TY - JOUR
AU - Thompson, Winston, E.
AB - Prohibitin (Phb1) is a highly conserved mitochondrial protein that is associated with granulosa cell differentiation, atresia, and luteolysis. Although prohibitin has been implicated in the suppression of apoptosis in mammalian cells, its specific role in programmed cell death in granulosa cells is unknown. In the present study, we examined the role of prohibitin in mediating staurosporine (STS) and serum withdrawal induced apoptosis in undifferentiated rat granulosa cells. Treatment of granulosa cells isolated from immature rat ovaries with STS and/or serum withdrawal induced a rapid decrease in the transmembrane potential of mitochondria, resulting in increased prohibitin content and induced apoptosis in a time- and dose-dependent manner. Infection of granulosa cells with a Phb1 adenoviral construct resulted in overexpression of prohibitin that markedly attenuated the ability of STS and serum withdrawal to induce apoptosis via the intrinsic apoptotic pathway. To determine the site of action of Phb1, granulosa cells were transfected with a prohibitin-eGFP fusion construct, and the fusion protein expression patterns were analyzed by fluorescence microscopy and Western blot analysis of cell fractionated samples. These studies indicated that the prohibitin-eGFP fusion protein moved from the cytoplasm into the mitochondria. However, no prohibitin-eGFP fusion protein was observed in the nucleus in response to the STS-induced apoptotic stimulus. This result was corroborated by Western blot analysis with green fluorescent protein-specific antibody. Furthermore, the prohibitin-eGFP fusion protein also inhibited programmed cell death. These results provide evidence that prohibitin could serve an antiapoptotic role in undifferentiated granulosa cells. OVARIAN GRANULOSA CELLS play an important physiological role in supporting the development and selection of the ovarian follicle by controlling oocyte maturation and by producing the steroid hormones, estradiol and progesterone, that are critical for maintenance of the ovarian cycle (1–5). For these reasons, analyses of the molecular events occurring during granulosa cell development are pivotal to our understanding of how these cells contribute to the modulation of processes critical for oocyte development. In humans, the prohibitin gene PHB1 is located on chromosome 17q21 close to the ovarian and breast carcinoma susceptibility gene (BRCA1) locus (6, 7). Prohibitins are a highly conserved family of proteins that are thought to play roles in cell cycle control (8–13), differentiation (14–18), senescence (19–22), and antiproliferative activity (8, 23–26). The rat and mouse prohibitin (Phb1) sequences are identical and differ from the human sequence by a single amino acid (9). A growing body of evidence has implicated a role for Phb1 in mitochondrial structure, function, and inheritance (15, 16, 27–33). Both rat and human Phb1 possess a short transmembrane helix near their N termini that may be integrated into the mitochondrial membranes. Previous studies in our laboratory (14) identified and characterized Phb1 in rat granulosa cells isolated from preantral and early antral follicles. We have shown that Phb1 expression in ovarian tissue is regulated in an age- and stage-specific manner. Furthermore, Phb1 is predominantly localized to the inner mitochondrial membrane of rat granulosa cells (15, 16). More recently, we demonstrated that the expression levels of the 30-kDa prohibitin protein (Phb1) increased during gonadotropin-induced granulosa cell differentiation. Additionally, Phb1 levels also increased during activated apoptosis in rat granulosa cells (16, 18). These studies suggested that Phb1 may play a critical regulatory role in granulosa development. We hypothesize that prohibitin acts as a suppressor of granulosa cell apoptosis. To test this hypothesis, a recombinant adenovirus vector was used to overexpress the 30-kDa prohibitin protein in rat granulosa cells stimulated to undergo apoptosis by staurosporine (STS) and serum withdrawal. In this study, we report that incubation of immature rat granulosa cells with STS as well as serum withdrawal activates apoptosis via the intrinsic pathway. Furthermore, overexpression of Phb1 delayed the onset of apoptosis by inhibiting the release of cytochrome c, caspase-3 cleavage, translocation of phosphatidylserine to the outer surface of the cell membrane, and mitochondrial depolarization. Materials and Methods Granulosa cell