The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells

Wang, Zhao Fang, Wang Qinghua

Description

Excessive loss of pancreatic β-cells, mainly through apoptosis, contributes to the development of diabetic hyperglycemia. Oxidative stress plays a major role in the process of β-cell apoptosis due to low expression level of endogenous antioxidants in the β-cells. Peroxiredoxins (PRDX) are a family of peroxide reductases which uses thioredoxin to clear peroxides. Several members of PRDX have been found in β-cells and recent studies suggested that these antioxidant enzymes possess protective effects in β-cells against oxidative stress mediated apoptosis. In this study, we aimed to investigate the role of PRDX2 in modulating β-cell functions. We detected the expression of PRDX2 both at the transcript and protein levels in the clonal β-cells INS-1 and MIN6 as well as rodent islets. Western blot showed that treatment of MIN6 β-cell line with proinflammatory cytokines, palmitic acid or streptozotocin dose- or time-dependently increased apoptosis, which was associated with reduced endogenous expression levels of PRDX2. To examine the role for PRDX2 in the apoptotic stimuli-induced β-cell apoptosis, we used plasmid overexpression and siRNA knockdown strategies to investigate whether the elevation or knockdown of PRDX2 affects stimuli-induced apoptosis in the β-cells. Remarkably, overexpression of PRDX2 in MIN6 cells significantly attenuated the oxidative stresses mediated apoptosis, as evaluated by cleaved caspase 3 expression, nuclear condensation and fragmentation, as well as FACS analysis. Conversely, attenuation of PRDX2 protein expression using siRNA knockdown exaggerated the cell death induced by proinflammatory cytokines and palmitic acid in the MIN6 cells. These results suggest that PRDX2 may play a protective role in pancreatic β-cells under oxidative stress.

