A Carboxyl Ester Lipase (CEL) Mutant Causes ChronicPancreatitis by Forming Intracellular Aggregates ThatActivate Apoptosis*

Received for publication, April 22, 2016, and in revised form, September 9, 2016 Published, JBC Papers in Press, September 20, 2016, DOI 10.1074/jbc.M116.734384

Xunjun Xiao‡, Gabrielle Jones‡, Wednesday A. Sevilla‡, Donna B. Stolz§, Kelsey E. Magee‡, Margaret Haughney‡,Amitava Mukherjee‡, Yan Wang‡, and Mark E. Lowe‡1

From the ‡Department of Pediatrics, Children’s Hospital of Pittsburgh at University of Pittsburgh Medical Center, Pittsburgh,Pennsylvania 15224 and §Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Patients with chronic pancreatitis (CP) frequently havegenetic risk factors for disease. Many of the identified geneshave been connected to trypsinogen activation or trypsin inac-tivation. The description of CP in patients with mutations in thevariable number of tandem repeat (VNTR) domain of carboxylester lipase (CEL) presents an opportunity to study the patho-genesis of CP independently of trypsin pathways. We testedthe hypothesis that a deletion and frameshift mutation(C563fsX673) in the CEL VNTR causes CP through proteotoxicgain-of-function activation of maladaptive cell signaling path-ways including cell death pathways. HEK293 or AR42J cells weretransfected with constructs expressing CEL with 14 repeats inthe VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onsetdiabetes of youth with a deletion mutation in the VNTR(MODY)). In both cell types, CEL MODY formed intracellularaggregates. Secretion of CEL MODY was decreased comparedwith that of CEL14R. Expression of CEL MODY increasedendoplasmic reticulum stress, activated the unfolded proteinresponse, and caused cell death by apoptosis. Our results dem-onstrate that disorders of protein homeostasis can lead to CPand suggest that novel therapies to decrease the intracellularaccumulation of misfolded protein may be successful in somepatients with CP.

Pancreatitis is an inflammatory disease with significanthealth and economic burdens (1, 2). There are no therapies toprevent recurrent episodes or progression to chronic disease.The absence of effective therapies stems in part from our lim-ited understanding about the pathophysiology of pancreatitis.The prevailing model holds that intracellular trypsinogen acti-vation in the acinar cell and failure of protective mechanismsresponsible for trypsin inactivation are central to pathogenesis(3). Accumulating evidence suggests that the acinar cellresponds to injury through multiple pathways and that trypsinactivation is not the only potential player in this process (4, 5).The recent clinical reports of patients who develop chronic

pancreatitis in association with mutations in a pancreatic diges-tive lipase, carboxyl ester lipase (CEL),2 offers a unique oppor-tunity to study the pathogenesis of chronic pancreatitis inde-pendently of trypsin (6 –9). CEL has no known role intrypsinogen activation or trypsin degradation, nor does CELrequire activation by trypsin for function. Thus, CEL variantslikely cause disease through a trypsin-independent mechanism.Understanding the molecular details of the pathophysiologyunderlying CEL variant-associated disease can provide insightinto novel therapies for chronic pancreatitis.

CEL is expressed in the pancreas and lactating mammaryglands (10). The protein consists of two domains, an N-termi-nal �/�-hydrolase fold and a C-terminal domain containing avariable number of proline-rich tandem repeats (VNTR) (11).The number of tandem repeats in humans ranges from three to28 (12, 13). About 40% of humans are homozygous for 16repeats (12). The proline-rich repeats are heavily O-glycosy-lated and are important for protein stability (14). Furthermore,the number of repeats influences intracellular processing ofCEL and may alter stability in an aqueous milieu after secretion(15, 16). The number of tandem repeats does not associate withincreased risk for chronic pancreatitis (17).

Conversely, alterations in the VNTR sequence can causepancreatic disease. A hybrid allele formed by recombinationbetween CEL and the adjacent pseudogene, CELP, encodes afusion protein of CEL and the abnormal VNTR of CELP (9). Thepresence of the hybrid allele increased the risk for chronic pan-creatitis in a northern European population about 5-fold (9).Interestingly, the hybrid allele did not associate with chronicpancreatitis in an Asian population (18).

In another study, mutations in CEL were identified in twoNorwegian families with pedigrees of nonketotic, autosomaldominant diabetes (7). Each disease-associated mutationcauses a frameshift in the VNTR, resulting in an altered aminoacid sequence and premature truncation of CEL. All of the car-

* This work was supported by National Institutes of Health Grant DK808820.The authors declare that they have no conflicts of interest with the con-tents of this article. The content is solely the responsibility of the authorsand does not necessarily represent the official views of the National Insti-tutes of Health.

1 To whom correspondence should be addressed: Children’s Hospital of Pitts-burgh, One Children’s Hospital Dr., 4401 Penn Ave., Pittsburgh, PA 15224.Tel.: 412-692-5412; Fax: 412-692-8906; E-mail: loweme2@upmc.edu.

2 The abbreviations used are: CEL, carboxyl ester lipase; ATF4, activating tran-scription factor 4; BiP, binding immunoglobulin protein; CEL14R, wild-typeCEL with 14 repeats in the VNTR; MODY, maturity onset diabetes of youthwith a deletion mutation in the VNTR; ER, endoplasmic reticulum; CHOP,C/EBP homologous protein; GRP94, glucose-regulated protein 94; LDH,lactate dehydrogenase; PARP, poly(ADP-ribose) polymerase; NF-�B,nuclear factor �B; UPR, unfolded protein response; VNTR, variable numberof tandem repeat(s); XBP1, X-box-binding protein-1; qRT-PCR, quantitativeRT-PCR; TEM, transmission electron microscopy; XBP1U, unspliced XBP1;XBP1S, spliced XBP1.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 44, pp. 23224 –23236, October 28, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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riers have pancreatic exocrine insufficiency evidenced by lowfecal elastase or steatorrhea and irreversible anatomicalchanges in the pancreas (such as fibrosis and fat infiltration) bycomputed tomography and histopathology. Abdominal painwas not a prominent feature of the carriers. Still, mild, recurrentabdominal pain was present in almost 60% of the carriers (7).Hence, all mutation carriers meet criteria for chronic pancrea-titis. The diabetes is likely a consequence of chronic pancreati-tis rather than an isolated effect of the CEL variant. Two re-cent publications reported the cellular fate of c.1686delT(p.C563fsX673) CEL also called CEL MODY, the most com-mon variant associated with chronic pancreatitis (19, 20). Thefirst publication demonstrated insoluble intracellular andextracellular aggregates of CEL MODY in HEK293 cells (19).Expression of CEL MODY variant modestly activated the pro-tein kinase RNA-like endoplasmic reticulum kinase/eukaryotictranslation initiation factor 2A arm of the unfolded proteinresponse (UPR) pathway but did not inhibit growth or cause celldeath in HEK293 cells (19). The authors speculated that theUPR offset the toxic consequences arising from intracellularaccumulation of CEL MODY and was not the cause of cellinjury. The authors also concluded that the formation of insol-uble aggregates causes disease through an undefined mecha-nism. In a later study, aggregates of CEL MODY were observedon the cell surface and inside cytoplasmic vacuoles (20). Theauthors speculated that many of the vacuoles resulted fromendocytosis of secreted CEL MODY. Furthermore, exposure ofpancreatic exocrine and endocrine cell lines to conditionedmedium from stably transfected HEK293 cells expressing CELMODY showed a significant decrease in viability. The authorsspeculated that extracellular aggregates of CEL MODY or theirdegradation products caused pancreatic cell injury. In thisalternate model, CEL MODY has a direct cytotoxic effect onbeta cells.

