Ó Springer 2006
Biogerontology (2006) 7:63–67 DOI 10.1007/s10522-005-6038-x
Research article
Organ- and tissue-specific alterations in the anti-apoptotic protein Bcl-2 in CD1 female mice of different ages Hugo Lo´pez-Araiza1, Jose´ L. Ventura2, Norma E. Lo´pez-Diazguerrero1, Humberto Gonza´lez-Marquez1, Marı´ a C. Gutie´rrez-Ruı´ z1, Alejandro Zentella2 & Mina Ko¨nigsberg1,* 1 Departamento. Ciencias de la Salud, DCBS, Universidad Auto´noma Metropolitana-Iztapalapa, 55-535, 09340, Mexico City, D.F., Me´xico; 2Departamento. Bioquı´mica, Instituto Nacional de Ciencias Me´dicas y Nutricio´n ‘‘Salvador Zubira´n’’ & Departamento. Medicina Geno´mica y Toxicologı´a Ambiental, IIB, UNAM, Me´xico, D.F., Me´xico; *Author for correspondence (e-mail:
[email protected]) Received 28 October 2005; accepted in revised form 1 December 2005
Key words: aging, Bcl-2, longevity, oxidative stress, senescence Abstract The anti-apoptotic protein Bcl-2, which also has cytoprotective and antioxidant functions might be one of the crucial factors that altogether, establish how a cell may deal with stress and damage, contributing to longevity. Among the controversial issues to understand Bcl-2 functions in vivo, is to establish its content and variation in tissues during an organismal lifespan. In this work we analyzed the changes of Bcl-2 levels in lung, liver, heart, kidney, spleen and brain homogenates obtained from CD1 mice throughout their lifespan (newborn to 24 months). A tendency of increment was observed in all the organs analyzed, except brain where Bcl-2 was not detected. Bcl-2 over-expression during aging could be interpreted as a protective mechanism preventing cell death, despite the overall accumulated cell damage.
Introduction Aging is a complex and inevitable process, in which a large number of factors may induce a progressive decline of the biological and physiological functions of an organism, that eventually leads to a gradual decrease in the reproductive rate and an increase in mortality (Martin et al. 1996; Kirkwood and Austad 2000). Mammalian cells can respond to cellular injury or stress in at least two different ways: by turning on a mechanism of programmed death, called apoptosis (Kerr et al. 1972) or by entering a state of arrested growth and altered function, termed cellular or replicative senescence (Campisi 2005). How and why do the cells enter one of these two states is still not clear, but now it is known that the pathways followed might share some regulatory molecules. One of these molecules is Bcl-2, originally identified as the product of a human
lymphoma oncogene (Tsujimoto et al. 1987), now recognized as a representative of a family of proteins that regulate cell death (Cory et al. 2003). Besides its anti-apoptotic function, Bcl-2 has also been accepted to play a cytoprotective and antioxidant role (Hockenbery, et al. 1993; Jang and Surh 2003) linked to a cell cycle regulatory activity (Borner 1996; Vairo et al. 2000; Kim 2005). All the above indicates that the amount of Bcl-2 in a cell or tissue and most likely of other members of the same family, might be one of the crucial factors that altogether, establish the way in which a cell may possibly deal with stress and damage, contributing to longevity. Bcl-2 content has been analyzed, both in vivo and in vitro, and contrasting results have been published. It has been reported that in human
64 mononuclear cells there is no change in Bcl-2 levels with age (Monti et al. 2000), while others have reported that Bcl-2 content in rat T cells and in rat heart decreases with age (Pahlavany and Vargas 2001; Phaneuf and Leeuwenurg 2002), and increases in mice lung (Konigsberg et al. 2004). These contradictory results could be explained on the basis of tissue specificity and differential aging rates between species. While monocytes and T cells are constantly replaced, cells from the lung and the heart have a slower rate of exchange. Yet, a closer analysis of the Bcl-2 content in different tissues during the life span of the same species is still required. A study like that will provide a global view that could help to understand why and how these age-related changes might occur. To our knowledge, this report is the first one that examines the content of a surviving protein, like Bcl-2, in six murine tissues with a slow rate of cell exchange throughout lifespan.
