Pro-apoptotic Bcl-2 Family Protein Bad Research Articles

BAD-Mediated Neuronal Apoptosis and Neuroinflammation Contribute to Alzheimer's Disease Pathology

Alzheimer's disease is the most common progressive neurodegenerative disease. However, the underlying molecular mechanism is incompletely understood. Here we report the identification of the pro-apoptotic BCL-2 family protein BAD as a key regulator for neuronal apoptosis, neuroinflammation and β-amyloid (Aβ) clearance in Alzheimer's disease. The pro-apoptotic activity of BAD is significantly increased in neurons of patients with Alzheimer's disease and 5XFAD mice. Conversely, genetic disruption of Bad alleles restores spatial learning and memory deficits in 5XFAD mice. Mechanistically, phosphorylation and inactivation of BAD by neurotropic factor-activated Akt is abrogated in cortical neurons in patients with Alzheimer's disease and 5XFAD mice, thereby triggering mitochondrial translocation of hypo-phosphorylated BAD to promote neuronal apoptosis and neurodegeneration. Through reactive oxygen species (ROS)-oxidized mitochondrial DNA (mtDNA) axis, BAD also promotes activation of microglial NLRP3 inflammasome, thereby skewing microglia toward neuroinflammatory microglia to inhibit microglial phagocytosis of Aβ plaques in 5XFAD mice. AAV-mediated Bad deletion in hippocampal neurons prevents neuronal apoptosis and rescues memory deficits in 5XFAD mice. In another Alzheimer's disease murine model, APP/PS1 mice, Bad loss also inhibits activation of microglial NLRP3 inflammasome and Aβ deposition, thereby restoring memory deficits. Our results support a model in which BAD contributes to memory deficits and neuropathology in Alzheimer's disease by driving neuronal apoptosis, neuroinflammation but suppressing microglial phagocytosis of Aβ plaques, suggesting that BAD is a potential therapeutic target for Alzheimer's disease.

BAD inactivation exacerbates rheumatoid arthritis pathology by promoting survival of sublining macrophages.

The resistance of synovial sublining macrophages to apoptosis has a crucial role in joint inflammation and destruction in rheumatoid arthritis (RA). However, the underlying mechanism is incompletely understood. Here we report that inactivation of the pro-apoptotic BCL-2 family protein BAD is essential for survival of synovial sublining macrophage in RA. Genetic disruption of Bad leads to more severe joint inflammation and cartilage and bone damage with reduced apoptosis of synovial sublining macrophages in collagen-induced arthritis (CIA) and TNFα transgenic (TNF-Tg) mouse models. Conversely, Bad3SA/3SA mice, in which BAD can no longer be inactivated by phosphorylation, are protected from collagen-induced arthritis. Mechanistically, phosphorylation-mediated inactivation of BAD specifically protects synovial sublining macrophages from apoptosis in highly inflammatory environment of arthritic joints in CIA and TNF-Tg mice, and in patients with RA, thereby contributing to RA pathology. Our findings put forward a model in which inactivation of BAD confers the apoptosis resistance on synovial sublining macrophages, thereby contributing to the development of arthritis, suggesting that BAD may be a potential therapeutic target for RA.

Cytoprotective effect of neuropeptides on cancer stem cells: vasoactive intestinal peptide-induced antiapoptotic signaling