In this study, we sought to rectify this controversy and testedan alternative hypothesis: CEL MODY causes pancreatitisthrough proteotoxic gain-of-function activation of maladaptivecell signaling pathways including cell death pathways. To iden-tify the CEL MODY-dependent adaptive cell signaling path-ways, we expressed CEL and CEL MODY in HEK293T cells andin a pancreatic acinar cell line, AR42J cells. We then determinedthe fate of the expressed proteins and measured activation ofthe UPR and downstream effects that activate inflammatoryand cell death pathways. Our findings indicate that CEL MODYforms insoluble intracellular aggregates that activate the UPRand trigger apoptosis.

Results

Expression and Activity of CEL VNTR Variants—To charac-terize the CEL MODY mutation associated with pancreatic dis-ease, we transfected Pichia pastoris GS115 yeast with vectorscontaining wild-type CEL14R or with a frameshift mutation inthe VNTR, CEL MODY. Immunoblotting and lipase activityassays both suggested impaired secretion of CEL mutant vari-ants by transformed yeasts with the most substantial secretiondefect observed for CEL MODY (data not shown). Even so, wewere able to purify each of the recombinant CEL variants fromculture medium. The proteins were purified as determined by

SDS-PAGE and protein staining (Fig. 1B). We assayed each CELvariant for activity against medium and long chain triglyceridesubstrates trioctanoin and triolein. The activity of the mutantvariants was indistinguishable from the activity of CEL14R,indicating that the structure of the VNTR did not influenceCEL activity (Fig. 1C).

Intracellular Accumulation of Aggregated CEL MODYExpressed in HEK293T Cells—We further investigated theimpact of mutations on CEL secretion in a mammalian cellsystem by transfecting HEK293T cells to transiently expressCEL protein variants. We focused only on CEL MODY becausethere is more clinical information on patients with this partic-ular mutation compared with the other variants (6, 7, 21). Sev-enty-two hours after transfection, no lipase activity against tri-octanoin was detected in the medium from cells transfectedwith a control plasmid. In contrast, lipase activity was easilydetectable in the medium from cells expressing the CEL vari-ants. The medium from cells expressing CEL14R had signifi-cantly more activity than did the medium from cells expressingCEL MODY (8.7 � 0.4 versus 0.7 � 0.2 units/ml) (p � 0.01).Western blotting analysis confirmed the presence of both CELvariants in the medium. The amount of CEL14R was 10-foldhigher than the amount of CEL MODY (Fig. 2, A and B) (p �0.001). In contrast, the amount of intracellular CEL MODY wasnearly 2-fold higher than the amount of CEL14R (p � 0.001).Furthermore, unlike CEL14R, the majority of intracellular CELMODY was present in a detergent-insoluble fraction, and onlya small fraction of CEL MODY was soluble. Altogether, theseresults suggest that the CEL MODY mutation causes CEL pro-tein to accumulate as insoluble aggregates inside cells, whichleads to a secretion defect.

Because the frameshift introduces 10 additional cysteine res-idues in the VNTR, we investigated whether the formation ofinappropriate disulfide bonds contributed to CEL MODYaggregation. To test this hypothesis, we separated the deter-gent-soluble and -insoluble fractions of CEL MODY andCEL14R by SDS-PAGE under non-reducing and reducing con-ditions and looked at the pattern of bands by protein immuno-blotting (Fig. 2C). Under both conditions, a band correspond-ing to the expected mass of CEL14R was present in both thedetergent-soluble and -insoluble fractions. In the samples con-taining CEL MODY, a faint band of the expected mass wasdetected in both fractions on non-reducing gels, whereas amuch stronger band was detected in both fractions when sep-arated by reducing gels. The results are consistent with thehypothesis that the mechanism of insoluble aggregation of CELMODY is the formation of inappropriate disulfide bonds.

Expression of CEL MODY Activates the UPR—Because mostof the intracellular CEL MODY is present as an insoluble aggre-gate, we next determined whether CEL MODY expressioncaused ER stress and activated the UPR. First, we measured theexpression of the major ER chaperones BiP and GRP94 by pro-tein immunoblotting (Fig. 3A). Only BiP was significantlyincreased in cells expressing CEL14R and CEL MODY com-pared with control-transfected cells (p � 0.001). However,there was a distinctive distribution of BiP in extracts from cellsexpressing the two CEL variants (Fig. 3B). In cells expressingCEL14R, BiP was mostly present in the soluble fraction with

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trace amounts in the insoluble fraction. Conversely, in cellsexpressing CEL MODY, BiP was mostly in the insoluble frac-tion with only a small amount in the soluble fraction. Theseresults suggest that BiP may attempt to aid folding or degrada-tion of the aggregation-prone CEL MODY protein but thenbecomes trapped upon substrate aggregation. Trapping of pro-tein quality control factors by an aggregating substrate is alsoevident in cytoplasmic Lewy bodies (22).

We next investigated whether CEL MODY expression trig-gered the UPR in HEK293T cells by measuring X-box-bindingprotein-1 (XBP1) mRNA splicing. Like BiP, the overexpressionof both CEL variants increased XBP1 mRNA splicing asopposed to the control (Fig. 3, C and D). The ratio of XBP1mRNA splicing was significantly higher in the cells expressingCEL MODY than in cells expressing CEL14R (p � 0.002). Col-lectively, these findings support the hypothesis that CELMODY expression causes ER stress and triggers the UPR.

