Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Collaborative Specialization

Developmental Biology

Supervisor

Pin, Christopher L.

2nd Supervisor

Stathopulos, Peter B.

Co-Supervisor

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.

Summary for Lay Audience

The pancreas is important for regulating blood glucose and producing and delivering substances necessary for digestion. A specific cell type, called the pancreatic acinar cell, has the primary function of producing and secreting enzymes that digest food particles you ingest. Enzymes are released from these cells when you eat, which requires a specific signal that can be transmitted inside of the cell. Ionized calcium (Ca2+) is required to transmit this signal inside of the cell. The pancreas can become inflamed, which is known as pancreatitis, if the Ca2+ signal is not transmitted properly. The mechanisms and molecules that regulate Ca2+ levels in pancreatic acinar cells are not well defined. Recently, a new Ca2+ regulating protein, termed secretory pathway Ca2+ ATPase 2 C (SPCA2C) was identified. Previous research in breast cancer cells suggested that the related SPCA2 protein increases Ca2+ levels inside cells, leading to the disease. Thus, the goal of this thesis was to determine the effects of SPCA2C on Ca2+ regulation in various mammalian and human cell types.

In this study, the protein sequence of SPCA2C was first determined. Subsequently, SPCA2C was expressed in cells to assess whether it is able to regulate Ca2+ levels. Changes in intracellular Ca2+ levels were also measured during a stimulation of enzyme release. In cells isolated from pancreatic cancer patients, the expression of ATP2C2, messenger nucleic acid transcript from which the SPCA2 protein is made, was analyzed. The level of expression was compared to cellular Ca2+ levels to ascertain how SPCA2C influences Ca2+ homeostasis in pancreatic disease.

I found that expressing SPCA2C in cells increased intracellular Ca2+ levels and the amount of Ca2+ signal after stimulation for exocytosis. In pancreatic cancer cells, increased expression of ATP2C2 increased the amount of Ca2+ that was being taken up by the cell. These findings indicate that SPCA2C influences Ca2+ homeostasis through multiple mechanisms. I also identified numerous candidate protein binding partners of SPCA2C that may be involved in regulating intracellular Ca2+ levels. Collectively, my work has uncovered a novel molecular mechanism by which pancreatic acinar cells regulate Ca2+ levels, laying the foundation for future hypothesis-based research.

Available for download on Friday, January 01, 2021

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