cultures Primary granulosa cells were isolated from immature (23 d old) Sprague Dawley rat (Charles River Laboratories Inc., Wilmington, MA) ovaries as previously described (14). In brief, ovaries were cleared from the surrounding fat, placed in ice-cold McCoy’s medium, and punctured with 27-gauge needles. Ovaries were then incubated in nonenzymatic Cell Dissociation Solution (Sigma, St. Louis, MO) at 37 C for 10 min, followed by 5 min incubation at room temperature in hypertonic sucrose in McCoy’s medium. The granulosa cells were harvested by gently pressing the ovaries against the wall of a polypropylene test tube with a Telflon pestle in ice-cold McCoy’s medium with 1% BSA and filtered through 50-μm nylon mesh. Cells were pelleted by centrifugation at 4 C, resuspended in 4F medium [15 mm HEPES, pH 7.4, DMEM/F-12 with transferrin (10 μg/ml), insulin (2 μg/ml), hydrocortisone (40 ng/ml), low density lipoprotein (10 μg/ml), high density lipoprotein (30 μg/ml), and gentamicin (10 μg/ml)], and counted to determine viability by the trypan blue exclusion technique (14). Before plating granulosa cells (5 × 105 per well in six-well plates), the culture dishes were coated with 20 μg Cell Attachment Matrix (Enactin-Collagen IV-Laminin; UBI, Lake Placid, NY) for 2 h at 37 C and washed with DMEM-F12. All animal care handling procedures in the present study were approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health and the U.S. Department of Agriculture. Induction of apoptosis Culturing granulosa cells in serum-free medium in the presence of STS resulted in the induction of apoptosis (34). For STS dose- and time-response studies, cells were initially cultured for 24 h in 4F medium and subsequently treated for 3 h in the presence of different concentrations of STS (0.5, 1, 2, and 4 μm) at different times (0.3, 1, 2, and 3 h) in 4F medium. Controls were similarly treated in the absence of STS. Total cells (attached and detached) were isolated after STS treatment, and the percentage of apoptosis was determined by nuclear staining with Hoechst 33248 stain (12.5 ng/ml; Sigma) as described by Sasaki et al. (35) and caspase-3 activity assay as described below. The effect of STS (1 μm) on nuclear morphology of granulosa cells during apoptosis was assessed by evaluating chromatin condensation and fragmentation into apoptotic bodies. For serum withdrawal studies, granulosa cells were initially cultured in 4F medium containing 10% fetal bovine serum (FBS) for 24 h. Subsequently, medium was changed to DMEM-F12 without fetal calf serum and apoptosis was determined at 0, 3, 6, 12, and 24 h after serum withdrawal. Apoptosis was assessed using the annexin V Vybrant apoptosis assay kit according to the manufacturer’s instructions (Molecular Probes, Inc., Eugene, OR) and caspase-3 activity assay as described below. Total cells (attached and detached) were labeled with annexin V, and positive cells were counted for each field examined (at least 10 fields per plate). The percentage of annexin V-positive cells was calculated as the number of annexin V-positive cells divided by the total number of cells. The assay was repeated on three granulosa cell preparations. The data were plotted as percent apoptotic cells against time in hours. Caspase enzymatic activity Caspase-3 activity was measured using a colorimetric assay kit (CaspACE-colorimetric; Promega, Madison, WI). After apoptotic induction of granulosa cells with 1 μm STS or serum withdrawal, cells were pelleted by centrifugation at 200 × g for 10 min at 4 C, washed with ice cold PBS, and resuspended in the CaspACE cell lysis buffer (5.0 × 105 cells/ml). The cell lysates were incubated on ice for 15 min and centrifuged at 15,000 × g at 4 C for 20 min. The supernatant was collected and stored at −80 C. The protein concentration was measured in the resulting supernatant using Bradford protein assay (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA) with a BSA reference standard. The caspase-3 assay was performed in 96-well plates. Fifty micrograms of extract protein were added together with the acetyl-Asp-Glu-Val-Asp p-nitroaniline (Ac-DEVD-pNA) substrate. The plate was incubated at 37 C, and the pNA released by active caspase was measured at 405 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). A pNA calibration curve was obtained using a pNA standard diluted in dimethylsulfoxide. Caspase-3 activity was calculated in picomoles per hour per microgram of protein and plotted as percentage of control. Western blot