CEL MODY Expression Induces Cell Death—The UPR caneither be adaptive to restore normal ER function and to pro-mote cell survival, or it can be maladaptive and activate cell

death pathways (23). First, we studied whether the expressionof CEL MODY triggered cell death by measuring lactate dehy-drogenase (LDH) activity in the medium at 48, 72, or 96 h aftertransfection (Fig. 4A). At each time point, the activity of LDH inthe medium from cells transfected with CEL MODY was signif-icantly higher than that from cells transfected with CEL14R orthe mock control (p � 0.001). The difference increased withtime. The LDH activity for the latter two groups did not differsignificantly throughout the experiment period. The LDHactivity was consistently higher in the medium from cells trans-fected with CEL MODY, independent of the initial seeding den-sity (ranging from 0.2 to 1.0 � 106 cell/ml) (Fig. 4B). This find-ing ruled out cell overpopulation as a potential contributingfactor for elevated LDH activity in the medium from cells trans-fected with CEL MODY. Interestingly, applying additionalenvironmental stress to the transfected cells including coldstress, glucose deprivation, oxidative stress with menadione,and hypoxia mimicked by treatment with cobalt chloride didnot increase the rate or amount of LDH release (data notshown). The results suggest that the UPR in cells expressing

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CEL14R GAPPVPPTGDSGAPPVPPTGDSGAPPVPPTGDSGAPPVPPTGDSGAPPVPPTGDSGAPPVPPTGDSC563fsX673 GPPPCRPRVTPAPPCRPRVTPGPPPCRPRVTPAPPPCRPRVTPGPPP

(CEL MODY)13 14

CEL14R GAPPVPPTGDSGAPPVPPTGDS

FIGURE 1. VNTR amino acid sequence, expression, and assay of VNTR variants. A, the VNTR amino acid sequence for CEL with 16, 14, and three repeats andthe sequence of the VNTR from patients with pancreatic disease are presented with the single-letter amino acid code (7). B, a GelCode Blue-stained SDS-polyacrylamide gel of CEL variants expressed in P. pastoris and purified to near homogeneity. Sections of the same gel are presented after the removal of a lanecontaining a sample from cells expressing CEL with the VNTR deleted. C, activity against trioctanoin and triolein of the VNTR variants is presented. The assayswere done as described under “Experimental Procedures.” Error bars represent S.D.

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CEL MODY is overwhelmed, and cell death pathways areactivated.

Others have reported that prolonged or profuse UPR cancause cell death through apoptosis (24). We therefore nextexamined whether apoptosis was triggered by expression ofCEL MODY by Western blotting analysis of cleaved PARP, amarker of apoptosis; staurosporine-treated cells were used as apositive control (Fig. 4C). Only a faint band representing thecleaved PARP was detected in cells transfected with vector con-trol and CEL14R. In contrast, the abundance of cleaved PARPwas 10-fold higher in the cells expressing CEL MODY com-pared with CEL14R (p � 0.001) with the level at roughly 60% ofthat in the cells treated with staurosporine (Fig. 4D). Further-more, propidium iodide staining at 24, 72, and 96 h showed thatcell death through necrosis did not occur in cells expressingCEL MODY (data not shown). The results demonstrate that theexpression of CEL MODY caused apoptotic cell death.

Insoluble Aggregates of CEL MODY Accumulate in AR42JCells—We next characterized CEL MODY in AR42J cells, amodel system for pancreatic acinar cells (25). These cells syn-thesize and store a number of pancreatic exocrine proteins insecretory granules that are secreted through both a basal and aregulated secretory pathway. The acinar phenotype of AR42Jcells is enhanced by treatment with dexamethasone. We foundthat AR42J cells are difficult to transfect with plasmids but arereadily transduced with adenovirus. Therefore, we transduceddexamethasone-treated AR42J cells with control, CEL14R, orCEL MODY adenovirus. The samples were harvested 72 h aftertransduction. First, real time quantitative PCR analysis showedthat the abundance of mRNA encoding CEL was not statisti-cally higher in the CEL MODY-transduced cells compared withCEL14R (p � 0.05) (Fig. 5A). This insured comparable levels oftransduction with each CEL variant. We then measured thelevels of CEL protein in the medium, in whole cell lysates, and inthe soluble and insoluble intracellular fractions (Fig. 5, B and C).The amount of CEL14R in the medium was 40% higher than theamount of CEL MODY (p � 0.001). The total intracellular lev-els of the two variants were similar, but CEL14R was mostlypresent in the soluble fraction, whereas CEL MODY was moreabundant in the insoluble fraction (p � 0.001). The results fromAR42J cells and HEK293T cells are comparable except theAR42J cells exhibit slightly improved secretion and slightlymore soluble CEL MODY protein compared with HEK293Tcells.

We next investigated whether CEL MODY aggregates aredisulfide-bonded as found in HEK293T cells. As aforemen-tioned, we separated the insoluble fraction of CEL MODY andCEL14R by SDS-PAGE under non-reducing and reducing con-ditions and performed immunoblotting analysis (Fig. 5D).Under both conditions, a band corresponding to the expectedsize of CEL14R was present. In the samples containing CELMODY, a faint band of the expected size was detected amongthe smear under non-reducing condition, whereas a muchstronger band was detected under reducing condition. Theresults are consistent with what we found in HEK293T cells.Importantly, our findings support the hypothesis that aggrega-tion of CEL MODY arises from the formation of inappropriatedisulfide bonds.

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FIGURE 2. Expression of CEL14R and CEL MODY in HEK293T cells. A, thecells were transfected with the indicated plasmid as described under “Exper-imental Procedures.” Samples were obtained from the medium and from theintact cells. In separate experiments, the intracellular CEL was separated intosoluble and insoluble fractions as described under “Experimental Proce-dures.” The proteins in each sample were separated by SDS-PAGE, and immu-noblotting was performed with CEL antiserum. Sections of the same gel arepresented after the removal of a lane containing a sample from cells express-ing CEL with the VNTR deleted. B, the amount of immunoreactive protein wasquantified by densitometry and presented in bar graphs. The medium andsoluble fractions contained significantly more CEL14R (p � 0.001). The totalintracellular sample and the insoluble fraction contained significantly moreCEL MODY (p � 0.001) (n � 6). C, cells were transfected with the indicatedplasmids, and the insoluble fraction was isolated. The samples were sepa-rated by SDS-PAGE with and without �-mercaptoethanol, and CEL wasdetected by immunoblotting. A representative example of three separateexperiments is shown. The arrow indicates the location of CEL MODY. Errorbars represent S.D. *** indicates p � 0.001.

