The Secretory Pathway Calcium ATPase 2C promotes increased cytosolic calcium levels

Melissa Fenech, The University of Western Ontario

Abstract

Pancreatic acinar cell exocytosis requires precise calcium (Ca²⁺) signals. When cytosolic Ca²⁺ levels remain high, cellular functions are disrupted, which is associated with initiation of pancreatitis. Ca²⁺ signals are achieved through regulating endoplasmic reticulum (ER) stores, Ca²⁺ATPases, and store-operated Ca²⁺ entry (SOCE). However, how these pathways interact to create precise signals is not well understood. In a pancreatic model of dysregulated Ca²⁺ homeostasis, secretory pathway Ca²⁺ATPase 2 (SPCA2) expression is significantly decreased. In the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. Recent studies indicate a role for SPCA2 in increasing Ca²⁺ influx involving Orai1 in breast cancer. The goal of this study was to determine the effects of SPCA2C on Ca²⁺ homeostasis.

Here, epitope-tagged SPCA2C was expressed in HEK293A cells. Co-immunofluorescence (Co-IF) determined subcellular localization. Using Fura2 imaging, cytosolic Ca²⁺ concentration [Ca²⁺] was examined during rest, SOCE and secretagogue-stimulated signaling. Effects of SPCA2C over-expression were also examined in 266.6 pancreatic acinar cells. In pancreatic cancer-derived cells, the abundance of the SPCA2C gene transcript (ATP2C2C) was assessed. Effects of SPCA2C over-expression was examined in cancer-derived cells to understand the functional role of SPCA2C in pancreatic pathology. Finally, an unbiased protein proximity assay was used to evaluate the SPCA2C interactome and broaden the understanding of SPCA2C function.

In HEK293A cells, exogenous SPCA2C expression localized to ER and Golgi compartments and increased resting cytosolic [Ca²⁺], Ca²⁺ release in response to carbachol, ER Ca²⁺ stores, and Ca²⁺ influx. Co-immunoprecipitation (co-IP) detected Orai1-SPCA2C interactions. However, SPCA2C's effect on cytosolic and ER Ca²⁺ levels are Orai1-independent. In pancreatic cancer, cell lines with increased endogenous ATP2C2 expression had increased ER Ca²⁺ stores and increased constitutive Ca²⁺ influx, while exogenous over-expression of SPCA2C increased resting Ca²⁺ levels. Examination of identified putative protein interactions using Gene Ontology (GO) and Reactome analysis predicted endoplasmic reticulum cellular compartment localization and Ca²⁺ ion homeostasis as a pathway SPCA2C may be involved in. These findings indicate SPCA2C influences Ca²⁺ homeostasis through multiple mechanisms. By delineating the Atp2c2c sequence and functionally characterizing SPCA2C's role in Ca²⁺ regulation, I have uncovered a novel mechanism that pancreatic acinar cells use to regulate cytosolic and ER Ca²⁺ levels.