Stanniocalcin 2 alters PERK signalling and reduces cellular injury during cerulein induced pancreatitis in mice
© Fazio et al; licensee BioMed Central Ltd. 2011
Received: 20 January 2011
Accepted: 5 May 2011
Published: 5 May 2011
Stanniocalcin 2 (STC2) is a secreted protein activated by (PKR)-like Endoplasmic Reticulum Kinase (PERK) signalling under conditions of ER stress in vitro. Over-expression of STC2 in mice leads to a growth-restricted phenotype; however, the physiological function for STC2 has remained elusive. Given the relationship of STC2 to PERK signalling, the objective of this study was to examine the role of STC2 in PERK signalling in vivo.
Since PERK signalling has both physiological and pathological roles in the pancreas, STC2 expression was assessed in mouse pancreata before and after induction of injury using a cerulein-induced pancreatitis (CIP) model. Increased Stc2 expression was identified within four hours of initiating pancreatic injury and correlated to increased activation of PERK signalling. To determine the effect of STC2 over-expression on PERK, mice systemically expressing human STC2 (STC2 Tg ) were examined. STC2 Tg pancreatic tissue exhibited normal pancreatic morphology, but altered activation of PERK signalling, including increases in Activating Transcription Factor (ATF) 4 accumulation and autophagy. Upon induction of pancreatic injury, STC2 Tg mice exhibited limited increases in circulating amylase levels and increased maintenance of cellular junctions.
This study links STC2 to the pathological activation of PERK in vivo, and suggests involvement of STC2 in responding to pancreatic acinar cell injury.
The stanniocalcin (STC) family of secreted glycoproteins consists of two members - STC1 and STC2 - that have 35% conserved identity and conservation of exon/intron boundaries, indicating that they are paralogs . STC was first discovered in fish and shown to be involved in calcium homeostasis through effects on calcium influx [2, 3]. Although terrestrial mammals rarely experience hypercalcemia, alterations in subcellular compartmentalization of Ca2+ are found under pathological conditions such as the accumulation of improperly folded proteins. STC2 expression is upregulated upon induction of hypoxic or endoplasmic reticulum (ER) stress in N2a neuroblastoma cells . This increase in Stc2 expression was reliant on the concomitant increase of Activating Transcription Factor (ATF4) , a protein integral to (PKR)-like endoplasmic reticulum kinase (PERK) signalling, which is part of the unfolded protein response (UPR). To date, no relationship between PERK signalling and STC2 activation has been identified in vivo.
The UPR is activated when ER homeostasis is disrupted. Alterations to proper functioning of ER machinery arise when protein load exceeds folding capacity, such as when luminal ER Ca2+ concentrations are perturbed. The UPR consists of three signalling pathways that are individually transduced by ATF6, inositol-requiring enzyme 1 (IRE1) or PERK [6, 7]. Each transducer is maintained in a repressed state by binding of the ER chaperone protein glucose regulated protein 78 (GRP78/BiP) [6, 7]. When unfolded proteins accumulate, they bind to GRP78/BiP resulting in its release from the UPR transducers [8, 9]. In the case of PERK, dissociation of GRP78/BiP leads to its homodimerization and autoactivation . Activation of PERK results in phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) , limiting its ability to contribute to the protein translational complex, thereby inhibiting global protein translation and alleviating the protein load on the cell . Phosphorylation of eIF2α also selectively upregulates translation of mRNAs from short upstream open reading frames . The predominant target mRNA for peIF2α is Atf4, which subsequently increases the expression of the transcriptional repressor Atf3, and growth arrest and DNA damage-inducible (Gadd) 34 [14, 15]. GADD34 (alternatively known as Myd116; myeloid differentiation, primary response gene 116, or Ppp1r15a; protein phosphatase 1, regulatory [inhibitor] subunit 15A) negatively regulates PERK signalling by combining with protein phosphatase 1 to dephosphorylate eIF2α, thereby restoring general translation [16, 17].
PERK is expressed in the exocrine and endocrine pancreas under normal conditions [11, 18] and Perk -/- mice experience deterioration of glycemic control and exocrine insufficiency over time . Exocrine specific deletion of PERK revealed cellular disorganization and degranulation, increased serum amylase levels and increased cell death . Our laboratory identified rapid activation of PERK signalling following induction of pancreatitis by supramaximal secretagogue stimulation and correlated decreased activation of the pathway in mice that exhibit increased sensitivity to pancreatic injury . Together, these observations indicate that PERK signalling has important roles in both the physiology and pathology of the exocrine pancreas.