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Expression of CEL MODY in AR42J Cells Increases ER Stressand Triggers the UPR—To determine whether CEL MODYexpression also triggered ER stress and UPR activation in AR42Jcells, we first analyzed the ultrastructure of the ER in cellsexpressing a green fluorescent protein (GFP) control, CEL14R,or CEL MODY by transmission electron microscopy. A welldocumented marker of ER stress is pronounced dilation of theER (20). In cells expressing GFP or CEL14R, the ER was notdilated (Fig. 6A). In contrast, cells expressing CEL MODYshowed marked dilation of the ER cisterna with granular mate-rial. The findings suggest the increased insoluble aggregates ofCEL MODY reside in the ER and consequently lead to ER stress.Second, we measured the levels of several major chaperones incells expressing CEL14R or CEL MODY (Fig. 6, B and C). Totalintracellular BiP protein and mRNA were both significantlyincreased in cells expressing CEL MODY compared withCEL14R (p � 0.01 for protein and p � 0.001 for mRNA). Fur-thermore, levels of GRP94 and calreticulin were also increased

in cells expressing CEL MODY in comparison with CEL14R(p � 0.01). As in HEK293T cells, BiP was predominantly pres-ent in the insoluble fraction from AR42J cells expressing CELMODY (Fig. 6B). Third, we investigated whether the increasedER stress in cells expressing CEL MODY up-regulated the UPRby measuring XBP1 mRNA splicing. Although the overexpres-sion of CEL14R increased XBP1 mRNA splicing compared withcontrol, XBP1 mRNA splicing was increased significantlyfurther in cells expressing CEL MODY compared with cellsexpressing CEL14R (p � 0.01) (Fig. 6D). Altogether, these find-ings clearly indicate that the expression of CEL MODY resultsin ER stress and triggers the UPR.

CEL MODY Also Leads to the Death of AR42J PancreaticAcinar Cells—As observed in HEK293T cells, the expression ofCEL MODY caused increased cell death in AR42J cells (Fig. 7A).Cell death was minimal in cells transduced with GFP, whereasabout 5% of cells expressing CEL14R died over 72 h. About 30%of the cells expressing CEL MODY underwent cell death, which

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FIGURE 3. Markers of ER stress in HEK293T cells transfected with CEL14R or CEL MODY expression plasmids. A, total cell extracts from cells transfectedwith control plasmid (mock), CEL14 R, or CEL MODY were separated by SDS-PAGE, and immunoblotting was performed with antiserum against BiP or GRP94.The amount of protein was determined by densitometry. The amount of BiP in the cells expressing CEL14R or CEL MODY was significantly greater than in cellstransfected with a plasmid control (p � 0.001) (n � 8). B, total cell extracts of cells transfected with a control plasmid or the CEL14R or CEL MODY expressionplasmid were separated into the soluble and insoluble fractions, and BiP was detected by immunoblotting. Sections of the same gel are presented after theremoval of a lane containing a sample from cells expressing CEL with the VNTR deleted. C, total RNA was isolated from cells transfected with the control plasmidor the CEL14R or CEL MODY expression plasmid. The amount of mRNA encoding the spliced and unspliced forms of XBP1 mRNA was determined by RT-PCR.GAPDH was examined as a loading control. The samples were separated on 2% agarose gels and stained with ethidium bromide. D, the amounts of XBP1U andXBP1S were determined by densitometry, and the results were plotted as the ratio of XBP1S/XBP1U. The ratio is significantly higher in the CEL MODY-expressing cells compared with the CEL14R-expressing cells (p � 0.01) (n � 6). Error bars represent S.D.

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was significantly higher compared with CEL14R (p � 0.001).Next, we evaluated several biomarkers associated with the acti-vation of apoptosis to determine whether CEL MODY-induceddeath occurred through apoptosis. First, we evaluated themRNA levels of ATF4 and CHOP in transduced cells. ATF4, abasic leucine zipper domain transcription factor, regulates UPRtarget genes including CHOP that mediate ER stress-mediatedapoptosis. The levels were similar between the cells transducedwith the control or CEL14R adenovirus (Fig. 7, B and C). Thelevels of ATF4 and CHOP mRNA were significantly increasedin the cells expressing CEL MODY (p � 0.007 and p � 0.01,respectively). Second, we evaluated PARP cleavage. Cellsexpressing CEL MODY had about 2-fold more cleaved PARPcompared with cells expressing CEL14R (p � 0.001) and about70% of the amount in the cells treated with staurosporine (Fig.

7D). Cleaved PARP was not detected in control cells. Collec-tively, these results provide strong evidence that AR42J cellsexpressing CEL MODY undergo apoptosis.

Induction of NF-�B in AR42J Cells Expressing CEL MODY—One of the pathological consequences of ER stress is the activa-tion of NF-�B (23). Additionally, NF-�B is activated in experi-mental models of both acute and chronic pancreatitis (26, 27).NF-�B regulates genes that control the inflammatory responseand cell survival. To determine whether expression of CELMODY induced NF-�B expression, we co-transduced cells withan adenovirus expressing luciferase under the control of theNF-�B promoter and with adenovirus expressing GFP, CEL14R,or CEL MODY (Fig. 7E). Only the cells expressing CEL MODYshowed an increase in luciferase activity, indicating that NF-�Bexpression was turned on in the CEL MODY cells.

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FIGURE 4. Activation of apoptosis and cell death in HEK293T cells transfected with the CEL14R or CEL MODY expression plasmid. A, cells weretransfected with the control plasmid or the CEL14R or CEL MODY expression plasmid. Aliquots of the medium were removed at the indicated times, and thecumulative LDH concentration was determined. At each time point, the amount of LDH in the CEL MODY medium was significantly higher than the amount inthe CEL14R medium (p � 0.001) (n � 8). There was no difference in the LDH content in the medium from control and CEL14R cells. B, cells were plated at theindicated densities and transfected with the control, CEL14R, or CEL MODY vectors 12 h after plating. Aliquots of each medium were removed at 72 h andassayed for LDH activity. The results are the average of three determinations at each cell density. C, total cellular extracts were separated by SDS-PAGE, andPARP was detected by immunoblotting. A representative blot is shown. Sections of the same gel are presented after the removal of a lane containing a samplefrom cells expressing CEL with the VNTR deleted. D, the amount of cleaved PARP relative to the empty vector sample is presented as a bar graph. The amountof cleaved PARP in the cells transfected with CEL MODY is significantly greater than the amount in the CEL14R-transfected cells. (p � 0.001) (n � 6). Error barsrepresent S.D.

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Discussion

In this study, we investigated the trafficking and function ofCEL MODY in HEK293T cells, a human epithelial cell line, andin AR42J cells, a rat pancreatic acinar cell line. The behavior andconsequences of the disease-causing mutant CEL protein weresimilar in both cell lines. The findings provide additional insightinto the molecular details of the pathophysiology underlyingchronic pancreatitis present in patients carrying this mutation.The biochemical characterization of purified recombinant CELvariant proteins suggested that their enzymatic activity againsttriglycerides was similar. However, CEL MODY was poorlysecreted from the transfected cell lines and largely retainedinside the cells, and the majority of the intracellular mutantprotein was present in detergent-insoluble aggregates, which isin line with previous reports (19). In addition, the retention of

mutant CEL inside cells caused ER stress and activated theUPR, which resulted in NF-�B activation and apoptosis in bothcell lines. Therefore, our results support the hypothesis thatCEL MODY causes chronic pancreatitis through proteotoxicgain of function.

