Regulation of anti-oxidant and anti-apoptotic genes by progesterone in cardiomyocytes

• Main Author: Morrissy, S J
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2007
• Subjects: Pharmacology
• ISBN: 9780549328148; 0549328149

The anthracycline quinone, doxorubicin (Adriamycin) is an antineoplastic agent that has substantial therapeutic activity against a broad variety of human cancers. Unfortunately, the use of this agent is limited by its cardiac toxicity, which is associated with free radical formation leading to apoptotic cell death. The goal of this work is to improve our understanding about doxorubicin induced cardiomyopathy and to identify compounds to limit doxorubicin induced cardiomyopathy. The knowledge gained here will have a generalized impact on all cardiac diseases involving oxidative stress and apoptosis. We show that doxorubicin induced apoptosis in primary neonatal rat cardiomyocytes can be attenuated by progesterone (PG). The anti-apoptotic action of PG was blocked by a progesterone receptor antagonist, Mifepristone (MF), indicating a progesterone receptor dependent pathway. Affymetrix gene analyses found that PG treated cardiomyocytes increased the expression of 180 genes. Among the genes upregulated is NAD(P)H: Quinone Oxidoreductase-1 (NQO1) gene. NQO1 is a flavo-enzyme that can catalyze a two-electron reduction of Dox to a more stable hydroquinone, thereby acting as a defense mechanism against oxidative stress. The induction of NQO1 mRNA and NQO1 activity in cardiomyocytes was observed in a dose and time-dependent manner with PG treatment and was blocked by MF. Induction of NQO1 by b-naphoflavone, an inducer of NQO1, resulted in a decrease in caspase-3 activity. However, inhibition of NQO1 by dicoumarol did not attenuate the cytoprotective effect of PG. This data indicates that although induction of NQO1 can decrease Dox induced apoptosis, this is not the primary mechanism of cytoprotection induced by PG. Microarray analyses revealed that PG induced an increase of Bcl-X L mRNA. Inhibiting the expression of Bcl-XL using siRNA reduced the anti-apoptotic effect of PG, suggesting that Bcl-XL is a key player in PG induced cytoprotection. Western blot analyses indicated that PG induced the expression of Bcl-XL in a dose and time dependent manner consistent with the protective effect of PG. Induction of Bcl-X L by PG was blocked by cyclohexamide, but was not blocked by Actinomycin D indicating that a transcriptional independent mechanism is responsible for the induction of Bcl-XL by PG. The activity of a bcl-x 3'UTR reporter was induced by PG and blocked by MF. These data suggest that PG may induce stabilization of the Bcl-X mRNA. We further explored the mechanism of PG induced Bcl-XL gene expression by comparing the effect of PG to two other steroids: corticosterone (CT) and retinoic acid (RA). Both CT and RA attenuate Dox induced apoptosis in cardiomyocytes. CT, but not RA or PG induced the activity of a GRE reporter plasmid. Analysis of the 5' region of the Bcl-XL promoter indicated that RA and CT, but not PG induced the activity of the 0.9kb region of the Bcl-XL promoter. The induction of the 0.9kb reporter plasmid by CT was glucocorticoid receptor dependent, since it was inhibited by MF. The Bcl-XL promoter does not contain any glucocorticoid or retinoid response elements, but does have AP-1 and NFkB response elements. CT, but not RA or PG induced the activity of an AP-1 reporter plasmid. RA, but not CT or PG induced the activity of an NFkB reporter plasmid. The induction of the 0.9kb Bcl-XL reporter plasmid by CT was blocked by expression of a dominant negative c-jun, TAM67 as well SB202190 indicating a nongenomic effect of CT in activating the Bcl-XL promoter through a p38 MAPK mediated AP-1 mechanism. Therefore although all three types of nuclear receptor ligands induce bcl-xL expression, the effect of CT is mediated by transcriptional activation by AP-1 signaling while NF-kB transcription factor appears to be involved in RA induced bcl-xL transcription. The lack of bcl-xL promoter activation by PG again suggests that PG induces bcl-xL by a nontranscriptional mechanism.