calreticulin-dependent manner ().
Two mechanisms are previously described for cell-surface calreticulin exposure (8
). The first pathway is dependent on the PERK arm of the UPR, and results in the specific co-translocation of calreticulin-ERp57 complexes to the cell surface (8
). The second suggested pathway, relevant to apoptotic cells, involves the binding of cytosolic calreticulin to phosphatidylserine on the inner leaflet of the plasma membrane prior to apoptotic exposure of phosphatidylserine-calreticulin complexes on the cell surface (40
). Neither of these pathways seems related to the mechanisms of surface exposure of calreticulin in viable cells subjected to ER calcium depletion, which display a general loss of ER retention of various ER chaperones. The thapsigargin-induced mode of calreticulin surface expression is independent of calreticulin-ERp57 binding (). Rather, we have recently shown that induction of a generic polypeptide-binding site on calreticulin facilitates its cell-surface expression under calcium-depleting conditions (21
Our data indicate a secretory route for ER proteins (), as also suggested for the calreticulin-ERp57 co-translocation mechanism (8
). It is noteworthy that several of the ER resident proteins detectable in supernatants of thapsigargin-treated cells () are known calcium binding proteins of the ER (48
). As is the case with calreticulin (49
), ER retention of BiP, PDI and gp96 may in part involve calcium-dependent processes. However, calcium-depletion alone appears insufficient to induce secretion of the ER chaperones (). Rather, the combined effects of KDEL receptor saturation and calcium-depletion appear to be the mechanism that drives secretion of the ER chaperones in thapsigargin-treated cells. Similar mechanisms may be relevant to the extracellular localization of ER chaperones under physiological protein misfolding conditions, known to induce the UPR and alter ER calcium homeostasis. To investigate this possibility, we compared chaperone secretion in wild type and cog/cog
mice thyroids, which express a misfolded, mutant thyroglobulin that accumulates in the ER (50
). In preliminary findings, we observed enhanced secretion of calreticulin, PDI and gp96 into the apical follicular lumen of cog/cog
mice thyroids compared to their wild type counterparts, correlating with significant enhancement in total chaperone levels in the cog/cog
thyroid lysates (Jeffery, Kellogg, Arvan and Raghavan, unpublished observations).
Previous studies in HeLa cells have shown that NF-κB activation in response to agents that induce ER stress involves the release of calcium from the ER and the subsequent generation of ROS (reviewed in (51
)). This mechanism likely accounts for sterile IL-6 production in response to direct thapsigargin treatments of BMDC (). Thapsigargin is a stronger activator of the sterile IL-6 response than tunicamycin () likely due to the rapid of effect of thapsigargin on cytosolic calcium elevation, whereas elevation of cytosolic calcium in response to tunicamycin treatment may be more modest as well as kinetically delayed. Many recent studies have shown synergies between TLR-derived signals and transcription factors induced by the UPR, including XBP-1 and C/EBP homologous protein (CHOP). Of the cytokines measured in and , XBP-1 has been previously shown to synergize with TLR signals in enhancing IL-6 production by murine macrophages (41
). In human, monocyte-derived DC, CHOP binding to the IL-23p19 promoter enhanced IL-23 production in response to TLR ligation (43
). Similar mechanisms could account for the synergy of ER stress signals and LPS in IL-23 production by BMDC (). We found that thapsigargin also strongly synergizes with LPS in the generation of IL-1β and IL-12p70. Recent findings have shown that high avidity ligation of ITAM-containing receptors such as DAP12 can synergize with TLR signals to activate pro-inflammatory gene expression (reviewed in (52
)). High avidity ligation of DAP12 triggers an acute, transient calcium increase (reviewed in (52
)). Here we show that, compared to tunicamycin, thapsigargin treatment strongly enhances various pro-inflammatory responses to LPS (), consistent with similar findings from previous studies (45
). These results point towards direct synergies between cytosolic calcium and TLR responses, and broader synergy between intracellular calcium and TLR signaling compared to those between the UPR and TLR signaling. Interestingly, it has been suggested that ER calcium depletion can occur under certain conditions of ER overload and protein polymerization, even in the absence of classical UPR induction (54
). Thus, findings described here may be relevant to a better understanding of inflammatory protein folding disorders that do not induce a classical UPR pathway.