Given the relationship between PERK and STC2 in vitro, the objectives of this study were to (1) determine if a similar relationship exists in the exocrine pancreas and (2) gain insight into a role for STC2 as part of the UPR. Our results revealed a correlation of Stc2 expression with PERK signalling in vivo only after initiating pancreatic injury. Transgenic over-expression of STC2 in mice (STC2 Tg )  resulted in altered PERK signalling and decreased signs of acinar cell damage associated with cerulein-induced pancreatitis (CIP). These observations suggest that STC2 is linked to PERK signalling in acinar cells and may have a role in limiting damage during pancreatic injury.
To understand why activation of PERK and eIF2α appear reduced in STC2 Tg tissue, we examined regulators of these two proteins. The phosphorylation of eIF2α is reversed by GADD34 through its interaction with protein phosphatase 1 (PP1) and recent evidence indicates that calcineurin binds PERK and stimulates its autophosphorylation . In STC2 Tg pancreatic tissue, the levels of GADD34 protein were significantly higher than wild type tissue (Figure 3C). Conversely, STC2 Tg calcineurin levels were considerably lower than WT counterparts. No changes in the ER resident protein calregulin were observed between genotypes (Figure 3C). Based on these results, it appears that over-expression of STC2 affected expression of mediators and feedback mechanisms in PERK signalling.
STC2 is a secreted protein that, when expressed to high levels in mice, has profound effects leading to growth restriction . However, ablation of Stc2 in mice results in limited phenotypic alterations suggesting that STC2 functions may fall outside normal physiology . In support of such a hypothesis, in vitro analysis has identified increased Stc2 accumulation following treatment with thapsigargin or tunicamycin, agents that activate ER stress pathways . STC2 is also up-regulated in response to hypoxic insult and is expressed to higher levels in many tumor-derived cell lines [4, 33]. This suggests that STC2 may be involved in responding to external challenges that activate cell stress pathways. In this study, we have identified increased Stc2 accumulation within four hours of inducing pancreatic injury that correlated with activation of the PERK signalling. Importantly, forced expression of STC2 in mice altered PERK signalling and reduced cellular damage in response to pancreatic injury. These results suggest a role for STC2 responding to and mediating the effects of cell stress in pancreatic tissue.
Studies assessing the expression of Stc2 have revealed a limited expression pattern in adult tissues. In the human pancreas, Stc2 is specifically expressed in alpha cells of the islets of Langerhans and not in acinar tissue . Our mRNA analysis revealed murine pancreatic expression of Stc2 only after induction of pancreatitis by supramaximal secretagogue stimulation. Unfortunately, IHC on CIP-treated tissue sections did not detect STC2 protein (data not shown) indicating that STC2 may be expressed at lower than detectable levels or that it is rapidly secreted into the circulation. While we have not definitively identified acini as the origin of Stc2 expression, Atf3, a downstream mediator of PERK signalling, accumulated only in acinar cells during CIP, suggesting that CIP treatment leads to a cell autonomous activation of Stc2. As observed in HEK293 and N2a neuroblastoma cells, the increased accumulation of Stc2 is consistent with activation of PERK signalling in response to injury . Induction of Stc2 mRNA levels was not observed after CIP in Mist1 -/- mice, which do not activate PERK or ATF3, and experience increased severity of CIP . These findings support the activation of Stc2 as a protective mechanism, potentially as a mediator of the UPR.
While previous studies have correlated increased expression of STC2 as part of the UPR, the effects of STC2 on PERK signalling have not been examined. We have now shown that systemically high levels of STC2 reduce PERK activation. STC2 Tg mice showed decreased phosphorylation of PERK and eIF2α in pancreatic tissue suggesting that continued exposure to STC2 may lead to activation of negative feedback mechanisms. Since no change in total eIF2α is observed, this difference is peIF2α is specific to the phosphorylation event and not decreased eIF2α accumulation. While we showed a decrease in pPERK, total PERK levels were not assessed and this could account for possible differences in activation of the pathways as well.
We have identified at least two effectors of the PERK signalling pathway that were changed in STC2 Tg mice. First, GADD34, which combines with protein phosphatase 1 to reduce eIF2α phosphorylation , accumulated to higher levels. Second, we observed significantly decreased accumulation of calcineurin, which stimulates PERK autophosphorylation, thereby increasing activity . Whether these changes are reflective of a direct role for STC2 in their regulation or a response to other events caused by over exposure to STC2 is unclear. However, the altered expression of these factors suggests a role for STC2 in regulating Ca2+ homeostasis since both calcineurin and PERK are affected by altered Ca2+ sequestration in the ER. These data suggest that constitutive exposure to STC2 results in depression of the activity of key factors regulating the UPR.