analysis Granulosa cell protein extracts obtained from different treatment conditions were subjected to one- or two-dimensional gel electrophoresis as described previously (16). Primary antibodies used were rabbit polyclonal prohibitin (1:1000; Neomarks, Fremont, CA), mouse monoclonal cleaved caspase-3 (1:1000; Cell Signaling, Beverly, MA), mouse monoclonal cytochrome c (1:1000; Cell Signaling), cyclophilin-a (1:1000; Neomarks), mouse monoclonal lamin A/C (1:1000; Upstate Cell Signaling Solution, Lake Placid, NY), mouse monoclonal porin (1:1000; Invitrogen, Carlsbad, CA), and mouse monoclonal green fluorescent protein (GFP) (1:500; Abcam, Cambridge, MA). Membranes were incubated with the appropriate secondary antibody for 2 h at room temperature, and antibody binding was detected by chemiluminescence (Pierce, Rockford, IL). Results of representative chemiluminescence were scanned and densitometrically analyzed using a G4 Apple Machintosh Computer (G4; Apple Computer Inc., Cupertino, CA) equipped with a ScanJet 6100C Scanner (Hewlett-Packard Co., Greeley, CO). Quantification of the scanned images was performed according to the NIH Image version-1.61 software (National Institutes of Health, Bethesda, MD). Generation of recombinant adenoviral and transfer plasmid vectors The prohibitin gene PHB1 cDNA was amplified by PCR using total RNA from rat ovarian cells. The specific primers used were: (5′-GTCGACCATGGCTGCCAAAGT-3′, 5′-AAGCTTGGGGTGGGAGCAGAAGGAA-3′) containing SalI and HindIII sites (underlined), respectively. The PCR product was subcloned into a pGEM-T-Easy vector (Promega) to create pGEM-T-Easy-PHB1. The plasmids pGEM-T-Easy-PHB1 and pAdTrack-cytomegalovirus (CMV; AdEasy system; with or without GFP and a CMV promoter) were digested using SalI and HindIII and religated, resulting in pAdTrack-CMV-PHB1-GFP and pAdTrack-CMV-PHB1. The AdEasy system was used for generation of the recombinant adenoviruses. The resultant plasmids encoded a PHB1 gene under the control of a CMV promoter followed either with or without GFP gene under the control of a second CMV promoter. These plasmids were cotransformed into electro-competent BJ5183 bacteria with pAdEasy-1 (containing the viral backbone) and selected on Kanamycin LB plates. These plasmids were amplified and purified using a plasmid maxiprep system (Qiagen, Valencia, CA). The complete adenovectors were linearized and used for transfection of Ad293 cells (human embryonic kidney cell line), where viral particles were further amplified, purified, and tittered according to GFP-positive units (36). Phb1-eGFP fusion protein was generated from the prohibitin gene PHB1 amplified by PCR using rat ovarian cDNA template as described above. The PCR product was subcloned into a pEGFP-C1 vector (Clontech, Mountain View, CA) to create pEGFP-C1 full-length PHB1. Adenoviral and plasmid transfection of granulosa cells Granulosa cells were cultured on six-well culture dish in 4F medium. Subsequently, medium was removed and cells were washed twice with Opti-MEM (antibiotics-free) and infected with adenoviral vector at a multiplicity of infection (MOI) of 1, 5, 10, 40, and 80 pfu/cell. Medium was replaced with 4F medium supplemented with 10% FBS after 2 h of incubation with occasional rocking. Twenty-four hours after exposure to different concentrations of adenoviruses, the medium was removed and replaced with 4F medium without FBS also in the presence and absence of 1 μm (dose determined from separate experiments) of STS. For serum withdrawal treatment, the medium was replaced with DMEM-F12. Granulosa cells were transfected at an efficiency of 60–70% with a plasmid encoding GFP-tagged prohibitin using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 1 μg of DNA was incubated in 600 μl of Opti-MEM, whereas 6 μl of LipofectAmine 2000 was added and left at room temperature for 25 min. The cultured granulosa cells were washed once with Opti-MEM. The DNA-lipid mix was added to the plates and incubated for 4 h, followed by replacement of 1 ml of 4F medium. The transfected granulosa cells were then maintained in culture at 37 C until used for protein expression or immunofluorescence microscopy. Immunofluoresence microscopy Immunofluorescence microscopy for granulosa cells was performed by the method described by Dixit et al. (17). For cytolocalization of exogenously expressed Phb1, granulosa cells were transfected with Phb1-eGFP cDNA and maintained in 4F medium for 24 h. Annexin V levels were also evaluated in the granulosa cells after