Materials and Methods Chemicals All chemicals and reagents were of the highest analytical grade and the majority was purchased from Sigma (St. Louis MO). The reagents obtained from other sources are detailed along the text. Animals CD-1 female mice from the closed colony of breeding at the Universidad Auto´noma Metropolitana-Iztapalapa (UAM-I) were used for this work. CD1 mice average life span in lab is 2 years (Lang 1995). Animals were sacrificed at different ages and their organs were frozen at )80 °C until processed. Mice were always caredfor according to the principles of the Mexican official standard 062-ZOO-1999. Bcl-2 content in tissue homogenates Bcl-2 was determined by Western blot independently in lung, liver, heart, spleen, kidney and brain homogenates at different ages. Organs were
obtained from newborn, 1, 2, 4, 6, 8, 12, 16 and 24 months mice, and homogenated in lysis buffer (50 mM Tris–HCl pH 8.0, 120 mM NaCl, 0.5% NP40, 100 mM NaF, 0.2 mM NaVO3, 10 lg/ml aprotinin, 5 mM PMSF, 10 lg/ml leupeptine). Homogenates were incubated at 4 °C for 5–10 min, and centrifuged at 14000 rpm at 4 °C for 20 min. The supernatant was collected for determination and Western blot. Protein concentration was determined using a commercial Bradford reagent (BioRad, Hercules CA, USA). Western blot analysis Tissue lysates were independently separated on 13% SDS-PAGE and transferred to a nitrocellulose membrane (BioRad, Hercules CA, USA). Membranes were blocked with TBS-Tween 0.1% containing 5% non-fat milk for 1 h, and incubated with a-Bcl-2 antibody (Neomarker, Fremont CA, USA) for 2 h. The membranes were washed three times with TBS-Tween and incubated with a horseradish peroxidase-conjugated a-mouse IgG secondary antibody (Pierce, Rockford IL, USA) for 1 h. Following three washes, the blots were developed using a commercial chemiluminiscent reagent (SupersignalÒ Pierce, Rockford IL, USA). Data analysis For Western blots, at least three experiments were performed using organs form different donor animals. Organs from up to six animals were pooled together for the younger ages (newborn to 4 months), and single organs were homogenized individually for the older ones. Results To determine Bcl-2 content during the CD1 mouse lifespan, Western blots of different tissue lysates were performed at various stages between newborn and 24 months of age. The cell line L929 previously transfected with pBabe–Puro–Bcl-2 (Go´mez and Zentella 1996) was used as a positive control of Bcl-2 presence. The fact that in the tissue lysates there is a combination of cell types made it impractical the use of a housekeeping protein as a control.
65 Figure 1 shows representative Western blots obtained with the six tissue lysates. Bcl-2 could not be detected in any tissues in newborns. Interestingly, the age of appearance differed among the organs. A tendency of increment was observed in all the organs analyzed, except brain. In liver, lung and spleen, the maximum Bcl-2 levels were found at 24 months of age, while the maximum in heart was at 16 and in kidney at 8. The case of the brain homogenates is very interesting, because although the positive control was always present, the Western blots for the homogenates of the animals at the different ages did not show any bands for Bcl-2. The results of densitometric analysis were normalized against the amount of Bcl-2 present at the earliest time point in life of each tissue. The average of the fold increase of at least three inde-
pendent experiments is presented in Table 1. In the lung the maximum increase in Bcl-2 content was almost 2.5 fold, very similar to the one obtained for the kidney (2.0 fold). While in spleen the augment was 4.0 fold and in liver and heart Bcl-2 increased 5.0 fold. Discussion There is increasing experimental evidence indicating that the cellular pathways related to survival, programmed cell death and senescence, share numerous molecules with more than one function (Balducci and Ershler 2005; Song et al. 2005). One of those multifaceted proteins is Bcl-2. This anti-apoptotic protein has cytoprotective and antioxidant functions that have been linked with a cell cycle regulatory activity (Vairo et al. 2000;
Figure 1. Western blot of tissue homogenates. The figure illustrates representative experiments obtained with tissue lysates as described in materials and methods. The results of densitometric analysis were normalized against the amount of Bcl-2 present at the earliest time point in life for each tissue.
