THE BIK PICTURE: REDUCED DAPK EXPRESSION IN BMF-DEFICIENT AIRWAY EPITHELIAL CELLS PREVENTS BIK-MEDIATED CALCIUM RELEASE AND CELL DEATH

Neal E. Lacey

Date of Award

2016

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Clore, Amy

Keywords

Biks, Calcium Kinetics, Cell Death, DAPK

Area of Concentration

Molecular and Cellular Biology

Abstract

Rationale: Chronic bronchitis is commonly linked to chronic obstructive pulmonary disease and both diseases are caused by dysregulation of tissue homeostasis. Apoptosis and autophagy are highly regulated processes that play a large role in this homeostasis. In human airway epithelial cells of the lung, Bik expression induces cell death, but Bmf deficient cells overexpressing this protein die to a lesser extent than wild type. Bmf is an autophagy inhibitor and Bik is pro-apoptotic, though both are BH3-only Bcl-2 family proteins. Endoplasmic reticulum-anchored Bik must interact with DAPK, ERK1/2 and Bak as a part of the BDEB complex to tether the mitochondria to the ER, initiate calcium release and cause efficient cell death. Suppression of DAPK levels due to increased autophagy flux, or the rate at which cellular contents are degraded, in Bmf-deficient cells may their reduced cell death when overexpressing Bik. This hypothesis was tested in the present study. Methods: The ER is the primary site for calcium storage as high cytosolic concentrations of calcium can trigger apoptosis. To learn more about the ER Bik-induced cell death pathway we investigated ER-calcium kinetics during Bik-induced cell death in human airway epithelial cells. Calcium kinetics were determined using fluorimetry or flow cytometry in the presence of a calcium-sensitive dye. To determine if DAPK is regulated by autophagy, wild type and Bmf-deficient cells were exposed to known inducers of autophagy (such as wood smoke) and DAPK protein expression levels were analyzed using western blot analysis. Results: Our studies indicate that Bik causes significant increases in intracellular calcium concentration and that autophagic conditions decrease DAPK protein expression. Tracheal epithelial cells in Bmf-deficient mice express reduced DAPK levels compared to wild type cells. Conclusions: The present study suggests that Bik mediates calcium release from the ER for mitochondrial uptake to cause cell death. Bmf-deficient cells may be less susceptible to Bik-induced cell death because increased autophagy flux decreases DAPK expression. This study may have implications in the context of regulation of airway epithelial cell numbers in response to injury to the lung.

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