In both transfected cell lines, CEL MODY formed detergent-insoluble aggregates. The mechanism underlying the aggrega-tion of CEL MODY was partially investigated in this study.Unlike CEL14R, CEL MODY was primarily detected at theexpected size on SDS-PAGE only when a reducing agent wasincluded, indicating that disulfide bonds likely contribute to theformation of aggregates. Of note, the frameshift caused by thesingle base deletion introduces 10 additional cysteine residuesin the VNTR region of CEL MODY as opposed to no cysteineresidues in the CEL14R VNTR (Fig. 1A). Nevertheless, it

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FIGURE 5. Expression of CEL14R and CEL MODY in AR42J cells transduced with adenovirus constructs. A, total RNA was isolated from cells transduced witha control or a CEL14R- or CEL MODY-containing adenovirus. The amount of mRNA encoding human CEL and GAPDH was determined by qRT-PCR as describedunder “Experimental Procedures.” There is no significant difference in the amount of mRNA encoding CEL14R and CEL MODY (n � 6). B, the cells weretransfected with the indicated adenovirus as described under “Experimental Procedures.” Samples were obtained from the medium and from the intact cells.In separate experiments, the intracellular CEL was separated into soluble and insoluble fractions as described under “Experimental Procedures.” The proteinsin each sample were separated by SDS-PAGE, and immunoblotting was performed with CEL antiserum. C, the amount of immunoreactive protein wasquantified by densitometry and presented in bar graphs. The medium and soluble fractions contained significantly more CEL14R (p � 0.001). The totalintracellular sample and the insoluble fraction contained significantly more CEL MODY (p � 0.001) (n � 8). D, cells were transduced with the indicatedadenoviral constructs, and the insoluble fraction was isolated. The samples were separated by SDS-PAGE in the presence or absence of �-mercaptoethanol,and CEL was detected by immunoblotting. Representative gels from three separate experiments are shown. Error bars represent S.D.

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remains possible that other structural or biophysical features ofthe acquired VNTR sequence in CEL MODY contribute to pro-tein misfolding and aggregation.

Overexpression of either CEL14R or CEL MODY increasedER stress likely in response to the increased requirements forprotein synthesis. Multiple criteria suggested important differ-ences in the response of cells transfected with CEL14R com-pared with cells transfected with CEL MODY with the latterinducing cell death. First, the ER in AR42J cells expressing CELMODY was markedly dilated, a hallmark of ER stress. Second,the expression of CEL MODY in AR42J cells up-regulated theexpression of several major ER chaperones beyond the levels incells expressing CEL14R. Third, the intracellular distribution of

BiP was quite different between cells expressing CEL14R andCEL MODY. The majority of BiP was in the insoluble fractionin CEL MODY cells, whereas virtually all of the BiP was in thesoluble fraction in CEL14R cells. Our studies do not distinguishbetween BiP binding to the aggregates to effect disaggregationor BiP sequestration in the aggregates. Presumably, the bindingof BiP to insoluble CEL MODY depletes the pool of soluble BiP,which provides a feasible explanation for the differential distri-bution of BiP in cells expressing CEL MODY compared withcells expressing CEL14R. Fourth, cells expressing CEL MODYshowed evidence of a maladaptive UPR as evidenced byincreased levels of spliced XBP1 mRNA and activation ofinflammatory and apoptosis.

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FIGURE 6. ER stress response in AR42J cells transduced with control, CEL14R, or CEL MODY adenovirus. A, transmission electron micrographs of cellstransduced with control, CEL14R, or CEL MODY adenovirus. Two separate experiments with CEL MODY are shown. The arrows mark ER. B, the ER stress markersBiP, calreticulin, and GRP94 were detected by immunoblotting from lysates from cells transduced with control, CEL14R, or CEL MODY adenovirus. Wedetermined the amount of BiP in the total cell extract as well as in the soluble and insoluble intracellular fractions. GAPDH was used as the loading control. C,left panel, the relative BiP protein abundance in the total cellular extract and the soluble fraction is presented as a ratio to the BiP content in the control cells.The insoluble fraction was not quantitated because no detectable BiP was present in the CEL14R samples. BiP was significantly higher in the total cellularextract of cells transduced with CEL MODY compared with cells transduced with CEL14R (p � 0.01) (n � 6). The insoluble fraction is not plotted because BiP wasnever detected in the cells expressing CEL14R. Middle panel, the amount of BiP mRNA was determined by qRT-PCR and plotted relative to the control sample.There was significantly more BiP mRNA in cells expressing CEL MODY than in cells expressing CEL14 R (p � 0.001) (n � 4). Right panel, the relative abundanceof GRP94 and calreticulin determined by immunoblotting. There was significantly more GRP94 and calreticulin in the cells expressing CEL MODY than in cellsexpressing CEL14R (p � 0.01) (n � 6). D, total RNA was isolated from cells transduced with the control adenovirus or the CEL14R or CEL MODY adenovirus. Theamount of mRNA encoding the spliced and unspliced forms of XBP1 mRNA was determined by RT-PCR. GAPDH was used as a loading control. The samples wereseparated on 2% agarose gels and stained with ethidium bromide. In AR42J cells, the hybrid form of XBP1 mRNA (XBP1H) was detected (44). The amounts ofXBP1U and XBP1S were determined by densitometry, and the results plotted as a ratio of XBP1S/XBP1U. The ratio is significantly higher in the CEL MODY cellscompared with the CEL14R cells (p � 0.01) (n � 6). Error bars represent S.D. NS, not significant. **, p � 0.01; ***, p � 0.001.

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Expression of CEL MODY alone was sufficient stress to acti-vate NF-�B and apoptosis. In fact, treatment of the transfectedcells with various metabolic stressors did not increase cell deathover that induced by CEL MODY. The observation that a defectin protein homeostasis alone can trigger cell death opens new

windows in understanding the pathogenesis of pancreatitis.The concept that perturbations of protein homeostasis causedisease in the exocrine pancreas fits well with the function ofthe acinar cell. Pancreatic acinar cells are specialized for proteinsynthesis, storage, and secretion. They synthesize more protein