serum withdrawal. Granulosa cells were grown on cell attachment matrix-coated (Upstate, Lake Placid, NY) coverslips. After the appropriate treatment, cells were fixed with 3% paraformaldehyde in PBS for 30 min, followed by PBS wash and subsequent treatment with cold absolute methanol for 5 min at −20 C. After treatment with 50 mm NH4Cl (to reduce auto-fluorescence of aldehyde groups in immunofluoresence microscopy) in PBS for 10 min, the cells were washed in PBS and then permeabilized with 0.2% Triton X-100 for 5 min. After blocking nonspecific binding using blocking buffer (0.2% Triton X-100 in PBS) containing 10% bovine serum, the slides were incubated overnight with either antirabbit prohibitin (1:200), antimouse cytochrome c (1:200), or antirabbit GFP (1:200; Abcam) at 4 C in blocking buffer. The cellular localization of Phb1, cytochrome c, and Phb1-eGFP fusion protein were visualized with an Alexa Fluor 555-conjugated-goat-antirabbit IgG or Alexa Fluor 488-conjugated-goat-antimouse IgG at 2 μg/ml, respectively (Molecular Probes, Inc.). Thereafter, coverslips were washed and subsequently incubated in 4′,6-diamidino-2-phenylindole for nuclear staining at a concentration of 1 μg/ml for 15 min. After thorough rinsing, coverslips were mounted on slides containing Mowoil. Negative controls were performed omitting the primary antibody or using an isotype-matched control antibody derived from the same species. Mounted slides were examined using an Olympus BX41 microscope equipped with an Optronics Magnafire digital camera and Prior Proscan motorized driven stage (Olympus, Melville, NY). Alexa Fluor 488 has a laser absorption and emission spectrum profile similar to fluorescein, whereas Alexa Fluor 555 has a profile similar to Texas Red. For each image, specific antibody staining was merged with nuclear staining (blue) using Soft Imaging System software that caused virtually no pixel shifting during image merger and resulted in shades of red, green, and blue. Representative photomicrographs were arranged using Adobe PhotoShop (Adobe, San Jose, CA) without any further adjustment to maintain the true nature of the findings. Time-lapse microscopy To determine whether the fusion Phb1 protein translocates from the cytoplasm to the nucleus or mitochondria during activated apoptosis, granulosa cells were transfected with Phb1-eGFP cDNA for 24 h and subsequently treated with 1 μm STS. Time-lapse fluorescence microscopy was performed on transfected cells treated with STS for 6 h. Images were captured every 10 min from a 37 C heated inverted Olympus microscope stage using Soft Imaging System software package (Universal Imaging Corp., Downington, PA). Isolation of S-100 fraction and mitochondria S-100 (cytosolic) fractions and mitochondria were prepared as previously described (17). Briefly, primary granulosa cells were harvested in PBS at 4 C. Cell pellets were resuspended in 5 vol buffer A [20 mm HEPES-KOH (pH 7.5), 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonylfluoride, 1% aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstain A, and 250 mm sucrose] and held at 4 C for 10 min. Cells were homogenized, and nuclei and cellular debris were removed by centrifugation at 500 × g for 10 min at 4 C. Mitochondria were collected from the supernatant by centrifugation at 10,000 × g for 15 min at 4 C, resuspended in buffer A, and held at −80 C. Cytosolic proteins were extracted by centrifugation of the mitochondrial supernatant at 100,000 × g for 1 h at 4 C. Protein levels of cellular fractions were analyzed by Western blot. The relative purity of each fraction was determined by specific subcellular protein markers, such as porin and lamin A/C for mitochondria and nuclei, respectively. Assessment of mitochondrial membrane potential Serum withdrawal cell samples were stained with 10 μg/ml JC-1 (5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetra-ethyl-benz-imidazolo-carbo-cyanine iodide; Molecular Probes, Inc.) in DMEM-F12 medium for 10 min. Granulosa cells were stained and maintained in high potassium buffer [137 mm KCl, 3.6 mm NaCl, 0.5 mm MgCl2, 1.8 mm CaCl2, and DMEM-F12 medium at pH 7.2 (16)] to dissipate plasma membrane potentials. Images were captured using an inverted Olympus microscope stage using Soft Imaging System software package. Statistical analysis All experiments were replicated a minimum of three times, unless otherwise stated. Data are expressed as mean ± sem of three experiments. Statistical analysis was performed by one-way ANOVA using