A truncated bacterially-expressed calreticulin construct was shown to induce TNF-α production by murine macrophages and human PBMC (10
). N-terminal truncation of calreticulin induces self-association likely via the exposure of hydrophobic surfaces (10
), and it is possible that protein truncation induces enhanced binding to bacterial pathogen-associated molecular patterns. We show here that full-length extracellular calreticulin derived from an endogenous source does not enhance the production of various pro-inflammatory cytokines in a thapsigargin-treated BMDC context (, , S2C-D and S3
). These findings are consistent with a previous report that a secreted form of calreticulin does not induce pro-inflammatory cytokine production by BMDC (55
). Calreticulin is known to be present in the extracellular environment under a number of physiological conditions, and a number of extracellular functions are described for calreticulin (reviewed in (3
)). Given these extracellular roles of the protein, the lack of innate immune induction by endogenously-derived, extracellular calreticulin is perhaps not surprising.
Calreticulin-high, thapsigargin-treated, target cells were phagocytosed more efficiently than untreated cells, and enhanced phagocytosis was partly dependent on calreticulin expression by the target cells (). Correlating with undetectable levels of surface calreticulin in UV-treated cells, we did not observe an effect of calreticulin on the phagocytosis of UV-treated cells (). In the UV-treated apoptotic cell context, other changes are expected that promote phagocytic uptake (reviewed in (56
)), including the exposure of phosphatidylserine, which likely explain why significant uptake was observed in the absence of any calreticulin expression by the target cells (). Correspondingly, UV-treated target cells had a higher level of total association with BMDC than any other target cell group (Fig. S4
). This is likely the result of molecules that are upregulated on the surface of apoptotic cells and responsible for bridging apoptotic phagocytic cargo to potential phagocytes (reviewed in (56
)). It is important to consider that increases in surface calreticulin by a magnitude measurable by flow cytometry were, in general, difficult to detect in apoptotic cells (-). Stronger non-specific protein binding to apoptotic cells could render a specific signal more difficult to detect by flow cytometry. Microscopic observations could allow for calreticulin redistribution or upregulation to be more readily visualized on apoptotic cells, as described in other studies (5
). It is also important to note that cell-surface calreticulin functions in the context of several other key eat-me and don’t-eat-me signals in viable and apoptotic cells (5
). The data of show that tunicamycin-treated cells displayed enhanced phagocytic uptake compared to untreated cells, although this uptake was not calreticulin-dependent. This observation correlates with the absence of detectable surface calreticulin in tunicamycin-treated cells (). The same level of uptake was seen with thapsigargin-treated, calreticulin-deficient cells (). It is possible that interference with proper protein folding resulting from tunicamycin and thapsigargin treatments also cause decreased surface expression of don’t-eat-me signals like CD47 (5
) and plasminogen activator inhibitor-1 (PAI-1) (57
). This potential decreased surface expression of don’t-eat-me signals may explain the increased level of uptake of tunicamycin-treated and thapsigargin-treated, calreticulin deficient cells by BMDC.
In contrast to some previous findings (7
), we show here that thapsigargin induces cell-surface calreticulin, and that thapsigargin promotes phagocytic uptake of cells via mechanisms that are, in part, calreticulin-dependent. Thus the inability of thapsigargin-treated cells to induce anti-tumor immunity with sufficient potency (7
), is likely not related to an absence of surface calreticulin per se
. Other immunogenic signals absent in thapsigargin-treated cells (Fig. S2E-F
), including HMGB1 (58
) and elevated levels of ATP (59
), may be required to confer stronger immunogenicity to tumor cells. Consequently, although thapsigargin has some potentially desirable attributes for a chemotherapeutic, including the abilities to induce stronger phagocytic uptake of treated cells by DC () and elicit stronger cytokine production by DC ( and ), these appear to be insufficient signals to confer anti-tumor immune protection. These observations are highly relevant to the ongoing clinical trials evaluating thapsigargin pro-drugs for the treatment of advanced solid tumors (ClinicalTrials.gov Identifier: NCT01056029, (60
)). Since phagocytic uptake and cytokine levels can influence T cell priming steps, our results highlight the importance of further research investigating the impacts of different forms of ER stress on the priming of CD8 and CD4 T cell responses.