Sujets

• Apoptosis
• Peroxiredoxin 2
• Oxidative stress

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Zhao and Wang Cell & Bioscience 2012, 2:22
http://www.cellandbioscience.com/content/2/1/22
Cell & Bioscience
RESEARCH Open Access
The protective effect of peroxiredoxin II on
oxidative stress induced apoptosis in pancreatic
β-cells
1,2 1,2*Fang Zhao and Qinghua Wang
Abstract
Excessive loss of pancreatic β-cells, mainly through apoptosis, contributes to the development of diabetic
hyperglycemia. Oxidative stress plays a major role in the process of β-cell apoptosis due to low expression level of
endogenous antioxidants in the β-cells. Peroxiredoxins (PRDX) are a family of peroxide reductases which uses
thioredoxin to clear peroxides. Several members of PRDX have been found in β-cells and recent studies suggested
that these antioxidant enzymes possess protective effects in β-cells against oxidative stress mediated apoptosis. In
this study, we aimed to investigate the role of PRDX2 in modulating β-cell functions. We detected the expression of
PRDX2 both at the transcript and protein levels in the clonal β-cells INS-1 and MIN6 as well as rodent islets.
Western blot showed that treatment of MIN6 β-cell line with proinflammatory cytokines, palmitic acid or
streptozotocin dose- or time-dependently increased apoptosis, which was associated with reduced endogenous
expression levels of PRDX2. To examine the role for PRDX2 in the apoptotic stimuli-induced β-cell apoptosis, we
used plasmid overexpression and siRNA knockdown strategies to investigate whether the elevation or knockdown
of PRDX2 affects stimuli-induced apoptosis in the β-cells. Remarkably, overexpression of PRDX2 in MIN6 cells
significantly attenuated the oxidative stresses mediated apoptosis, as evaluated by cleaved caspase 3 expression,
nuclear condensation and fragmentation, as well as FACS analysis. Conversely, attenuation of PRDX2 protein
expression using siRNA knockdown exaggerated the cell death induced by proinflammatory cytokines and palmitic
acid in the MIN6 cells. These results suggest that PRDX2 may play a protective role in pancreatic β-cells under
oxidative stress.
Keywords: β-cells, Apoptosis, Peroxiredoxin 2, Oxidative stress
Introduction Particularly the β-cells are more susceptible to oxidative
Excessive loss of pancreatic β-cell mass, mainly due to stress due to the fact that they express major antioxi-
apoptosis, is a major cause in the development of dia- dants such as superoxide dismutase, catalase and gluta-
betic hyperglycemia in both type 1 and type 2 diabetes thione peroxidase at low levels [1,8,9]. In the pancreatic
mellitus [1]. β-cell apoptosis is initiated by a variety of islets, superoxide dismutase expression is 30-40% com-
stimuli such as inflammatory cytokines, chronic hyper- pared with that of the liver, glutathione peroxidase ex-
glycemia and hyperlipidemia [2,3] and downstream pression is 15%, and catalase expression cannot be
effects such as endoplasmic reticulum stress [4] and detected [10].
mitochondrial dysfunction [5]. Oxidative stress plays a At the cellular level, oxidative stress-mediated β-cell
permissive role in the process of apoptosis leading to cell apoptosis can result from an imbalance between reactive
destruction in many types of cell lineages [6,7]. oxygen species (ROS) generation and its clearance by
antioxidants [9]. It has been demonstrated that proin-
* Correspondence: [e-mail] flammatory cytokines induced β-cell apoptosis is
1
Division of Endocrinology and Metabolism, the Keenan Research Centre in
mediated through elevation of ROS in the mitochondria
the Li Ka Shing Knowledge Institute, St. Michael's Hospital, 209 Victoria
via altered electron transport chain action [11], andStreet, Room 414, Toronto, ON, Canada M5B 1T8
2
Department of Physiology, University of Toronto, Toronto, ON, Canada increased nitric oxide (NO) production via activation of
© Zhao and Wang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.Zhao and Wang Cell & Bioscience 2012, 2:22 Page 2 of 9
http://www.cellandbioscience.com/content/2/1/22
inducible nitric oxide synthase (iNOS) [12]. The process Oxidative stress induced apoptosis and decreased PRDX2
is known to be involved with activation of the nuclear expression in β-cells
factor-κB (NF-κB) and the c-Jun N-terminal kinase To examine the PRDX2 expression during the process of
(JNK/SAPK) or the FAS-FAS ligand pathways [13]. In- oxidative stress-mediated apoptosis in the β-cells, MIN6
duction of ROS is found to be multilateral. Long chain cells were treated with or without the oxidative stress
saturated non-esterized fatty acids (NEFA) such as pal- agents PA, cytokines or STZ at indicated concentrations
mitic acid (PA) induces ROS production in the mito- and for the indicated times. Cell lysates were subjected
chondria through the electron transport chain [11,14]. to Western blot analysis using relevant antibodies. As
The long chain saturated NEFAs could also directly shown, incubation of MIN6 cells with tested oxidative
interact with respiratory chain proteins and increase the stress inducers resulted in significant apoptosis as deter-
oxygen radicals [15]. Streptozotocin (STZ) is a toxic mined by increased cleaved form of caspase-3 levels,
agent that causes β-cell death via DNA alkylation caus- which was associated with decreased levels of PRDX2
ing strand breaks and induction of apoptosis [16], and expression (Figure 2A-C). Densitometry analysis of the
local injection of STZ can produce oxidative stress in Western blots showed that the reduction of PRDX2
situ causing tissue or organ dysfunction [17]. Previous levels in the β-cells treated with various oxidative stress
studies suggested that STZ can increase production of agents were statistically significant (Figure 2A’-2 C’,
oxygen radicals [18], and induction H O and DNA frag- *p<0.05, n=3). These results indicate that endogenous2 2
mentation [19] in the pancreatic β-cells [16,20]. PRDX2 expression level is reduced upon oxidative stress
Peroxiredoxins (PRDX) are a family of antioxidant in the pancreatic β-cells.
enzymes which is capable of metabolizing hydrogen
peroxide [21]. PRDXs are thioredoxin-specific antioxi- Elevation of PRDX2 expression protects against oxidative
dants first identified in yeast and are found in archea, stress-induced apoptosis in the β-cells
prokaryotes as well as eukaryotes [22]. To date, six To determine the role of PRDX2 in the process of oxida-
members of PRDXs have been found to be expressed tive stress-induced β-cell apoptosis, we investigated
in mammalian cells, as well as in the pancreatic β- whether overexpression of PRDX2 would protect against
cells [23]. the apoptosis induced by oxidative stress stimulating
Previous studies have suggested that PRDX2 can regu- agents. Western blotting showed that elevation of
late many cellular functions such as cell proliferation PRDX2 by transfection displayed a significant reduction
and differentiation [24,25]. Through the clearance of in apoptosis induced by either PA, or the cytokines or
H O PRDX2 also play critical role in the modulation of STZ in the transfected MIN6 cells, as evaluated by the2 2,
cell survival [26]. A recent study demonstrated that at- levels of cleaved caspase-3 (Figure 3A, *p<0.05, n=3).
tenuation of PRDX2 inhibited proliferation and induced Apoptotic assays were conducted to examine the
apoptosis in granulosa cells. This was achieved through apoptosis at the cellular level using DAPI nuclear stain-
the modulation of the NF-κB/iNOS pathway [27]. In pri- ing. We found that the three oxidative stress agents pro-
mary cortical neurons, overexpression of PRDX2 pro- duced profound apoptosis as determined by condensed
tected against apoptosis through the suppression of the or fragmented nuclei in the mock transfected MIN6
apoptotic ASK-1 signalling pathway [28,29]. PRDX2 is cells, which was significantly reduced in the cells overex-
found to be relatively highly expressed in the pancreatic pressing PRDX2 (Figure 3B, *p<0.05, n=3). Consist-
islet, i.e. with up to 3 fold higher compared with the liver ently, FACS using propidium iodide (Figure 3C) and
[30]. However, the biological functions of PRDX2 in the Annexin V FITC (Figure 3D) also demonstrated that the
pancreatic β-cells are not known. In this study, we inves- oxidative stress induced β-cell apoptosis was remarkably
tigated PRDX2 expression and its role in modulating reduced in the cells overexpressing PRDX2 (*p<0.05,
β-cell survival and death in the mouse β-cell line MIN6. n=3-4).
These results suggest that elevation of PRDX2 protein
expression can attenuate the apoptosis induced by PA,
Results or the cytokines or STZ in MIN6 cell line, which is sug-
Expression of PRDX2 in pancreatic β-cells gestive of the protective effects of PRDX2 in the β-cells.
It has been previously reported that PRDX2 is
expressed in variety of cells and tissues [31]. To de- PRDX2 knockdown exaggerated oxidative stress-induced
termine whether PRDX2 is expressed in pancreatic β- apoptosis in the β-cells
cells, we performed RT-PCR and Western blot ana- We further conducted knockdown studies using siRNA
lysis. As shown, both PRDX2 transcripts and proteins method to verify the protective role of PRDX2 in