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From the * Departamento de Morfología y
Biología Celular, Facultad de Medicina. Universidad de Oviedo, Oviedo,
Spain; the Centro de Biología Molecular
"Severo Ochoa" CSIC/UAM Campus de la Universidad Autónoma
de Madrid, Cantoblanco, Madrid, Spain; and the
Servicio Regional de Investigacion y
Desarrollo Agroalimentario (SERIDA), Villaviciosa, Spain.
| Correspondence to: Dr Ana Coto-Montes, Departamento de Morfología y Biología Celular, Facultad de Medicina, C/Julián Clavería s/n, 33006 Oviedo, Spain (e-mail: acoto{at}uniovi.es). |
| Received for publication March 13, 2008; accepted for publication September 23, 2008. |
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Abstract |
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Key words: Beclin 1, cathepsin D, LC3
Programmed cell death (PCD) gives rise to 2 histological patterns as revealed by electron microscopy. Apoptosis, or type I PCD, is characterized by condensation of the cytoplasm and preservation of organelles, essentially with no autophagic degradation. In contrast, in autophagic cell death, or type II PCD, there is extensive autophagic degradation of the Golgi apparatus, polyribosomes, and endoplasmic reticulum preceding nuclear destruction (Bursch et al, 2000; Gozuacik and Kimchi, 2004). The mechanisms and molecules involved differ in the 2 types of cell death. The caspases, a family of cysteine proteases, play an essential role in inducing and carrying out apoptosis. When activated, caspases acquire the ability to cleave key intracellular substrates, giving rise to the biochemical and morphological changes associated with apoptosis (Roberg et al, 2002).
In the search for biochemical differences between the 2 PCD processes, recent investigations have suggested that the cytoskeleton has distinct fates during autophagic and apoptotic cell death. The preparatory process that apoptotic cells undergo includes depolymerization or cleavage of actin, cytokeratins, lamins, and other cytoskeletal proteins (Hengartner, 2000; Bursch, 2001). In contrast, in cells undergoing autophagic death, the cytoskeleton, although redistributed, is largely preserved (Bursch et al, 2000; Bursch, 2001). Furthermore, Kabeya et al (2000) have suggested that the microtubule-associated protein light chain 3 (LC3) is an ideal marker for the autophagic process. LC3 is converted into LC3-I, and part of this modified protein is subsequently converted into LC3-II, which is localized in autophagosomal membranes. The conversion of LC3-I to LC3-II is induced under starvation conditions, which are well known to induce autophagy.
Beclin 1 is a 60-kDa protein that has been implicated as an important regulator of macroautophagy. It was originally discovered during the course of a yeast 2-hybrid screen of a mouse brain cDNA library using human Bcl-2 as the bait (Liang et al, 1998). Coimmunoprecipitation studies suggest that the major physiological partner for Beclin 1 is the mammalian class III phosphatidylinositol 3-kinase Vps34 (Zeng et al, 2006). Expression of Beclin 1 in MCF7 mammary carcinoma cells increases their autophagic response to nutrient deprivation (Liang et al, 1999). Consistent with this observation, several studies have implicated Beclin as an essential gene for cell survival under adverse nutritional conditions. In addition to its specific role in adaptation to nutrient deprivation, accumulating evidence suggests that Beclin may play a more general role in cell survival during embryonic development.
Isahara and collaborators (1999) have described a pathway for cell death in which cathepsin D has a relevant role. These authors postulate that cathepsin D acts as a death factor in this pathway. This enzyme translocates from lysosomal compartments to the cytosol, and seems to be related to PCD (Ishisaka et al, 2001). Cathepsin D, the main lysosomal aspartic protease, is synthesized and translocated into the endoplasmic reticulum as a 52-kDa inactive proenzyme (preprocathepsin D). It is converted in the endosomal compartment into a slightly active 48-kDa single-chain form (procathepsin D; Demoz et al, 2006). In humans and most other species, this single-chain form undergoes further proteolytic processing in late endosomes and lysosomes, yielding the mature form. Mature cathepsin D is composed of a light (14 kDa) chain and a heavy (32 kDa) chain held together by disulfide bonds (Diment et al, 1989). However, extensive vacuolization resulting from autophagy interferes with trafficking of this enzyme through the endosome, increasing procathepsin D levels (Zeng et al, 2006). Therefore, indirect measures of autophagy may show a higher ratio of procathepsin D (52 and/or 46 kDa) to mature cathepsin D (33 kDa).