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FIGURE 7. Cell death and apoptosis in AR42J cells transduced with control, CEL14R, or CEL MODY adenovirus. A, cell death was determined as describedunder “Experimental Procedures.” The level of cell death was significantly higher in cells expressing CEL MODY than in cells expressing CEL14R (p � 0.001) (n �3). B, the amount of mRNA encoding ATF4 in each cell line was determined by qRT-PCR 48 h after transduction. The amount of ATF4 was significantly higher incells expressing CEL MODY than in cells expressing CEL14R (p � 0.007) (n � 4). C, the amount of mRNA encoding CHOP in each cell line was determined byqRT-PCR 48 h after transduction. The amount of CHOP was significantly higher in cells expressing CEL MODY than in cells expressing CEL14R (p � 0.01) (n � 3).D, total cellular extracts from cells transduced with control, CEL14R, or CEL MODY adenovirus or treated with staurosporine were separated by SDS-PAGE, andcleaved PARP was detected by immunoblotting. A representative blot is shown. The amount of cleaved PARP relative to the mock sample is presented as a bargraph. The amount of cleaved PARP in the cells transfected with CEL MODY is significantly greater than the amount in the CEL14R-transfected cells (p � 0.001)(n � 5). E, activation of the NF-�B promoter was determined in a luciferase assay as described under “Experimental Procedures.” There was significantly moreactivation of the NF-�B promoter in cells transduced with adenovirus expressing CEL MODY than in cells transduced with CEL14R (p � 0.001) (n � 3). There wasno difference among the mock, GFP, and CEL14R expressed proteins. Error bars represent S.D. **, p � 0.01; ***, p � 0.001.

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than other cells in the body. In particular, acinar cells synthesizelarge amounts of a small number of proteins, the 20 or so diges-tive enzymes (28). Because protein synthesis is inherentlyerror-prone, there is a constant flux of destabilized proteins,and any increase in destabilized proteins is potentially devastat-ing for acinar cells. However, pancreatic acinar cells haveadapted to handle a large flux of newly synthesized proteins inthe ER. In fact, five transcription factors known to regulategenes encoding key players in protein homeostasis are amongthe 22 most highly expressed transcription factors in acinarcells (29).

Because they make large quantities of select digestiveenzymes, pancreatic acinar cells are susceptible to injury fromsmall perturbations in the homeostasis of these proteins. Anychange that leads to the excessive accumulation of misfoldedproteins can trigger maladaptive cell signaling pathways (30).Recent studies of transfected cells expressing proteases or pan-creatic lipases containing polymorphisms associated withhigher risk for chronic pancreatitis (p.R116C in PRSS1, p.A73Tin chymotrypsin C, p.N256K in CPA1, and p.T221M in pancre-atic lipase) have demonstrated that the polymorphismsincreased protein misfolding and decreased secretion, leadingto ER stress and UPR activation (31–35). It was suggested thatthe ER stress response played a role in the pathogenesis ofchronic pancreatitis in patients with these polymorphisms. Ofthese examples, only the p.A73T chymotrypsin C mutant wasshown to activate apoptosis (36). It remains unclear whetherthe other mutations elicit a similar cascade of events as occursin cells expressing CEL MODY as shown in this study.

Our findings differ in important ways from previous studiesof CEL MODY (19, 20). Most importantly, previous studiesshowed that expression of CEL MODY in stably transfectedHEK293 cells activated an ER stress response without activat-ing cell death pathways. The activation of NF-�B was not exam-ined in these studies. The most likely explanation for the dis-crepancy between previous and our current work is therelatively lower expression levels of CEL variant proteins in pastwork. It is possible that the lower expression levels were a resultof the selection process, which may have resulted in enrichmentfor cells with lower expression levels because cells with higherexpression levels would trigger apoptosis and would not survivethe selection process. A similar explanation could also accountfor the results obtained in CEL MODY transgenic mice inwhich the level of mRNA encoding CEL MODY was less thanone-tenth of the endogenous mRNA that encodes mouse CEL(37). As a result, the CEL MODY transgenic mice failed tomimic the pancreatic pathophysiology seen in the patients.These results suggest that the physiological expression levels ofmutant CEL MODY protein are critical to study the cellularresponses induced by this mutant protein.

In another study, the same investigators found evidence thatCEL MODY is toxic to cells. They treated various cell linesincluding pancreatic acinar and beta cell model cell lines withconditioned medium from HEK293 cells expressing eitherCEL16R or CEL MODY. Interestingly, CEL MODY was takenup by all of the cell lines to a much greater extent than CEL16R.Prolonged exposure of the pancreatic exocrine and endocrinecell lines to conditioned medium containing CEL MODY

caused increased cell death via apoptosis relative to mediumcontaining CEL16R (20). The authors proposed that CELMODY endocytosis was an essential feature of the events lead-ing to cell death and that endocytosis plays a role in the devel-opment of pancreatic insufficiency and diabetes in patientscarrying the CEL variant. Our results suggest that ER stresstriggered by aggregated CEL MODY is more important ininducing cell death pathways in pancreatic acinar cells.

Consistent with our data, ER stress is an early pathogenicfeature present in a variety of models of experimentally inducedacute pancreatitis (27, 30, 38 – 40). For instance, the reductionof ER stress by tauroursodeoxycholic acid, a chemical chaper-one, lessened acinar cell damage and systemic inflammation inrat models of acute pancreatitis, suggesting ER stress as apotential contributing factor for pancreatitis (41). Ethanol-in-duced pancreatitis in mice triggers an ER stress response that isthought to be a protective mechanism that improves cell sur-vival (5).

In sum, our results provide direct evidence that a mutation inCEL is sufficient to cause ER stress, induction of the UPR,NF-�B activation, and acinar cell death. In our model, the accu-mulation of mutant protein CEL MODY overwhelms the firstline adaptive and protective mechanism offered by the ER stressresponse. Apoptosis may serve as a protective response todecrease the severity of cell injury by eliminating damaged cellswithout eliciting significant inflammation. As the purifiedCEL14R and CEL MODY proteins have similar functionalproperties, future studies should address the clearance of thetoxic mutant protein inside cells to alleviate its proteotoxicity.This knowledge has the potential to provide the basis for noveltherapeutic approaches to treat chronic pancreatitis.

Experimental Procedures

Plasmid DNA Constructions—All DNA manipulations werecarried out by standard methods unless stated otherwise. Thefull-length human CEL cDNA clone was purchased fromOpenBiosystems (clone ID 5187959; GenBankTM accessionnumber BC042510.1). The cDNA encodes for a 14-repeatVNTR, the variant present in the CEL MODY family. TheEcoRI/XhoI fragment containing the entire CEL cDNA codingregion was subcloned into yeast (pPICZA) and mammalian(pcDNA3) protein expression vectors. The desired mutationswere introduced by using customized primers and aQuikChange II XL site-directed mutagenesis kit (Stratagene). Ifstated, a FLAG tag was inserted in between human CEL secre-tory signal peptide and its mature polypeptide by overlap PCRto facilitate protein detection, and the insertion had no detect-able effect on the properties of the CEL variant proteins (datanot shown). Dideoxynucleotide sequencing validated all plas-mid DNA constructs.

