Bcl-2 Overexpression Protects Photooxidative Stress-Induced Apoptosis of Photoreceptor Cells via NF-κB Preservation

doi:10.1006/bbrc.2001.4501

Biochemical and Biophysical Research Communications

Volume 281, Issue 5, 16 March 2001, Pages 1304-1312

https://doi.org/10.1006/bbrc.2001.4501 Get rights and content

Abstract

We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-κB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-κB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-κB which revealed that protein levels of the RelA subunit of NF-κB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-κB protein levels and activity in the nucleus, indicating that preservation of NF-κB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis.

References (46)

R.R. Krishnamoorthy et al.
Photo-oxidative stress down-modulates the activity of nuclear factor-kappaB via involvement of caspase-1, leading to apoptosis of photoreceptor cells
J. Biol. Chem.
(1999)
D.M. Hockenbery et al.
Bcl-2 functions in an antioxidant pathway to prevent apoptosis
Cell
(1993)
J.E. Thompson et al.
I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B
Cell
(1995)
U. Zabel et al.
Purified human I kappa B can rapidly dissociate the complex of the NF-kappa B transcription factor with its cognate DNA
Cell
(1990)
Z.-G. Liu et al.
Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death
Cell
(1996)
M. Grilli et al.
NF-kappa B and Rel: Participants in a multiform transcriptional regulatory system
Int. Rev. Cytol.
(1993)
M. Mandal et al.
Bcl-2 prevents CD95 (Fas/APO-1)-induced degradation of lamin B and poly(ADP-ribose) polymerase and restores the NF-kappaB signaling pathway
J. Biol. Chem.
(1996)
D. de Moissac et al.
Bcl-2 activates the transcription factor NFkappaB through the degradation of the cytoplasmic inhibitor IkappaBalpha
J. Biol. Chem.
(1998)
E.P. Dixon et al.
Bcl-Xshort is elevated following severe global ischemia in rat brains
Brain Res.
(1997)
N. Agarwal et al.
Immunocytochemical colocalization of clusterin in apoptotic photoreceptor cells in retinal degeneration slow rds mutant mouse retinas
Biochem. Biophys. Res. Commun.
(1996)

N. Agarwal et al.

Possible involvement of Bcl-2 pathway in retinoid X receptor alpha-induced apoptosis of HL-60 cells

Biochem. Biophys. Res. Commun.

(1997)

R.S. Roque et al.

Retina-derived microglial cells induce photoreceptor cell death in vitro

Brain Res.

(1999)

Y. Ma et al.

Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells

Free Radical Biol. Med.

(1998)

S.C. Cosulich et al.

Bcl-2 regulates amplification of caspase activation by cytochrome c

Curr. Biol.

(1999)

D.S. Papermaster et al.

Death at an early age. Apoptosis in inherited retinal degenerations

Invest. Opthalmol. and Visual Sci.

(1995)

M.O. Tso et al.

Apoptosis leads to photoreceptor degeneration in inherited retinal dystrophy of RCS rats

Invest. Opthalmol. Visual Sci

(1994)

A.S. Abler et al.

Photic injury triggers apoptosis of photoreceptor cells

Invest. Opthalmol. Visual Sci.

(1994)

D.L. Vaux et al.

Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells

Nature

(1988)

Hawkins, C. J., and Vaux, D. L.Analysis of the role of bcl-2 in apoptosis. Immunol. Rev.142, 127–39,...

S.J. Korsmeyer et al.

Bcl-2/Bax: A rheostat that regulates an anti-oxidant pathway and cell death

Semin. Cancer Biol.

(1993)

Z.N. Oltvai et al.

Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death

Cell

(1993)

M. Sioud et al.

Analysis of the NF-kappa B p65 subunit, Fas antigen, Fas ligand and Bcl-2-related proteins in the synovium of RA and polyarticular JRA

Clin. Exp. Rheumatol.

(1998)

Cited by (65)

