Abstract
Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.
Highlights
Pancreatitis is an inflammatory disease with significant health and economic burdens [1, 2]
Expression and Activity of carboxyl ester lipase (CEL) variable number of proline-rich tandem repeats (VNTR) Variants—To characterize the CEL MODY mutation associated with pancreatic disease, we transfected Pichia pastoris GS115 yeast with vectors containing wild-type CEL with 14 repeats in the VNTR (CEL14R) or with a frameshift mutation in the VNTR, CEL MODY
We investigated the trafficking and function of CEL MODY in HEK293T cells, a human epithelial cell line, and in AR42J cells, a rat pancreatic acinar cell line
Summary
Expression and Activity of CEL VNTR Variants—To characterize the CEL MODY mutation associated with pancreatic disease, we transfected Pichia pastoris GS115 yeast with vectors containing wild-type CEL14R or with a frameshift mutation in the VNTR, CEL MODY. Because the frameshift introduces 10 additional cysteine residues in the VNTR, we investigated whether the formation of inappropriate disulfide bonds contributed to CEL MODY aggregation To test this hypothesis, we separated the detergent-soluble and -insoluble fractions of CEL MODY and CEL14R by SDS-PAGE under non-reducing and reducing conditions and looked at the pattern of bands by protein immunoblotting (Fig. 2C). The activity of LDH in the medium from cells transfected with CEL MODY was significantly higher than that from cells transfected with CEL14R or the mock control (p Ͻ 0.001). The LDH activity was consistently higher in the medium from cells transfected with CEL MODY, independent of the initial seeding density (ranging from 0.2 to 1.0 ϫ 106 cell/ml) (Fig. 4B).
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