The pro‐cell death Bcl‐2 family member, BNIP3, is localized to the nucleus of human glial cells: Implications for glioblastoma multiforme tumor cell survival under hypoxia

Abstract

The Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) is a hypoxia-inducible proapoptotic member of the Bcl-2 family that induces cell death by associating with the mitochondria. Under normal conditions, BNIP3 is expressed in skeletal muscle and in the brain at low levels. In many human solid tumors, BNIP3 is upregulated in hypoxic regions but paradoxically, this BNIP3 expression fails to induce cell death. Herein, we have determined that BNIP3 is primarily localized to the nucleus of glial cells of the normal human brain, as well as in the malignant glioma cell line U251. Upon exposure of U251 cells to hypoxia, BNIP3 expression in the cytoplasm increases and localizes with the mitochondria, contributing to induction of cell death. In contrast, when BNIP3 is forcibly over expressed in the nucleus, it fails to induce cell death. Expression of N-terminal BNIP3 (lacking the transmembrane and conserved domains) in U251 cells blocks hypoxia-induced cell death acting as a dominant negative protein by binding to wildtype BNIP3 and blocking its association with the mitochondria. In glioblastoma multiforme (GBM) tumors, BNIP3 expression is increased in hypoxic regions of the tumor and is primarily localized to the nucleus in 80% of tumors. Hence, BNIP3 is sequestered in the nucleus within the brain but under hypoxic conditions, BNIP3 becomes primarily cytoplasmic, promoting cell death. In GBMs, BNIP3 expression is increased but it remains sequestered in the nucleus in hypoxic regions, thereby blocking BNIP3's ability to associate with the mitochondria, providing tumor cells with a possible survival advantage. ' 2005 Wiley-Liss, Inc.Key words: BNIP3; astrocytes; glial cells; glioblastoma multiforme; hypoxia; mitochondria; cell death; nuclear localization The Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) is a pro-cell death Bcl-2 family member that localizes to the mitochondria upon overexpression, causing opening of the permeability transition (PT) pore, loss of mitochondrial membrane potential (Dwm) and reactive oxygen species (ROS) production resulting in cell death. 1 BNIP3 also induces an autophagic response in cells contributing to cell death. 2,3 BNIP3-induced cell death is independent of mitochondrial cytochrome c release and caspase activation. 1,4 The BNIP3 protein has 4 major domains: a PEST domain that targets BNIP3 for degradation, a putative BH3 (Bcl-2 homology 3) domain that is homologous to other Bcl-2 family members, a CD (conserved) domain that is conserved from C. elegans to humans, and a TM (transmembrane) domain, which targets BNIP3 to the mitochondria and is essential for BNIP3-induced cell death. [5][6][7] Deletion of the CD or BH3 domains fails to affect BNIP3's ability to induce cell death, while deletion of the TM domain blocks BNIP3-induced cell death because of its inability to associate with the mitochondrial membrane. 8 BNIP3 is expressed in normal tissues such as skeletal muscle and brain and its expression is increased under hypoxic conditions correlating with hypoxia-induced...

HIF-1α activation by a redox-sensitive pathway mediates cyanide-induced BNIP3 upregulation and mitochondrial-dependent cell death

“…Expression of the transmembrane domain deleted form of BNIP3 (BNIP3ΔTM) reduced the cyanide-induced cell death. BNIP3ΔTM functions as a dominant negative mutant to block integration of wildtype BNIP3 into the mitochondrial membrane [43]. In summary, it was shown that in cyanide-induced cell death, oxidative stress initiates upregulation of BNIP3 expression via a HIF-1α signaling regulated transcription.…”

Cardiac reanimation: targeting cardiomyocyte death by BNIP3 and NIX/BNIP3L

“…More importantly, they also suggest that during ischemic stress, factors other than simple upregulation are important in generating the full apoptotic response to BNIP3 in ischemic injury. Whether this is hypoxia/acidosis-mediated post-translational modification and mitochondrial translocation of BNIP3 as has been suggested (Kubasiak et al, 2002;Kubli et al, 2008), or some other as-yet unidentified factor such as nuclear translocation that is seen in ischemic brain injury (Schmidt-Kastner et al, 2004;Burton et al, 2005), is not currently known.…”

The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death

“…There are multiple mechanisms that could control BNIP3 expression localization and function in tumors that are discussed below. The expression of BNIP3 is upregulated in primary prostate tumors, 56 glioblastoma multiforme (GBM) tumors, 57 endometrial cancer, 58 cervical tumors, 59 ductal carcinoma in situ (DCIS), 60 invasive breast carcinomas, 61 lung tumors, 62 ependymoma, 63 follicular lymphomas 64 and gastric adenocarcinomas. 65 (Table 1).…”

“…BNIP3 is observed to be localized to the cytoplasm, the nucleus or the perinuclear space. BNIP3 is expressed in the nucleus in noncancerous primary human astrocyte cultures, normal brain sections 57 and chondrocytes of brachial cartilage, and also in the cytoplasm in the normal bronchial epithelium. 62 Expression in tumors has been observed to be exclusively nuclear, exclusively cytoplasmic or both nuclear and cytoplasmic.…”

“…The amount of nuclear versus cytoplasmic BNIP3 expression in different tumor types is shown in Table 2 and discussed below. Nuclear BNIP3 is detected in GBM tumors, 57 nonsmall cell lung tumors, 62 cervical tumors, 59 breast tumors 61 and prostate tumors, 56 and a perinuclear BNIP3 staining pattern is observed in pancreatic tumors 72 and colorectal adenocarcinomas. 69 In prostate tumors, there is a correlation between cytoplasmic BNIP3 expression and Gleason score, age AR and GLUT1 expression, but nuclear BNIP3 did not correlate with any clinicopathological factors.…”