An evaluation of selected biomarkers of apoptosis (eg, caspase-3 activity) and autophagy (eg, Beclin 1 and LC3-II expressions), as well as ultrastructural studies, were performed in the present study to demonstrate the hypothesized primary role of autophagy in androgen-dependent PCD, as observed in the flank organs of male and female Syrian hamsters. The data collected in this study are important because they allow us to show that autophagy can act as an androgen-dependent mechanism, and this role is supported by the fact that autophagy acts differently in female vs male flank organs. This information can be used for improving knowledge about hair growth mechanisms, because it is known that the flank organ responds to androgen and antiandrogen treatments (Chen et al, 1996).
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Materials and Methods |
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The Oviedo University Local Animal Care and Use Committee approved the experimental protocol. All experiments were carried out according to the Spanish Government Guide and the European Community Guide for Animal Care.
The animals were anesthetized and decapitated and their flank organs rapidly procured. For the biochemical studies, the flank organs were homogenized in a Polytron homogenizer at 4°C in 1 mL of lysis buffer (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 5 mM dithiothreitol) supplemented with protease inhibitors (1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/mL aprotinin). Tissue homogenates were then centrifuged at 3000 x g for 6 minutes at 4°C. Supernatants were collected and centrifuged again under the same conditions. The protein contents of the supernatants were determined by the Bradford (1976) method.
Caspase-3 Activity
Caspase-3 activity was determined using a colorimetric assay according to
the manufacturer's instructions (CASP-3-C; Sigma, St Quentin-Fallavier,
France). In this assay system, the colorimetric substrate
(acetyl-Asp-Glu-Val-Asp-p-nitroanilide) is hydrolyzed by caspase-3, and
p-nitroaniline (pNA) is released from the substrate. To account for
nonspecific hydrolysis of the substrate, the control reaction mixture
contained tissue homogenates, substrate, and the specific caspase-3 inhibitor
Ac-DEVD-CHO in assay buffer. Both mixtures were monitored for 5 hours at
37°C by a plate reader (ELx800 UV; Bio-Tek Instruments, Winooski,
Vermont). Data obtained using the caspase-3 inhibitor were subtracted from
data obtained without the caspase-3 inhibitor to correct for nonspecific
hydrolysis. Blanks were incorporated and caspase-3 protein was used as a
positive control. Data were normalized by protein content of each sample. The
caspase-3 activity was expressed as nmol of pNA released per minute per
milligram of protein (Tomas-Zapico et al,
2005).
Cathepsin D Activity
The aspartate-protease cathepsin D (EC 3.4.23.5) was assayed
spectrophotometrically (Uvikon 930; Kontron Instruments, Madrid, Spain) at 280
nm according to the method described by Takahashi and Tang
(1981) with minor
modifications (Schreurs et al,
1995), using hemoglobin as the substrate. Two hundred microliters
of tissue homogenate (see above) was pipetted with 500 µL of substrate
solution (3% hemoglobin in 200 mM acetic acid) in microcentrifuge tubes,
followed by incubation for 30 minutes at 37°C. The reaction was stopped by
adding 500 µL of 15% trichloroacetic acid, and the tubes were kept at
4°C for 30 minutes and then centrifuged at 12 000 x g for 5
minutes. The optical densities of the supernatants were used to calculate
cathepsin levels, expressed as enzyme units per milligram of protein.
Detection of Autophagic Markers by Western Blotting
Tissue samples were fractionated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis and transferred to polyvinylidene difluoride membranes.
