Publication Date

2012-12-12

Availability

Embargoed

Embargo Period

2014-12-12

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2012-11-06

First Committee Member

Lawrence H. Boise

Second Committee Member

Carlos T. Moraes

Third Committee Member

Gennaro D'Urso

Fourth Committee Member

Theodore J. Lampidis

Fifth Committee Member

Jonathan Kaufman

Abstract

The caspase family of proteins consists of upstream initiator caspases that activate downstream effector caspases, which are responsible for the proteolytic cleavage of many hundreds of proteins during apoptosis. Caspases 7 and 3 cleave a variety of proteins that lead to the hallmarks of apoptosis including, nuclear condensation, DNA fragmentation and membrane blebbing. Classically, caspase 7 and 3 were thought of as redundant because they share a 49.2 % amino acid sequence identity, a 62.0 % amino acid sequence similarity and have the same preferred cleavage motif (DEVD). However, recent data have demonstrated that caspase 7 and 3 are distinct proteases that have differential activity toward a variety of substrates. Therefore, we examined the distinct roles of these caspases during serum withdrawal induced cell death by using effector caspase knockout MEFs. Here, we find that during serum withdrawal induced cell death caspase 7 deficient MEFs display no ROS production and are not resistant to intrinsic apoptosis stimulation, but remain attached to the ECM. In contrast, caspase 3 deficient MEFs display increased ROS production and are less sensitive to intrinsic cell death stimulation. These data suggest that while caspase 3 is the dominant executioner caspase, caspase 7 plays a distinct role in the degradation phase of apoptosis. We also demonstrate that caspase 7 and 3 have distinct non apoptotic functions that regulate processes such as morphology, adhesion and motility. Caspase 7 deficient MEFs display an elongated and spread out morphology with increased actin stress fibers, while caspase 3 deficient MEFs display only an elongated morphology with no increase in the size of the cell footprint in contact with the ECM. Also, caspase 7 deficient MEFs and caspase 3 deficient MEFs display an increase in adhesion. Interestingly, previous studies have shown that caspase 3 deficient MEFs display a delay in the upstream events of apoptosis such as bax activation, cytochrome c release and MOMP. Therefore, we hypothesized that the delay in these events was controlled by increased adhesion and that in the absence of caspase 3, cells have increased adhesion signaling and a higher apoptotic threshold. Consistent with this, we find that when caspase 3 deficient MEFs are subjected to anoikis conditions, they have no resistance to cell death. Since there is crosstalk between the pathways that regulate morphology and adhesion, and migration, we examined the role of caspase 7 and 3 during in vitro wound healing. Caspase 7 deficient MEFs close wounds as efficiently as WT MEFs, however they display an increase in average velocity and a decrease in directional migration. In contrast, caspase 3 deficient MEFs do not close wounds as efficiently as WT MEFs and this can be attributed to a decrease in average velocity and a decrease in directional migration. Interestingly, we demonstrate that changes in morphology and motility in caspase deficient MEFs revert back to a WT phenotype by reconstituting the MEFs with the appropriate caspase or catalytically inactive caspase, suggesting that caspase 7 and 3 regulate these processes through a mechanism that is independent from their catalytic activity. To further investigate the role of caspase 3 in adhesion and motility, we examined adhesion signaling and production and secretion of the ECM component, fibronectin. In the absence of exogenous factors from serum, caspase 3 deficient MEFs adhere more quickly than WT MEFs and display increased P FAK expression and increased production and secretion of fibronectin. Taken together, these data suggest that caspase 7 and 3 are distinct proteases that have complementing roles during apoptosis and have novel non apoptotic roles in regulating morphology, adhesion and motility through a mechanism that is independent of their catalytic activity.

Keywords

caspase, apoptosis, adhesion, motility, morphology

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