In light of decreased PERK and eIF2α activation, it was surprising that increased accumulation of ATF4 was observed in the pancreas of STC2 Tg mice. Phosphorylation of eIF2α is known to stimulate translation of Atf4 mRNA, leading to increased ATF4 protein accumulation. However, increased ATF4 expression, and the subsequent increase in GADD34 could be mediated by increased stability of the ATF4 protein. Recent studies have shown that ATF4 is stabilized by interaction with p300 through inhibition of ubiquitination , and that association with p300/CBP enhances ATF4 transcriptional ability [34, 35]. It is possible that in STC2 Tg mice, stabilization of ATF4 is enhanced regardless of decreased phosphorylation of eIF2α, accounting for its increased expression. Although there is little known about non-peIF2α related transcriptional activation of ATF4, increased transcriptional activation by a yet to be identified factor cannot be ruled out.
As would be expected from increased ATF4 expression, STC2 Tg mice also exhibited an increased level of autophagy, a downstream consequence of elevated ATF4 levels. Autophagy is important for maintenance of cellular homeostasis through balancing synthesis and degradation or recycling of cellular components. Independent studies have indicated that either phosphorylation of eIF2α or expression of ATF4 is required for induction of autophagy in vitro [28, 36, 37]. The increased expression of ATF4 in STC2 Tg pancreatic tissue may be the cause of increased autophagy. Interestingly, we did not observe increased acinar cell apoptosis (data not shown), which is another downstream result of increased UPR. Therefore, it appears that only certain parts of the UPR have been activated in STC2 Tg mice.
Whether STC2 is protective in nature is still unclear since studies have shown it has both protective  and detrimental roles [4, 33] in cancer progression in vitro. Our results suggest a protective advantage following exocrine pancreatic injury in vivo. STC2 Tg mice exhibit a decreased proportional increase in serum amylase levels, increased maintenance of cellular junctions, decreased apoptosis and necrosis and decreased activation of CPA in exocrine pancreatic tissue, all suggestive of decreased pancreatitis severity. In addition, the enhanced autophagy observed in STC2 Tg acinar tissue would facilitate more rapid degradation of resident digestive enzymes upon injury, thereby limiting damage to cellular contents. This would account for increased maintenance of cellular junctions. However, to truly understand the role of STC2 in pancreatic injury, further experiments in mice lacking Stc2 mice should be performed.
In conclusion, this is the first study to correlate PERK signalling and STC2 in vivo, and suggests two novel roles for STC2, as a downstream effector of PERK signalling and possible protective factor during pancreatic injury. This is also the first time that STC2 has been shown to affect proteins that regulate Ca2+-mediated response in the cell.
For characterization of Stc2 expression during cerulein-induced pancreatitis (CIP), wild type and Mist1 -/- mice were maintained on a C57/Bl6 background. For analysis of pancreatitis severity, STC2 Tg and wild type mice are maintained on a C57/Bl6 × CBA background. Mice carrying a targeted ablation of the Mist1 gene (Mist1 -/- )  and transgenic mice that express STC2 (STC2 Tg ) from the CMV promoter + chicken β-actin promoter , have previously been described. All experiments were approved by the Animal Care Committee at the University of Western Ontario (Protocol #116-2008) and mice handled according to regulations stipulated by the Canadian Council on Animal Care.
Induction of pancreatitis
To induce pancreatic injury, 2-4 month-old female mice were given up to seven hourly intraperitoneal injections of cerulein (50 μg/kg body weight; Sigma-Aldrich). As a control, mice were injected with 0.9% saline. Mice were sacrificed 1, 4 or 8 hours after initial cerulein injection and pancreatic tissue from each was immediately processed to isolate RNA, protein, and tissue sections for histological or immunohistochemical analysis. To assess serum amylase, blood was obtained via cardiac puncture, placed on ice for 20 minutes and centrifuged at 4°C, 2500 rpm for 15 minutes. Serum amylase was quantified using a Phadebas amylase assay (Magle Life Sciences, Lund, Sweden) as per manufacturer's instructions.
Tissue preparation and histology
List of Primary Antibodies Used in Fazio et al (2011)
Santa Cruz Biotechnology (Santa Cruz, CA, USA)
Wu et al. (2010). Nature. 465: 368-372
Santa Cruz Biotechnology
Matsushita et al. (2009). Mol. Cell. Biol. 29: 5843-5857.
Sigma (St. Louis, MO, USA)
Santa Cruz Biotechnology
AbD Serotec (Oxford, UK)
Transduction Laboratories (San Jose, CA, USA)
Millipore (Temecula, CA, USA)
Invitrogen (Carlsbad, CA, USA)
Harding et al. (2009). PNAS. 106(6), 1832-1837
Cell Signalling (Beverly, MA, USA)
Nanbo et al. (2002) EMBO J. 21, 954-965.
Santa Cruz Biotechnology
Makris et al. (2000). Mol. Cell. 5: 969-979.