The membranes were blocked with 5% skimmed milk in phosphate-buffered saline
containing 0.05% Tween-20 solution and incubated with primary antibodies
against anti-mouse cytokeratins (Sigma), anti-goat LC3 (sc-16756, Santa Cruz,
Santa Cruz, California), anti-goat Cathepsin D (sc-6486, Santa Cruz),
anti-goat Beclin 1 (sc-10086, Santa Cruz), and β-actin (sc-1615, Santa
Cruz), and with the corresponding horseradish peroxidase–conjugated
secondary antibodies (Sigma and Santa Cruz, for cytokeratin, LC-3, Beclin1,
and β-actin). The membranes were developed using Western Blotting Luminol
Reagent (Santa Cruz).
Digital images were analyzed with a Chemidoc XRS system (Biorad, Hercules, California), which permits quantification of band intensity. Routinely, protein load was monitored by the Bradford method and phenol red staining of the blot membrane.
Ultrastructural Studies
Flank organs for morphological studies were lightly fixed by immersion in a
solution containing 1.5% glutaraldehyde and 2.5% paraformaldehyde in phosphate
buffer 0.1 M (pH 7.4). Fixation was prolonged overnight using fresh fixative
at 4°C. The tissue was then postfixed in 1% OsO4 for 2 hours.
After dehydration in a graded acetone series, the tissue fragments were
embedded in Taab 812 (TAAB Laboratories Equipment Ltd, Aldermaston, United
Kingdom) and semithin sections (1 µm) were stained with toluidine blue.
Ultrathin sections were collected on cooper grids, stained with uranyl
acetate–lead citrate, and examined using a Zeiss EM-109 transmission
electron microscope (Zeiss, Oberkochen, Germany) operating at 80 kV. Negatives
were scanned by HP Scanjet 3970 (Palo Alto, California) and imported by Adobe
Photoshop 7.0.1 (San Jose, California), and the images were incorporated into
the figures using Corel Draw X3 (Ottawa, Canada).
Statistical Analysis
Data are presented as 
± SD
calculated for at least 3 separate experiments, each performed in triplicate.
The normality of the data was established by the Kolmogorov-Smirnov test.
Statistical comparisons between sexes were performed using Student's
t test for data showing a normal distribution and the nonparametric
Mann-Whitney test for nonnormally distributed data. The level of significance
was set at P < .05.
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Results |
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Ultrastructural Studies— Electron microscopy revealed that the studied cells of the flank organ in both sexes of Syrian hamsters do not undergo apoptosis because we were not able to observe, in any case, apoptotic nuclei in cells from flank organs of both sexes. This is a qualitative study, so the presence of undetected apoptotic nuclei could be possible.
Study of Autophagy
Cathepsin D Activity—
Cathepsin D activity was assayed using its corresponding substrate in flank
organs procured from male and female Syrian hamsters. The results showed
statistical differences (P 
.05) between male and female flank
organs in relation to cathepsin D activity, because this protease activity was
always 4 times higher in males than in females
(Figure 1).
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Detection of Autophagic Markers by Western Blotting— Western blot analysis of flank organ extracts was performed using antibodies against cytokeratins, LC3, cathepsin D, Beclin 1, and β-actin.
The cytokeratin pattern was analyzed in untreated male and female flank organs. The study was developed using a wide range of anticytokeratin antibodies that recognize various cytokeratins, including the type II neutral-to-basic cytokeratin subfamily and the type I acidic subfamily. Male and female Syrian hamsters displayed a similar cytokeratin pattern (Figure 2A). The main cytokeratin detected was cytokeratin 8 (CK8). Additional bands were detected below the expected molecular weight for cytokeratins (33 and 22 kDa). In all cases, female hamsters expressed higher amounts of these proteins than males. Quantification of Western blot bands supported these data. Immunoblot analysis for β-actin demonstrated that there was no degradation of this protein, which is characteristic of autophagy (Figure 2B).
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We also carried out an immunoblot analysis for Beclin 1. Both male and female flank organs showed a band of 60 kDa corresponding to Beclin 1. This band was clear in females but only slightly visible in males (Figure 3). Quantification of Western blot bands supports these data.