Expression, Purification, and Characterization of Recombi-nant Proteins—Recombinant human CEL protein variants wereproduced as secretory proteins in P. pastoris GS115 and puri-fied as described in detail previously (34). Six micrograms ofeach purified protein was resolved by 4 –15% SDS-PAGE andstained with GelCode Blue stain reagent (Pierce) to evaluatetheir homogeneity and integrity. The lipase activity was carriedout with 10 �g of purified protein and trioctanoin or triolein as

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substrate in assay buffer containing 12 mM sodium cholateusing the pH-stat method (34). The lipolytic activities areexpressed in international lipase units/mg of enzyme. One unitcorresponds to 1 �mol of fatty acid released/min.

Recombinant Adenovirus—The cDNAs for human CEL14R,which is a normal variant of CEL, and CEL MODY were sub-cloned into the pVQAd CMV K-NpA shuttle vector at KpnI/XbaI sites, and the recombinant adenoviruses were then cus-tom-made by Viraquest. Adenovirus expressing enhanced GFPwas purchased from Viraquest.

Cell Culture of HEK293T and AR42J Cells—Both cell lineswere purchased from ATCC and maintained at 37 °C in a 5%CO2-humidified incubator following the manufacturer’s in-structions with HEK293T cells in DMEM � 10% FBS andAR42J cells in F-12K � 20% FBS. The transfection of HEK293Tcells was carried out using FuGENE 6 (Roche Diagnostics) fol-lowing the protocol described previously (34). Unless otherwisestated, culture medium and cells were harvested following rou-tine procedures 48 or 72 h after transfection. Prior to transduc-tion, AR42J cells were seeded in 10-cm dishes at a density of 107

cells/dish in 10 ml of medium containing 100 nM dexametha-sone (Sigma) and maintained for 24 h. The cells were trans-duced with 1 � 109 particles of recombinant adenovirus (mul-tiplicity of infection, 100) to express GFP, CEL14R, and CELMODY proteins. Samples were harvested for analysis 72 h aftertransduction unless stated otherwise.

Collection of Culture Media and Cells—Samples were har-vested as described previously (33). Briefly, 72 h after transfec-tion or transduction, conditioned medium were withdrawn andsubjected to 200 � g centrifugation for 5 min to obtain cell-freeconditioned medium. Collected cells were washed twice withice-cold PBS followed by 200 � g � 5-min centrifugation.Whole cell lysates were extracted using 1� Laemmli buffer fol-lowed by 3 � 15-s sonication. As for soluble and insoluble frac-tions, the soluble fraction was defined as the supernatant ofradioimmune precipitation assay buffer extracts of cells after16,000 � g centrifugation for 20 min. The final pellets wereresuspended in 1� Laemmli buffer and sonicated until no par-ticle was visible. This part was designated as the insoluble lysatefraction. The protein concentration of cell lysate samples wasdetermined by BCA Protein Assay (Thermo Scientific).

Western Blotting Analysis—Aliquots of conditioned medium(20 �l) or cell lysates (10 –50 �g for soluble fractions; 0.1–5 �gfor insoluble fractions) were prepared as described previouslywith the exception of the non-reducing SDS-PAGE in which2-mercaptoethanol was omitted (34). The prepared sampleswere resolved on 4 –15% Mini-PROTEAN TGX pre-cast gel(Bio-Rad) and transferred onto Immobilon PVDF membranes(Millipore). The blots were incubated sequentially in 5% nonfatmilk in TBS-Tween 20, with a primary antibody for 1 h at roomtemperature or overnight at 4 °C, and then with a correspond-ing secondary antibody conjugated with HRP at room temper-ature for 1 h. The HRP signal was detected using the Super-Signal West Femto chemiluminescent substrate (Pierce), andthe band density was quantified by densitometry. For CEL anal-ysis, medium (40 �l), insoluble (1.5 �g), and soluble (5 �g)samples were loaded. For other proteins, insoluble (1.2 �g) andsoluble (20 �g) samples were loaded.

Antibodies used in this study included rabbit polyclonal anti-body against human CEL (1:20,000). To create the antigen, weintroduced a premature stop codon into the cDNA encodingCEL by site-directed mutagenesis. The mutation introduced apremature stop codon prior to the VNTR at p.V562X, therebypreventing transcription of the entire VNTR. The cDNA wascloned into pPICZA and expressed in yeast as above. The pro-tein was purified from the medium. Rabbit antiserum againstthe protein was produced by Cocalico Biologicals (Stevens, PA).Rabbit anti-BiP (catalog number 3183; 1:1000), GRP94 (catalognumber 2104; 1:1000), PARP (catalog number 9545, 1:4000),and GAPDH (catalog number 5174, 1:4000) were purchasedfrom Cell Signaling Technology.

Quantitative and Semiquantitative PCR—For transducedAR42J cells, total RNA was purified using an RNeasy Plus Minikit (Qiagen) and reverse transcribed into cDNA using a HighCapacity cDNA Reverse Transcription kit (Applied Biosys-tems). XBP1 mRNA splicing was analyzed by RT-PCR and visu-alized by ethidium bromide staining after gel electrophoresis on2% agarose gels. The mRNA expression of human CEL, BiP,CHOP, ATF4, and GAPDH (endogenous control) was evalu-ated by qRT-PCR using the TaqMan Gene Expression Assay(Applied Biosystems) on a 7300 Real Time PCR System(Applied Biosystems) and primer/probes from Life Technol-ogy. The measurement of XBP1 mRNA splicing in transfectedHEK293T cells expressing the CEL variant proteins or controlwas carried out as described (34).

Assays for Cell Death—To determine cell death in HEK293Tcells, we measured LDH release into the medium following themanufacturer’s instructions (Sigma-Aldrich, catalog numberTOX7). For determination of the kinetics of LDH release fromHEK293T cells, we plated the cells in a 24-well culture dish.Cells were transfected with control plasmid, CEL14R, and CELMODY as described above. Another set of wells was not trans-fected. Medium (100 �l) was removed from the transfectedcells at the indicated times for the LDH assay. The medium wasremoved from a well of the untreated cells, and the cells werelysed with 1 ml of 1% Triton X-100 in PBS. A separate well washarvested for each time point. The insoluble material was pel-leted in a microcentrifuge. Total LDH activity in the cells wasdetermined by assaying 100 �l. The amount of LDH in themedium of the transfected cells divided by the total LDH wasplotted. To determine the effect of cell density on cell toxicity,we plated HEK293T cells in 6-well plates at the indicated den-sities. Transfections were done as above. A 100-�l aliquot ofmedium was assayed for LDH.