SPSS version 11.0 software (SPSS, Chicago, IL). Multiple comparisons were done by Newman-Keuls’ test, as well as Student t test. Differences were considered significant at P ≤ 0.05. Results Induction of apoptosis in granulosa cells by STS As shown in Fig. 1, A and B, immature rat granulosa cells treated with STS showed significantly high numbers of cells with apoptotic nuclear morphology when compared with controls. At 3 h after 1 μm STS exposure, 50 ± 3% of granulosa cells nuclei were fragmented vs. 2 ± 0.3% (P < 0.05, Fig. 1C) for control cells. STS treatment also resulted in cells exhibiting evidence of cell detachment, loss of cell processes, membrane shrinkage, as evidenced by curling of cells, and formation of apoptotic bodies (data not shown). Fig. 1. Open in new tabDownload slide Induction of apoptosis in granulosa cells by STS. Granulosa cells untreated (control) (A) or treated (B) with 1 μm STS for 3 h were fixed and stained with Hoechst 33248 to identify nuclei. Note the condensation and increased fluorescence of nuclei from STS-treated granulosa cells, as well as the appearance of nuclear fragmentation (arrows). The inset represents a higher magnification, demonstrating fragmented nuclei. Images were captured at ×400 magnification by an epifluorescene microscope. C, The percentage of apoptotic nuclei was assessed in untreated and treated (1 μm STS) granulosa cells at the indicated time periods (0–3 h). D, Caspase-3 activity in cytosolic protein extracts from granulosa cells untreated or treated with STS was measured using the spectrophotometric substrate DEVD-pNA. Graphically, activities are represented as a percentage of the control group, and all numerical values are represented as mean ± sem of three individual experiments (n = 3). An asterisk indicates a significant difference between the control and treated groups (P < 0.05). Fig. 1. Open in new tabDownload slide Induction of apoptosis in granulosa cells by STS. Granulosa cells untreated (control) (A) or treated (B) with 1 μm STS for 3 h were fixed and stained with Hoechst 33248 to identify nuclei. Note the condensation and increased fluorescence of nuclei from STS-treated granulosa cells, as well as the appearance of nuclear fragmentation (arrows). The inset represents a higher magnification, demonstrating fragmented nuclei. Images were captured at ×400 magnification by an epifluorescene microscope. C, The percentage of apoptotic nuclei was assessed in untreated and treated (1 μm STS) granulosa cells at the indicated time periods (0–3 h). D, Caspase-3 activity in cytosolic protein extracts from granulosa cells untreated or treated with STS was measured using the spectrophotometric substrate DEVD-pNA. Graphically, activities are represented as a percentage of the control group, and all numerical values are represented as mean ± sem of three individual experiments (n = 3). An asterisk indicates a significant difference between the control and treated groups (P < 0.05). To quantify the biochemical differences between normal and STS-treated granulosa cells, caspase-3 enzymatic activity was measured. Based on preliminary observations, a concentration of 1 μm STS was selected for treatment of granulosa cells to induce apoptosis (data not shown). The STS treatment for 2 and 3 h causes a significant (P < 0.05) increase in caspase-3 activity in granulosa cells compared with nontreated controls in a time-dependent manner (Fig. 1D). Prohibitin expression and activation of cleaved caspase-3 during apoptosis To determine whether the level of prohibitin expression can be correlated with STS-induction of apoptosis in granulosa cells, cultured cells were exposed to STS for the indicated times (1, 2, and 3 h) and at the indicated concentrations (0.5, 1, 2, and 4 μm) (Fig. 2, A–C). Western blot analyses were performed to characterize Phb1 content and correlate its expression pattern with the presence of cleaved caspase-3. Prohibitin and cleaved caspase-3 immunoreactive bands of approximately 30- and 17-kDa, respectively, were detected in granulosa cells treated with STS. Fig. 2. Open in new tabDownload slide Western blot analyses of protein levels for prohibitin and active caspase-3 in apoptosis granulosa cells induced by STS. Thirty micrograms of protein from time (1, 2, and 3 h)- and dose (0.5, 1, 2, and 4 μm)-dependent STS-treated granulosa cells were applied to each lane (3, 4, 5 and 6 ) and analyzed for prohibitin (30-kDa) expression protein levels and cleaved caspase-3 expression levels byWestern