Ultrastructural Studies— Through electron microscopy, it was revealed that the cells of the flank organ in both sexes of Syrian hamsters undergo autophagy. Flank organ cells in males had a cytoplasm filled with ribosomes and rough endoplasmic reticulum surrounding abundant mitochondria with clear cristae. Along the cytoplasm, there were prominent Golgi complexes with several autophagosomes that contained cytoplasmic components with indistinguishable structures. In the same cell, individual membrane-bound autolysosomes could be observed (Figure 4). Hence, some cells showed diminished electrodensity as a result of the degradation of cytoplasmic components, which is a hallmark of autophagic cell death (Figure 5). Moreover, when both the nuclear envelope and the plasma membrane lost their integrity, the cells exhibited intense vacuolization of the cytoplasm (Figure 6).
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Discussion |
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Caspases, a family of cysteine proteases, have been described to act as initiators of apoptosis, playing a central role in regulating this mechanism of PCD (Kitanaka and Kuchino, 1999). However, we observed that caspases were inactive in tissue from both sexes in the absence of external inhibition of caspase activities. Moreover, recent data suggest that different pathways of caspase-independent PCD can be activated, resulting mainly in a nonapoptotic form of cell death (Kitanaka and Kuchino, 1999; Bursch et al, 2000). The data shown in this article support the possibility that, in flank organs, a different type of PCD, such as autophagy, occurs. Our data do not exclude the possibility that autophagy and other kinds of apoptotic processes not mediated by caspases coexist (Joza et al, 2001).
Autophagy is a pivotal physiological process for survival during starvation, differentiation, and normal growth control, and may play a number of roles in other cellular functions via the turnover of cellular macromolecules and organelles (Kabeya et al, 2000). Autophagic vacuoles (autophagosomes) are initially formed from membranes of the endoplasmic reticulum that surround a region of cytoplasm. These structures, bounded by a double membrane, then develop into mature degradative vacuoles (autolysosomes) by progressive fusion with late endosomes and lysosomes. Accumulation of autophagosomes and autolysosomes is a morphological feature that is a marker of type II PCD, also referred to as autophagic cell death (Zeng et al, 2006).
LC3-II and Beclin 1 are classically described as autophagy markers (Zhu et al, 2007). Based on the results of this study, male flank organs show much lower levels of autophagy than do female organs, because they show reduced expression of Beclin 1 and an absence of LC3-II. However, using electron microscopy, we observed signs of extensive autophagy, including abundant autophagosomes derived from the rough endoplasmic reticulum. We saw normal nuclei in flank organ cells of both sexes. Because other parameters have pointed out clear sexual differences in autophagic processes of the flank organ, with higher development in females vs males, the combination of both biochemical and morphological data leads us to think that autophagy is more widespread in female flank organs than in male ones. This is in terms of affected area, and not in relation to the autophagic flux, which shows the same morphological characteristics in the flank organs of both sexes.
The autophagy process depends, to a large extent, on cytoskeletal proteins. The sequestration of parts of cytoplasm requires intermediate filaments (cytokeratin and vimentin), and the movement and fusion of lysosomes with the late autophagosomes requires the microtubular system. So, autophagic death needs a preserved cytoskeleton, although the cytoskeleton could be redistributed (Bursch et al, 2000; Bursch, 2001). Thus, we have also studied the cytokeratin pattern in the flank organ, as described by Bursch et al (2000), who indicated that the fate of the cytoskeleton is different between apoptosis and autophagy.
Cytoskeletal proteins appear to be preserved, at least in part, during autophagic-driven cell death. This pattern of change is clearly different from that ascribed to apoptosis (Bursch, 2001). However, all experiments focusing on preservation of the cytoskeleton in autophagy have been performed in vitro using specific PCD inductors, such as tamoxifen (Bursch et al, 2000). This type of study is difficult to perform in in vivo conditions because of a lack of controls. According to previous results, we obtained, in our model lacking of any form of induction, the same cytokeratin pattern in both sexes, although it was more intense in female organs than in male ones. Our Western blots indicate that most cytokeratins remained undegraded, such as the simple epithelial CK8, the most expressed cytokeratin. Moreover, the observed 23-kDa band was present only in females, supporting the hypothesis that autophagy takes up a bigger area of flank organs in females than in males.