66 Table 1. Fold increase average.
Lung Liver Heart Spleen Kidney
1
2
4
6
8
12
16
24
1.0 – – – –
1.09±0.05 – – 1.0 1.0
1.56±0.07 1.0 – 1.12±0.07 1.08±0.10
1.36±0.03 2.26±0.25 – 1.10±0.10 1.26±0.20
1.5±0.01 3.06±0.20 1.0 1.23±0.15 1.96±0.70
1.59±0.04 1.73±0.25 1.04±0.06 1.83±0.50 1.96±0.25
2.21±0.27 4.06±0.20 1.24±0.11 3.6±0.62 1.88±0.20
2.33±0.20 4.4±0.36 4.66±0.87 1.33±0.25 1.63±0.30
The results in the table correspond to the fold increase average of at least three Western blots. The results of densitometric analysis were normalized against the amount of Bcl-2 present at the earliest time point in life for each tissue.
Janumyan et al. 2003). However, most of the studies that have reported a Bcl-2 protective effect have been done overexpressing bcl-2. Therefore, an important issue to understand Bcl2 functions in vivo is to establish its content and variation in tissues during an organismal lifespan. In this work we have begun to approach this problem by analyzing the changes of Bcl-2 levels in six murine tissues with a slow rate of cell exchange throughout lifespan. Our results indicate that the maximum levels of Bcl-2 were found at 24 months of age in liver, lung and spleen while the maximum in heart was at 16 and in kidney at 8. Bcl-2 could not be detected in any tissues in newborns, and different ages of appearance were found. It is important to take into consideration that these data were obtained with whole organ homogenates, implicating that there are diverse cellular types contributing to Bcl-2 content, in a not necessarily equal proportion. The organs that maintain elevated levels of Bcl-2 during their lifespan are either constantly exposed to an oxygen tension, like the lung (Agusti and Rodriguez-Roisin 1993); or achieve a vast metabolic and energetic activity like the liver. In this work, we were not able to detect Bcl-2 in the brain homogenates, even though the positive control was always there. This finding suggests that in the CD1 mice brain there could also be other Bcl-2 family members that might be responsible for the survival functions. This idea is supported by the reports of different expression patterns of the Bcl-2 family during development (Yachnis et al. 1997; Hamner et al. 1999). It has also been suggested that Bcl-2 is present in brain only during neuronal development (Akhtar et al. 2004), and therefore other members of the family, like Bcl-xL, might be regulating cell survival during adult life (Parsadanian et al.
1998). More experiments have to be done in order to elucidate which one of them is playing the main role in the CD1 mice brain. One of the main contributions of this work is to support the idea that Bcl-2 related proteins might have different expression patterns related to tissue specificity that might differ between species. There is still limited and controversial information available for other tissue types, and nothing is known about why and how these agerelated changes occur. However, the finding that Bcl-2 levels rise with age in most of the tissues analyzed, is relevant because of the vast literature demonstrating that experimental over-expression of Bcl-2 confers protection against ROS. Bcl-2 over-expression during aging (16–24 months), can be interpreted as a protective mechanism preventing cell death, despite the overall accumulated cell damage. A physiological disadvantage of Bcl-2 over-expression would be the preservation of damaged cells that would have died otherwise and that might enter replicative senescence contributing to organ deterioration and aging (Patil et al. 2005). An analysis of the composition of the senescent cell population related to increased Bcl-2 expression is necessary to confirm this hypothesis. Our results indicate absence of Bcl-2 earlier in life, expression in the adult and over-expression at old age. The meaning of these observations remains unknown, and will have to wait for similar experiments in other species to interpret its significance.
Acknowledgements The authors would like to thank Dr. He´ctor Zayas and M. in Sc. Marı´ a del Carmen Escobar
67 for their technical advice. This work was supported by CONACyT’s grant No. 400200–5J34194-M. Norma E. Lo´pez-Dı´ azguerrero is a scholarship holder from CONACYT.
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