Novus Biologicals (Littleton, CO, USA)
ProSci Incorporated (Poway, CA, USA)
Abcam (Cambridge, MA, USA)
Santa Cruz Biotechnology
Denis et al. (2010). Neuroscience. 170(4):1035-44
Chemicon (Temecula, CA, USA)
TUNEL analysis was used to identify apoptotic cells using the In situ Cell Death Detection kit (Roche, Laval, QC, Canada) following manufacturer's instructions. Briefly, frozen sections were fixed, washed and permeabilized before labelling with TUNEL reaction mixture for 60 minutes. After washing, sections were counterstained with DAPI, coverslipped and visualized using a Leica DMIOS upright microscope and images were captured using OpenLab 4.0.3 Software (PerkinElmer, Waltham, MA, USA). Analysis of percent apoptotic cells was carried out by comparing TUNEL positive cells to total cell number. Cell counts based on DAPI were done using Image J software, and TUNEL positive cells were determined manually. Ten random fields of view from 4 sections per animal were used for analysis.
RNA isolation, Northern blotting and Real-Time RT-PCR
RNA was isolated from the splenic portion of the pancreas and processed using TRIzol (Invitrogen, Burlington, ON, Canada) as described . Northern blotting was performed as previously described . Thirty μg of RNA was resolved by electrophoresis on a 1% agarose/formaldehyde gel and blotted onto Hybond membrane (GE Healthcare, Baie d'Urfe, QC, Canada). Membranes were hybridized overnight at 42°C with an α-32PdCTP radiolabelled probe for Stc2 or for 18S rRNA as a loading control.
For RT-PCR, two μg of RNA was reverse transcribed using Improm-II reverse transcriptase and random primers (Promega, Madison WI, USA). RT-PCR for Stc2, Atf3, amylase and β-actin was performed with 1 μL of cDNA and Taq DNA polymerase (Promega, Madison WI, USA).
Protein isolation and immunoblot analysis
Protein extraction was carried out to obtain both nuclear and cytoplasmic fractions. Briefly, 5 volumes of buffer CE pH 7.6 (10 mm Hepes, 60 mM KCl, 1 mM EDTA, 0.075% v/v NP-40, 1 mM DTT, 1 mM PMSF) were added to whole pancreatic tissue prior to homogenization. Homogenized cells were centrifuged at 1000 rpm, 4°C for 4 minutes. The supernatant was removed and saved as cytoplasmic extract. The nuclear pellet was gently washed with CE buffer lacking NP-40 and centrifuged again. After removal of the supernatant, the nuclear pellet was covered with 1 pellet volume of NE buffer (20 mm Tris-HCl, 420 mM NaCl, 1.5 mm MgCl2, 0.2 mM EDTA, 1 mM PMSF, 25% w/v glycerol) and the NaCl concentration was adjusted to 400 mM for the entire volume. An additional pellet volume of NE buffer was added followed by vortexing for resuspension. The extract was incubated on ice for 10 minutes with intermittent vortexing. Both cytoplasmic and nuclear extracts were centrifuged at 14,000 rpm, 4°C, for 10 minutes to pellet any debris. Nuclear extracts were used for analysis of MIST1, PDX1, XBP1 and ATF4 levels, whereas cytoplasmic extracts were used for all other immunoblot analysis.
Electrophoresis and immunoblotting were carried out as previously described . For immunoblot analysis, 40 to 60 μg of pancreatic protein was resolved by SDS/PAGE and transferred to PVDF membrane (Biorad, Mississauga, ON, Canada). Blots were blocked with 5% non-fat dry milk (NFDM) and probed with primary antibodies diluted in 5% NFDM overnight at 4°C. For primary antibodies and their dilutions, see Table 1. Blots were washed three times in PBS supplemented with Tween-20 (PBS-T) then incubated with anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibody (1:10,000 or 1:2000, Jackson Labs, Bar Harbor, ME, USA) in 5% NFDM for one hour. Blots were subjected to another series of washes in PBS-T, incubated with Western Lightning chemiluminescence substrate (Perkin Elmer, Waltham MA, USA), exposed to X-ray film (Fisher Scientific, Ottawa, ON, Canada) and developed. Denistometry was performed on immunoblot autoradiographs by analysis with a FluorChem 8800 documentation system and accompanying FluorChem 8800 software (Alpha Innotech, San Leandro CA, USA).
All statistical analyses were performed using Graphpad Prism 4.02 (Graphpad Software, San Diego CA, USA). Carboxypeptidase cleavage and serum amylase levels were analyzed using a two-way ANOVA followed by a Bonferroni post-hoc test. Levels of phosphorylated eIF2α and LC3 cleavage were statistically analyzed using a Mann-Whitney test.
The authors would like to thank members of the Pin and DiMattia laboratories, especially Charis Johnson, Christin Giller and Yudith Ramos-Valdes for their support and suggestions in these studies. This work was funded by operating grants from the Canadian Institutes of Health Research (CP - MOP 53083; GD - MOP 53128). C.P. is supported by a salary award from the CIHR. E.F. is supported by a doctoral research award from the CIHR/CDHF.
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