Cancer stem cells (CSCs) are increasingly considered to be responsible for tumor initiation, metastasis and drug resistance. The drug resistance mechanisms activated in CSCs have not been thoroughly investigated. Although neuropeptides such as vasoactive intestinal peptide (VIP) can promote tumor growth and activate antiapoptotic signaling in differentiated cancer cells, it is not known whether they can activate antiapoptotic mechanisms in CSCs. The objectives of this study are to unravel the cytoprotective effects of neuropeptides and identify antiapoptotic mechanisms activated by neuropeptides in response to anticancer drug treatment in CSCs. We enriched and purified CSCs (CD44+/high/CD24−/low or CD133+ population) from breast and prostate cancer cell lines, and demonstrated their stemness phenotype. Of the several neuropeptides tested, only VIP could protect CSCs from drug-induced apoptosis. A functional correlation was found between drug-induced apoptosis and dephosphorylation of proapoptotic Bcl2 family protein BAD. Similarly, VIP-induced cytoprotection correlated with BAD phosphorylation at Ser112 in CSCs. Using pharmacological inhibitors and dominant-negative proteins, we showed that VIP-induced cytoprotection and BAD phosphorylation are mediated via both Ras/MAPK and PKA pathways in CSCs of prostate cancer LNCaP and C4-2 cells, but only PKA signaling was involved in CSCs of DUVIPR (DU145 prostate cancer cells ectopically expressing VIP receptor) and breast cancer MCF7 cells. As each of these pathways partially control BAD phosphorylation at Ser112, both have to be inhibited to block the cytoprotective effects of VIP. Furthermore, VIP is unable to protect CSCs that express phosphorylation-deficient mutant-BAD, suggesting that BAD phosphorylation is essential. Thus, antiapoptotic signaling by VIP could be one of the drug resistance mechanisms by which CSCs escape from anticancer therapies. Our findings suggest the potential usefulness of VIP receptor inhibition to eliminate CSCs, and that targeting BAD might be an attractive strategy for development of novel therapeutics.

Mechanistic Insights into the Antileukemic Activity of Hyperforin

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) - conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells - two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Mechanistic Insights into the Antileukemic Activity of Hyperforin

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) - conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells - two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Mechanistic Insights into the Antileukemic Activity of Hyperforin

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) – conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells – two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo. Keywords: Apoptosis, drug resistance, hyperforin, leukemia, proliferation, signaling

Phosphorylation of Bad at Thr-201 by JNK1 Promotes Glycolysis through Activation of Phosphofructokinase-1

The mitogen-activated protein kinase JNK1 suppresses interleukin-3 withdrawal-induced cell death through phosphorylation of the BH3-only pro-apoptotic Bcl-2 family protein Bad at Thr-201. It is unknown whether JNK1 regulates glycolysis, an important metabolic process that is involved in cell survival, and if so, whether the regulation depends on Thr-201 phosphorylation of Bad. Here we report that phosphorylation of Bad by JNK1 is required for glycolysis through activation of phosphofructokinase-1 (PFK-1), one of the key enzymes that catalyze glycolysis. Genetic disruption of Jnk1 alleles or silencing of Jnk1 by small interfering RNA abrogates glycolysis induced by growth/survival factors such as serum or interleukin-3. Proteomic analysis identifies PFK-1 as a novel Bad-associated protein. Although the interaction between PFK-1 and Bad is independent of JNK1, Thr-201 phosphorylation of Bad by JNK1 is required for PFK-1 activation. Thus, our results provide a novel molecular mechanism by which JNK1 promotes glycolysis for cell survival.

Transplacental exposure to the vacuolar-ATPase inhibitor bafilomycin disrupts survival signaling in β cells and delays neonatal remodeling of the endocrine pancreas

A wave of β cell apoptosis occurs around 2 weeks of age in rats and mice. We have previously reported that exposure in utero to bafilomycin, a plecomacrolide antibiotic that inhibits the vacuolar (v)ATPase enzyme and contaminates the human diet, delays this neonatal wave and accelerates diabetes in non-obese diabetic (NOD) mice. Here we exposed C57BL/6J mice in utero to bafilomycin and assessed the effects on islet morphology, apoptosis and activation of cell survival signaling in β cells. The neonatal wave of β cell apoptosis was associated with high expression and low phosphorylation of the pro-apoptotic Bcl-2 family protein Bad, whereas after weaning (3 weeks of age) Bad was down-regulated and β cell apoptosis was low. In contrast, in bafilomycin-exposed mice the frequency of apoptotic β cells and the expression of Bad remained high after weaning. Bafilomycin exposure also inactivated the insulin/IGF signaling pathway intermediate, FoxO1, and increased the insulin content in neonatal islets. Thus, exposure in utero to bafilomycin disrupts the regulation of Bad in neonatal β cells, increases cell survival signaling and delays the neonatal wave of apoptosis. Increased expression of Bad in adult β cells provides an explanation for accelerated diabetes in bafilomycin-exposed NOD mice, whereby disruption of neonatal islet-cell turnover may render the adult β cells more susceptible to induced cell death.

