Floribeth Aguilar

Antagonism of E2F-1 regulated Bnip3 transcription by NF-κB is essential for basal cell survival

The transcription factor E2F-1 drives proliferation and death, but the mechanisms that differentially regulate these divergent actions are poorly understood. The hypoxia-inducible death factor Bnip3 is an E2F-1 target gene and integral component of the intrinsic mitochondrial death pathway. The mechanisms that govern Bnip3 gene activity remain cryptic. Herein we show that the transcription factor NF-κB provides a molecular switch that determines whether E2F-1 signals proliferation or death under physiological conditions. We show under basal nonapoptotic conditions that NF-κB constitutively occupies and transcriptionally silences Bnip3 gene transcription by competing with E2F-1 for Bnip3 promoter binding. Conversely, in the absence of NF-κB, or during hypoxia when NF-κB abundance is reduced, basal Bnip3 gene transcription is activated by the unrestricted binding of E2F-1 to the Bnip3 promoter. Genetic knock-down of E2F-1 or retinoblastoma gene product over-expression in cardiac and human pancreatic cancer cells deficient for NF-κB signaling abrogated basal and hypoxia-inducible Bnip3 transcription. The survival kinase PI3K/Akt inhibited Bnip3 expression levels in cells in a manner dependent upon NF-κB activation. Hence, by way of example, we show that the transcriptional inhibition of E2F-1-dependent Bnip3 expression by NF-κB highlights a survival pathway that overrides the E2F-1 tumor suppressor program. Our data may explain more fundamentally how cells, by selectively inhibiting E2F-1-dependent death gene transcription, avert apoptosis down-stream of the retinoblastoma/E2F-1 cell cycle pathway.

A Novel Hypoxia-Inducible Spliced Variant of Mitochondrial Death Gene Bnip3 Promotes Survival of Ventricular Myocytes

Rationale: Alternative splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. Previously, we established hypoxia-inducible death factor Bnip3 as a critical component of the intrinsic death pathway. Objective: To investigate alternative splicing of Bnip3 pre-mRNA in postnatal ventricular myocytes during hypoxia. Methods and Results: We identify a novel previously unrecognized spliced variant of Bnip3 (Bnip3&Dgr;ex3) generated by alternative splicing of exon3 exclusively in cardiac myocytes subjected to hypoxia. Sequencing of Bnip3&Dgr;ex3 revealed a frame shift mutation that terminated transcription up-stream of exon5 and exon6 ablating translation of the BH3-like domain and critical carboxyl-terminal transmembrane domain crucial for mitochondrial localization and cell death. Notably, although the 26-kDa Bnip3 protein (Bnip3FL) encoded by full-length mRNA was localized to mitochondria and provoked cell death, the 8.2-kDa Bnip3&Dgr;ex3 protein encoded by the truncated spliced mRNA was defective for mitochondrial targeting but interacted with Bnip3FL resulting in less association of Bnip3FL with mitochondria and diminished apoptotic and necrotic cell death. Forced expression of Bnip3FL in cardiac myocytes or Bnip3−/− mouse embryonic fibroblasts triggered widespread cell death that was inhibited by coexpression of Bnip3&Dgr;ex3. Conversely, RNA interference targeted against sequences encompassing the unique exon2-exon4 junction of the Bnip3&Dgr;ex3 sensitized cardiac myocytes to mitochondrial perturbations and cell death induced by Bnip3FL. Conclusions: Given the otherwise lethal consequences of deregulated Bnip3FL expression in postmitotic cells, our findings reveal a novel intrinsic defense mechanism that opposes the mitochondrial defects and cell death of ventricular myocytes that is obligatorily linked and mutually dependent on alternative splicing of Bnip3FL during hypoxia or ischemic stress.

A tea/vanadate decoction delivered orally over 14 months to diabetic rats induces long-term glycemic stability without organ toxicity

Vanadium can induce potent hypoglycemic effects in type 1 and type 2 diabetes mellitus animals, but toxic adverse effects have inhibited the translation of these findings. Administration of vanadate in a black tea decoction has shown impressive hypoglycemic effects without evidence of toxicity in short-term studies. The purpose of this study was to investigate the hypoglycemic action and the toxic adverse effects of a tea/vanadate (T/V) decoction in diabetic rats over a 14-month treatment period. Streptozotocin-induced type 1 diabetes mellitus rats were orally gavaged with 40 mg sodium vanadate in a black tea decoction only when blood glucose levels were greater than 10 mmol/L. Glycemic status and liver and kidney function were monitored over 14 months. All of the diabetic rats in this treatment group (n = 25) required treatment with the T/V decoction at the start of the study to reduce blood glucose levels to less than 10 mmol/L. Diarrhea was uncommon among the T/V-treated animals during the first week of T/V treatment and was absent thereafter. There was no evidence of liver or kidney dysfunction or injury. From 2 to 6 months, fewer animals required the T/V treatment to maintain their blood glucose levels. After 9 months of treatment, none of the diabetic animals required any T/V to maintain their blood glucose levels at less than 10 mmol/L. Oral administration of a T/V decoction provides safe, long-acting hypoglycemic effects in type 1 diabetes mellitus rats. The typical glycemic signs of diabetes were absent for the last 5 months of the study.

Dissecting apoptosis and intrinsic death pathways in the heart.

The limited regenerative capacity of postnatal ventricular myocytes coupled with their meager ability for genetic manipulation has presented a major technical obstacle for deciphering apoptosis initiation and execution signals in the heart. In this report, we describe the technical approaches used to study the intrinsic death pathways in postnatal ventricular myocytes during acute hypoxic injury. Discussed are methods for hypoxia, recombinant adenovirus-mediated gene transfer, cellular viability assays using the vital dyes calcein acetomethoxyester and ethidium homodimer-1, analysis of nuclear morphology by use of Hoechst dye 33258, and assessment of the state of the mitochondrial permeability transition pore. Our work has established that hypoxia triggers perturbations to mitochondria consistent with loss of mitochondrial membrane potential, permeability transition pore opening, and apoptotic cell death by the intrinsic pathway.