Because our experiments were conducted using in vivo conditions, we cannot compare our results to a baseline control (at time 0) as with cell cultures. Nevertheless, the cytokeratins determined here confirm previous findings wherein CK8 appeared in autophagy. This cytokeratin has been observed in MCF-7 cells incubated with tamoxifen (Bursch et al, 2000) and more recently in the Syrian hamster Harderian gland (Tomas-Zapico et al, 2005). Our Western blots of total actin revealed no signs of proteolytic cleavage, which is also consistent with the data reported by Bursch et al (2000).
Cathepsin D is a lysosomal aspartic proteinase, initially synthesized as an inactive precursor, procathepsin D, which upon arrival at the lysosome is proteolytically cleaved to yield the mature active enzyme (Neurath, 1991, 1994). Beclin 1, as part of 2 distinct protein complexes that contain the PI 3-kinase Vps34 (Kihara et al, 2001), causes vacuolation of late endosomal compartments, impeding the traffic of procathepsin D from late endosomes to lysosomes (Zeng et al, 2006) and, therefore, impedes lysosome development. The main proteolytic activation event is presumed to depend on the action of lysosomal cysteine proteinases. In agreement with preceding hypotheses and previous biochemical data, our results show that female cathepsin D activity is low in comparison to male cathepsin activity, that cathepsin D is present in males, and that there is higher expression of procathepsin D in both sexes. Therefore, the presence of either cathepsin D or procathepsin D could be used as indirect evidence of autophagy, and, once more, autophagic processes in flank organs are stronger in females than in males.
The flank organ contains highly developed clusters of sebaceous glands immersed in both sides of the back skin. The skin possesses all the enzymes required for transformation of the steroid precursors of adrenal origin into active androgens and estrogens, and these enzymes are expressed at a particularly high level in sebaceous glands as in the flank organ (Sansone-Bazzano and Reisner, 1974). Moreover, since 1972, it has been known that the flank organ contains a considerable amount of dihydrotestosterone compared with the prostate and seminal vesicle (Takayasu and Adachi, 1972). Likewise, the hamster flank organ is a scent gland that produces a high amount of pheromones that allow animals to distinguish males from females, and to identify the reproductive status of both sexes and whether an animal will be aggressive or submissive (Johnston and Rasmussen, 1984). The flank organ can also be considered a steroid-secreting organ because of its combination of sexual steroid transformation and steroidal pheromone production. This information is very important in relation to autophagy because this kind of PCD takes place in normal steroid-secreting cells at higher frequencies than in many other cells, probably as a mechanism to adapt to or even moderate the varying steroid-secreting level by changing the intensity of autophagic activity (Yi and Tang, 1995). In light of these findings, the autophagic processes that we have observed in the flank organ are very understandable. The preference, or even specificity, for destroying some of the mitochondria and smooth endoplasmic reticulum would be responsible for the turnover of steroid-producing organelles, and may consequently result in an influence upon the rate of steroid secretion. Likewise, our group has demonstrated autophagic processes in the Harderian gland, which is also present in Syrian hamsters and has a lot of similarities to the flank organ. This is also an organ that is a pheromone producer and sexual steroid transformer (Tomas-Zapico et al, 2005). This similarity between 2 organs that develop their functions in relation to sexual hormones seems to support the hypothesis that autophagy could be a common mechanism of detoxifying these kinds of organs.
However, sexual differences between female and male flank organs were unexpected. The decrease in flank organ size could be caused by orchidectomy, partially reversed by dehydroepiandrosterone and completely reversed by 4-enedione, testosterone, and dihydrotestosterone implants (Chen et al, 1996). Androgen hormones seem to play an important role in flank organ formation and structural maintenance. Therefore, this article shows that flank organs in Syrian hamsters develop autophagy, as PCD, at different degrees dependent on sex. Because the flank organ is an androgen-dependent organ, this is the first time that variations in androgen levels have been shown to be the most important factor for the development of autophagy, and this information could be very useful for gaining knowledge into the control mechanisms for hair growth.
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Acknowledgments |
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Footnotes |
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Supported in part by grants FISS-06-RD06/0013/0011 from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) and FEDER fund (European Union).
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, male
flank organ; 
, female flank organ. Values are