BAD Ser128 Is Not Phosphorylated by c-Jun NH2-Terminal Kinase for Promoting Apoptosis

The phosphorylation and regulation of the proapoptotic Bcl-2 family protein BAD by c-Jun NH2-terminal kinase (JNK) is controversial. JNK can suppress interleukin-3 withdrawal-induced apoptosis via phosphorylation of BAD at Thr201. However, it has also been reported that JNK promotes apoptosis through phosphorylation of BAD at Ser128. Here, we report that JNK is not a BAD Ser128 kinase. JNK phosphorylates murine BAD (mBAD), but not human BAD (hBAD), in which Ser91 is equivalent to Ser128 in mBAD. In contrast, Cdc2, which phosphorylates Ser128, phosphorylates both mBAD and hBAD. Replacement of Ser128 by alanine has no effects on BAD phosphorylation by JNK in vitro and in vivo. Two-dimensional phosphopeptide mapping in combination with phosphoamino acid analysis reveals that JNK does not phosphorylate BAD at Ser128. Elimination of Ser128 phosphorylation has no effects on the proapoptotic activity of BAD in apoptosis induced by UV via JNK or growth factor withdrawal. Thus, our results show that Ser128 is not phosphorylated by JNK for promoting cell death.

Akt/Bad signaling and motor neuron survival after spinal cord injury

The serine-threonine kinase Akt is a cell survival signaling pathway that inactivates the proapoptotic BCL-2 family protein Bad and promotes cell survival in cerebral ischemia. Involvement of the Akt/Bad signaling pathway after spinal cord injury (SCI) is, however, uncertain. Our results showed that phospho-Akt (serine-473) and phospho-Bad (serine-136) were significantly upregulated at 1 day after SCI. In addition, phospho-Akt and phospho-Bad were colocalized in motor neurons that survived SCI and inhibition of PI3-K reduced expression of phospho-Akt and phospho-Bad. Dimerization of Bad with 14-3-3 in the cytosol was increased whereas Bad/Bcl-XL binding in the mitochondria was decreased after SCI. We further found that reduced oxidative stress by SOD1 overexpression in rats enhanced the expression of phospho-Akt, phospho-Bad, Bad/14-3-3 binding and reduced Bad/Bcl-XL binding after SCI, as compared to wild-type rats. We conclude that oxidative stress may play a role in modulating Akt/Bad signaling and subsequent motor neuron survival after SCI.

Role of JNK activation in apoptosis: A double-edged sword

JNK is a key regulator of many cellular events, including programmed cell death (apoptosis). In the absence of NF-kB activation, prolonged JNK activation contributes to TNF-a induced apoptosis. JNK is also essential for UV induced apoptosis. However, recent studies reveal that JNK can suppress apoptosis in IL-3-dependent hematopoietic cells via phosphorylation of the proapoptotic Bcl-2 family protein BAD. Thus, JNK has pro- or antiapoptotic functions, depending on cell type, nature of the death stimulus, duration of its activation and the activity of other signaling pathways.

JNK Suppresses Apoptosis via Phosphorylation of the Proapoptotic Bcl-2 Family Protein BAD

JNK has been suggested to be proapoptotic, antiapoptotic, or have no role in apoptosis depending on the cell type and stimulus used. The precise mechanism of JNK action, under conditions when it promotes cell survival, is not entirely clear. Here, we report that JNK is required for IL-3-mediated cell survival through phosphorylation and inactivation of the proapoptotic Bcl-2 family protein BAD. IL-3 withdrawal-induced apoptosis is promoted by inhibition of JNK but suppressed by expression of a constitutively active JNK. JNK phosphorylates BAD at threonine 201, thereby inhibiting BAD association with the antiapoptotic molecule BCL-X(L). IL-3 induces BAD phosphorylation at threonine 201, and replacement of threonine 201 by alanine generates a BAD mutant, which promotes IL-3 withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK contributes to cell survival.

MEK and ERK protect hypoxic cortical neurons via phosphorylation of Bad.