Activation inhibitors of nuclear factor kappa B protect neurons against the NMDA-induced damage in the rat retina
2017, Journal of Pharmacological Sciences
We reported that high-mobility group Box-1 (HMGB1) was involved in excitoneurotoxicity in the retina. HMGB1 is known to activate nuclear factor kappa B (NF-κB). However, the role of NF-κB in excitotoxicity is still controversial. Here, we demonstrated that NF-κB activation induced by NMDA led to the retinal neurotoxicity. Male Sprague–Dawley rats were used, and NMDA (200 nmol/eye) and bovine HMGB1 (15 μg/eye) were intravitreally injected. Triptolide (500 pmol/eye), BAY 11-7082 (500 pmol/eye), and IMD-0354 (7.5 nmol/eye), NF-κB inhibitors, were co-injected with NMDA or HMGB1. Retinal sections were obtained seven days after intravitreal injection. Cell loss in the ganglion cell layer was observed in the HMGB1- and the NMDA-treated retina. All of the NF-κB inhibitors used in this study reduced the damage. BAY 11-7082 reduced the expression of phosphorylated NF-κB 12 h after NMDA injection, upregulation of GFAP immunoreactivity induced by NMDA 12 and 48 h after NMDA injection, and the number of TUNEL-positive cells 48 h after NMDA injection. The results suggest that NF-κB activation is one of the mechanisms of the retinal neuronal death that occurs 48 h after NMDA injection or later. Prevention of NF-kB activation is a candidate for the treatment of retinal neurodegeneration associated with excitotoxicity.
Screening and identification of neuroprotective compounds produced by Lactobacillus paracasei subsp. paracasei NTU 101
2016, Journal of Functional Foods
Citation Excerpt :
It was reported previously that an anti-apoptotic gene, B-cell lymphoma 2 (Bcl-2), is activated by NF-κB (Culmsee & Mattson, 2005). Bcl-2 is able to block a variety of apoptotic signals, and its anti-apoptotic function may depend on its interactions with the members of several antioxidant pathways (Crawford et al., 2001). Another member of the Bcl-2 family, Bcl-2-associated X (Bax), was described as a pro-apoptotic molecule, with apoptosis-stimulating functions (Brady & Gil-Gomez, 1998).
Ischaemia/reperfusion injury plays important roles in the morbidity and mortality associated with ischaemic strokes. We investigated the neuroprotective effects of ethanol extract fractions isolated form Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) fermentation products, against oxygen-glucose deprivation and reperfusion (OGD/R) in vitro. Pre-treatment with 1000 µg/mL water or ethanol extracts reversed OGD/R-induced cell death by 17% and 42%, respectively, while post-treatment with these extracts reversed OGD/R-induced cell death by 3% and 27%, respectively. A major neuroprotective compound extracted from NTU 101 fermentation products, glyceryl 1,3-dipalmitate (GD), was characterised using ultra-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. Pre-treatment with GD modulated the expression of inducible nitric oxide synthase, nuclear factor-kappa B, and Bcl-2-associated X, compared with OGD/R group. Our findings indicate the potential of the therapeutic application of GD in the prevention and treatment of cerebral ischaemic and neurodegenerative diseases.
Two molluscan BCL-2 family members from Manila clam, Ruditapes philippinarum: Molecular characterization and immune responses
2013, Fish and Shellfish Immunology
Citation Excerpt :
Moreover, elevated Bcl-2 and Mcl-1 expression was shown in LPS treated neutrophils [31]. Bcl-2 can regulate the NF-κB activity through the regulation of inhibitor of NF-κB (IκB) [32]. Previously we have showed that immune responses of Manila clam IκB in gills and hemocytes after LPS and V. tapetis challenges [20].
Apoptosis based immune responses are important component of host defense in mollusks. In this study, we have identified two novel molluscan BCL-2 cDNAs from Manila clam, Ruditapes philippinarum and named as RpBCL-2A and RpBCL-2B. There were four and three highly conserved BCL-2 homology (BH) regions in RpBCL-2A and RpBCL-2B, respectively suggesting these two genes could be different isoforms of anti-apoptotic BCL-2 family. Phylogenetic results revealed that Manila clam BCL-2 genes were clustered closely with invertebrate BCL-2 members. It gives evidence of their common origin and conserved features of invertebrate BCL-2 family. RpBCL-2A and 2B were expressed in tissue-specific manner showing the highest and lowest level of expression in gills and hemocytes, respectively. However there was no clear expression profile difference between two genes. After Vibrio tapetis challenge, transcriptional responses of RpBCL-2A and RpBCL-2B were induced in gills and hemocytes with high variation that could be due to effects of immune reactions of other host defense molecules.
Anticancer activity of esculetin via-modulation of Bcl-2 and NF-κB expression in benzo[a]pyrene induced lung carcinogenesis in mice
2013, Biomedicine and Preventive Nutrition
Citation Excerpt :
Bcl-2 over-expression has been reported to increase the activity of AKT (protein kinase B) and IKK resulting in increased NF-κB transcriptional activity in pancreatic cancer [40]. Studies have also shown that over-expression of Bcl-2 result, in increased NF-κB activity [41–44]. NF-κB also has anti-apoptotic function [45–47].
Esculetin (ESC), a coumarin derivative, has shown to possess antioxidant, anti-inflammatory, anticancer and chemopreventive properties. In the present study, we evaluated the chemopreventive activity of ESC against benzo[a]pyrene (B[a]P) induced lung carcinogenesis in Swiss albino mice by measuring antioxidant parameter, like lipid peroxidation, reduced glutathione (GSH) and superoxide dismutase (SOD); and levels of Bcl-2 and NF-κB protein in lung tissues. Lung carcinogenesis was induced by B[a]P (50 mg/kg, intraperitoneally). Pre-treatment and post-treatment groups were treated with ESC (50 mg/kg, per oral). Oxidative stress-induced by B[a]P was decreased by ESC. Anti-apoptotic protein Bcl-2 and pleotropic regulator NF-κB are found to possess oncogenic roles in B[a]P induced carcinogenesis. Western blot analysis study showed alleviated levels Bcl-2 and NF-κB in ESC pre-treated and post-treated groups in comparison to B[a]P treated group. These results suggest the anti-initiating and apoptosis inducing property of ESC on pre- and post-treatment in B[a]P induced lung carcinogenesis in Swiss albino mice.
Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27<sup>Kip1</sup>-dependent G1/S cell cycle arrest in conjunction with NF-κB activation
2009, Biochemical Pharmacology
2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21^Cip1/Waf1 and up-regulating p16^INK4A. Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27^Kip1. Bcl-2 and p27^Kip1 were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-κB activity and p50 levels were increased by 2-ME2 and suppression of NF-κB signaling reduced p27^Kip1 expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27^Kip1 in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27^Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.
Resveratrol suppresses interleukin-1β-induced inflammatory signaling and apoptosis in human articular chondrocytes: Potential for use as a novel nutraceutical for the treatment of osteoarthritis
2008, Biochemical Pharmacology
Citation Excerpt :
Second, Western blot analysis showed that resveratrol down-regulated the expression of MMPs, which are intimately linked with cartilage matrix degradation in OA [24,60,61]. Third, the expression of VEGF and COX-2, which are linked with angiogenesis and inflammation in cartilage [47–49,62,63], was significantly decreased by resveratrol. Finally, resveratrol clearly down-regulated NF-κB-regulated pro-apoptotic proteins, including caspase-3 and PARP in human chondrocytes.
Osteoarthritis is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective on pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Resveratrol is a phytoalexin stilbene produced naturally by plants including red grapes, peanuts and various berries. Recent research in various cell models has demonstrated that resveratrol is safe and has potent anti-inflammatory properties. However, its potential for treating arthritic conditions has not been explored. In this study we provide experimental evidence that resveratrol inhibits the expression of VEGF, MMP-3, MMP-9 and COX-2 in human articular chondrocytes stimulated with the pro-inflammatory cytokine IL-1β. Since these gene products are regulated by the transcription factor NF-κB, we investigated the effects of resveratrol on IL-1β-induced NF-κB signaling pathway. Resveratrol, like N-Ac-Leu-Leu-norleucinal (ALLN) suppressed IL-1β-induced proteasome function and the degradation of IκBα (an inhibitor of NF-κB) without affecting IκBα kinase activation, IκBα-phosphorylation or IκBα-ubiquitination which suppressed nuclear translocation of the p65 subunit of NF-κB and its phosphorylation. Furthermore, we observed that resveratrol as well as ALLN inhibited IL-1β-induced apoptosis, caspase-3 activation and PARP cleavage in human articular chondrocytes. In summary, our results suggest that resveratrol suppresses apoptosis and inflammatory signaling through its actions on the NF-κB pathway in human chondrocytes. We propose that resveratrol should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