We determined cell death in AR42J cells by measuring thenumber of living cells by the dehydrogenase activity against awater-soluble tetrazolium with Cell Counting Kit-8 (DojindoMolecular Technologies) (36). Seventy hours after viral trans-duction in a 24-well plate, 40 �l of the tetrazolium substrate wasadded to each well. After the incubation at 37 °C for 2 h, theabsorbance at 450 nm was measured using a Synergy H1 Hybridplate reader (BioTek). Absorbance values were normalized tocells transduced to express GFP.

Measurement of NF-�B Activity—NF-�B-luciferase activitywas assayed using a luciferase assay kit (E397A, Promega). Fifty-six hours after viral transduction to express GFP, CEL14R, or

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CEL MODY, AR42J cells were then transduced with Ad-NF-�B-luciferase (42). Sixteen hours later, cells were harvested andlysed using 5� reporter lysis buffer. The treatment of cells with500 �M taurolithocholic acid 3-sulfate for 2 h prior to sampleharvest served as a positive control. Samples were then rigor-ously vortexed and subjected to a 2-min 12,000 � g centrifuga-tion. The supernatants were plated in a 96-well plate, and theluciferase activities were measured using a Synergy H1 platereader.

Transmission Electron Microscopy (TEM)—Cell monolayersof AR42J cells were prepared for TEM as described previously(43). Briefly, the cells were washed with PBS and fixed with 2.5%glutaraldehyde in PBS for 1 h at room temperature. After wash-ing with PBS three times, the samples were postfixed in 1%osmium tetroxide and 1% potassium ferricyanide for 1 h atroom temperature. After washing with PBS and dehydration ina graded series of ethanol, the samples were infiltrated withPoly/Bed 812. Beam capsules filled with resin were placed onthe monolayer and allowed to polymerize. The beam capsuleswere removed from the culture dishes and sectioned en face forTEM. Sections were stained with 1% uranyl acetate and 1% leadcitrate. Images were acquired with a JEM-1400 transmissionelectron microscope.

Author Contributions—X. X. helped conceive and coordinate thestudy. He performed or directed construction of all plasmids, pre-pared recombinant proteins, helped with all cell studies, and directedothers involved in the work. He helped write the first draft of themanuscript. G. J. performed studies on AR42J cells including cellculture, transfection, immunoblotting, and PCR analysis. W. A. S.performed studies on HEK293T cells including cell culture, immu-noblotting, and cell death assays. D. B. S. was responsible for theelectron microscopy. K. E. M. and M. H. helped with studies inHEK293T cells including transfection and immunoblots. A. M.helped conceive studies evaluating cell death pathways and helpeddirect those studies. Y. W. also helped conceive studies evaluatingcell death pathways, helped direct those studies, and contributed tothe preparation of the figures. M. E. L. helped conceive the projectand directed all aspects of the project. He analyzed data, preparedfigures, and along with X. X. wrote the initial draft of the manuscript.All authors reviewed the results and approved the final version of themanuscript.

Acknowledgment—We thank Dr. Jeffrey Brodsky for comments on themanuscript.

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Mutation in CEL VNTR Causes Apoptotic Cell Death

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Magee, Margaret Haughney, Amitava Mukherjee, Yan Wang and Mark E. LoweXunjun Xiao, Gabrielle Jones, Wednesday A. Sevilla, Donna B. Stolz, Kelsey E.

Intracellular Aggregates That Activate ApoptosisA Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming

doi: 10.1074/jbc.M116.734384 originally published online September 20, 20162016, 291:23224-23236.J. Biol. Chem. 

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VOLUME 291 (2016) PAGES 23224 –23236DOI 10.1074/jbc.A116.734384

A carboxyl ester lipase (CEL) mutant causes chronicpancreatitis by forming intracellular aggregates thatactivate apoptosis.Xunjun Xiao, Gabrielle Jones, Wednesday A. Sevilla, Donna B. Stolz,Kelsey E. Magee, Margaret Haughney, Amitava Mukherjee, Yan Wang,and Mark E. Lowe

We recently learned that the cDNA encoding CEL purchasedfrom OpenBiosystems (clone ID 5187959; GenBankTM accessionno. BC042510.1) for this work contains a three-base pair in-frame deletion resulting in p.E365del. (The correct sequenceshould be 356NKGNKKVTEEDFYKLVSEFTITKGL380 and not356NKGNKKVTE-DFYKLVSEFTITKGL380.) p.E365 is conserved inmost primates. The residue is located in a surface loop of the CELglobular domain distant from the catalytic site and the bile acid-binding site. In our study, both CEL14R and CEL MODY containedp.E365del. The experimental variable was the sequence of the vari-able number of tandem repeat (VNTR) domain (Fig. 1A). Thus, thereported differences between CEL14R and CEL MODY reflect theeffect of the VNTR MODY mutation on CEL folding. In support ofthis contention, another group that expressed a form of CEL withp.E365 also found that CEL MODY forms significant levels of intra-cellular protein aggregates compared with CEL14R (Johansson, B. B.,Torsvik, J., Bjørkhaug, L., Vesterhus, M., Ragvin, A., Tjora, E., Fjeld, K.,Hoem, D., Johansson, S., Ræder, H., Lindquist, S., Hernell, O., Cnop, M.,Saraste, J., Flatmark, T., Molven, A., and Njølstad, P. R. (2011) Diabetesand pancreatic exocrine dysfunction due to mutations in the carboxyl esterlipase gene-maturity onset diabetes of the young (CEL-MODY): A proteinmisfolding disease. J. Biol. Chem. 286, 34593–34605).

Because our hypothesis was that CEL MODY causes disease by mis-folding and activating maladaptive cell signaling pathways, the presenceof p.E365del raises concerns that deletion may have affected proteinfolding efficiency and contributed to the unfolded protein response weobserved in our study. Based on our results with CEL14R, the effect ofp.E365del on the folding of CEL appears minimal. CEL14R was robustlysecreted and only formed low levels of detergent-insoluble intracellularaggregates (Figs. 2 and 5). CEL14R had good activity against triglycer-ides (Fig. 1) and was stimulated by bile acids (Xiao, X., and Lowe, M. E.,unpublished observations). The results with CEL14R also showed thatthe presence of p.E365del was not sufficient to trigger activation of celldeath and inflammatory pathways to the same level as seen with CELMODY (Figs. 4 and 7). Still, we cannot exclude the possibility that thepresence of p.E365del amplified the unfolded protein response elicitedby the aggregation of CEL MODY resulting in a more robust activationof cell death pathways. Given this new information, the conclusion that“a mutation in CEL is sufficient to cause ER stress, induction of the UPR,NF-�B activation, and acinar cell death” in the first sentence of the lastparagraph under “Discussion” is overstated. Even with this caveat, ourdata support the conclusion that disorders of protein homeostasis canhave a role in the pathophysiology of chronic pancreatitis.

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7744 J. Biol. Chem. (2017) 292(19) 7744 –7744

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.