We investigated the role of mitogen-activated protein kinase (MAPK) pathways in hypoxic neuronal injury using primary cultures from murine cerebral cortex. Hypoxia caused the death of approximately 50% of neurons at 16 h and approximately 65% of neurons at 24 h. This was associated with phospho-activation of the MAPK/extracellular signal-regulated kinase (ERK) kinase MEK1/2 and its downstream target ERK1/2, but not p38 MAPK or c-Jun N-terminal kinase (JNK), as detected by western blotting. The MEK1/2 inhibitor, PD98059, increased neuronal death in hypoxic cultures, suggesting that MEK1/2 promotes neuronal survival, whereas the p38 inhibitors, SB202190 and SB203580, had no effect. To identify downstream effects of ERK1/2 that might regulate hypoxic neuronal death, we measured hypoxia-induced phosphorylation of three ERK1/2 targets: the 90-kDa ribosomal protein S6 kinase (RSK), the transcription factor ELK1, and the pro-apoptotic Bcl-2 family protein Bad. We observed increased abundance of inactivated (phospho-)Bad, but no change in phospho-RSK or phospho-ELK1. Moreover, the MEK inhibitor PD98059 reduced phospho-inactivation of Bad in hypoxic cultures. These findings suggest that a cell-survival program involving phospho-activation of MEK1/2 and ERK1/2 and inactivation of Bad is mobilized in hypoxic neurons, and may help to regulate neuronal fate following hypoxic-ischemic injury.

P21-activated Protein Kinase γ-PAK Suppresses Programmed Cell Death of BALB3T3 Fibroblasts

In response to stress stimulants, cells activate opposing signaling pathways for cell survival and programmed cell death. p21-activated protein kinase gamma-PAK is involved in both cell survival and cell death pathways. Many stress stimulants activate gamma-PAK as a full-length enzyme and as a proteolytic fragment. Caspase-mediated proteolytic activation parallels cell death and appears to be a pro-apoptotic factor in stress-induced cell death. Here, we show that activation of full-length gamma-PAK promotes cell survival and suppresses stress-induced cell death. Expression of constitutively active gamma-PAK-T402E, which mimics activated full-length gamma-PAK, stimulates cell survival of BALB3T3 fibroblasts in response to tumor necrosis factor alpha, growth factor withdrawal, and UVC light. This stimulation of cell survival is mainly due to protection of cells from cell death rather than by stimulation of proliferation. Expression of gamma-PAK-T402E increases phosphorylation of the pro-apoptotic Bcl-2 family protein Bad and protects from cell death induced by ectopic expression of Bad. In response to tumor necrosis factor alpha, expression of gamma-PAK-T402E increases the early but reduces the late activation of ERK, JNK, and p38. Our results indicate that the ubiquitous gamma-PAK may have a crucial function in cell survival by regulating the pro-apoptotic activity of Bad and the stress-induced activation of ERK, JNK, and p38 pathways.

β-Adrenergic Pathway Induces Apoptosis through Calcineurin Activation in Cardiac Myocytes

Apoptosis of cardiac myocytes is one of the causes of heart failure. Here we examine the mechanism by which the activation of beta-adrenergic receptor induces cardiomyocyte apoptosis. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling and DNA ladder analyses revealed that isoproterenol (Iso) induced the apoptosis of cardiac myocytes of neonatal rats through an increase in intracellular Ca(2+) levels. The Iso-induced cardiomyocyte apoptosis was strongly inhibited by the L-type Ca(2+) channel antagonist nifedipine and by the calcineurin inhibitors cyclosporin A and FK506. Iso reduced the phosphorylation levels of the proapoptotic Bcl-2 family protein Bad and induced cytochrome c release from mitochondria to the cytosol through calcineurin activation. Infusion of Iso increased calcineurin activity by approximately 3-fold in the hearts of wild-type mice but not in the hearts of transgenic mice that overexpress dominant negative mutants of calcineurin. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling analysis revealed that infusion of Iso induced apoptosis of cardiac myocytes and that the number of apoptotic cardiomyocytes was significantly less in the hearts of the transgenic mice compared with the wild-type mice. These results suggest that calcineurin plays a critical role in Iso-induced apoptosis of cardiac myocytes, possibly through dephosphorylating Bad.