View all citing articles on Scopus

^☆: Abbreviations used: NF-κB, nuclear factor-κB; EMSA, electrophoretic mobility shift assay; TUNEL, terminal deoxynucleotidyl transferase mediated fluoresceinated dUTP nick end labeling, IκBα, Inhibitory subunit of NF-κB; MPT, mitochondrial permeability transition; AMD, age-related macular degeneration.

¹: To whom correspondence should be addressed at Department of Pathology and Anatomy & Cell Biology, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. Fax: (817) 735-2610. E-mail: [email protected].

View full text

Regular ArticleBcl-2 Overexpression Protects Photooxidative Stress-Induced Apoptosis of Photoreceptor Cells via NF-κB Preservation☆

Abstract

J. Biol. Chem.

Cell

Cell

Cell

Cell

Int. Rev. Cytol.

J. Biol. Chem.

J. Biol. Chem.

Brain Res.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Brain Res.

Free Radical Biol. Med.

Curr. Biol.

Death at an early age. Apoptosis in inherited retinal degenerations

Invest. Opthalmol. and Visual Sci.

Apoptosis leads to photoreceptor degeneration in inherited retinal dystrophy of RCS rats

Invest. Opthalmol. Visual Sci

Photic injury triggers apoptosis of photoreceptor cells

Invest. Opthalmol. Visual Sci.

Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells

Nature

Bcl-2/Bax: A rheostat that regulates an anti-oxidant pathway and cell death

Semin. Cancer Biol.

Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death

Cell

Analysis of the NF-kappa B p65 subunit, Fas antigen, Fas ligand and Bcl-2-related proteins in the synovium of RA and polyarticular JRA

Clin. Exp. Rheumatol.

Regular Article
Bcl-2 Overexpression Protects Photooxidative Stress-Induced Apoptosis of Photoreceptor Cells via NF-κB Preservation☆