blot analysis (A–C). Representative blots were scanned using the NIH Image software program computer-assisted analysis system for quantitative assessment of changes in protein levels. The bar graph represents the mean ± sem of results from three replicate experiments after normalization of data against cyclophilin-A protein. An asterisk and plus sign indicate a significant difference (P < 0.05) when compared with control. Fig. 2. Open in new tabDownload slide Western blot analyses of protein levels for prohibitin and active caspase-3 in apoptosis granulosa cells induced by STS. Thirty micrograms of protein from time (1, 2, and 3 h)- and dose (0.5, 1, 2, and 4 μm)-dependent STS-treated granulosa cells were applied to each lane (3, 4, 5 and 6 ) and analyzed for prohibitin (30-kDa) expression protein levels and cleaved caspase-3 expression levels byWestern blot analysis (A–C). Representative blots were scanned using the NIH Image software program computer-assisted analysis system for quantitative assessment of changes in protein levels. The bar graph represents the mean ± sem of results from three replicate experiments after normalization of data against cyclophilin-A protein. An asterisk and plus sign indicate a significant difference (P < 0.05) when compared with control. When granulosa cells were treated with 1 and 2 μm STS and were compared with controls (without any treatment) and dimethylsulfoxide, there was no change in Phb1 levels at 1 h (Fig. 2A). However, Phb1 content significantly increased in the STS-treated samples at 2 h (1–4 μm) (Fig. 2B). At 3 h the Phb1 content increased in 0.5, 1, and 2 μm STS-treated granulosa cells but decreased at a dose of 4 μm STS (Fig. 2C). To further confirm and correlate caspase activation during STS-induced apoptosis, we examined extracts for the cleaved form of caspase-3 by using Western blotting techniques. Cleaved caspase-3 protein levels were detected and increased in a dose-dependent manner between 2 and 3 h after STS treatment (Fig. 2, B and C). Cleaved caspase-3 protein levels were significantly (P ≤ 0.05) induced at 4 μm STS concentration at 3 h (Fig. 2C). Cleaved caspase-3 protein content was undetected in all the control granulosa cells cultures. Western blot analyses revealed a significant correlation (P < 0.05; r2 = 0.860) between Phb1 and cleaved caspase-3 protein expression levels at concentrations between 1 and 4.0 μm at 2 h and between 1 and 2.0 μm at 3 h. However, at 4.0 μm at 3 h, this observed correlation did not exist in response to STS-dependent induction of apoptosis. Overexpression of prohibitin inhibits STS-induced apoptosis in granulosa cells Primary granulosa cells were infected with adenovirus carrying PHB1 cDNA in sense orientation at a MOI of 1, 5, and 10 pfu/cell for 24 h. Immunofluorescence microscopy was used to observe prohibitin eGFP expression in infected cells. At a MOI of 10 pfu/cell, prohibitin eGFP expression was detected in greater than 95% of cultured cells (Fig. 3A). Next, the expression levels of exogenous Phb1 in the infected granulosa cells were examined by using Western-blotting techniques. As expected, antiprohibitin immunoreactive bands of approximately 30-kDa were detected in cells infected with adenovirus sense PHB1 eGFP cDNA. We also observed a concentration-dependent increase in adenovirus-expressed Phb1 in granulosa cells (Fig. 3B). As assessed by Western blotting, Phb1 levels were approximately 10-fold greater in granulosa cells infected with 5 pfu/cell, and about 20-fold greater with 10 pfu/cell, when compared with granulosa cells infected with 1 pfu/cell (Fig. 3B). Infection with vector without PHB1 did not show any change in prohibitin levels (data not shown). Fig. 3. Open in new tabDownload slide Effects of recombinant adenovirus-directed overexpression of prohibitin on procaspase-3 cleavages in granulosa cells. A, Overexpression of prohibitin (30-kDa) in granulosa cells by transient infection with recombinant adenovirus constructs containing prohibitin sense cDNA and eGFP (Ad-PHB1-eGFP). Granulosa cells were infected with the adenoviral prohibitin constructs for 2 h and cultured for 24 h. Live cell photographs were taken under an inverted epifluorescence microscope showing green fluorescence for the overexpressed eGFP-prohibitin for the different concentrations of adenovirus-PHB1-eGFP, respectively (MOI = 1, 5, and 10 pfu/cell). B, Western blot analysis of overexpressed prohibitin protein in granulosa cells. Equal amounts of protein (15 μg) from granulosa cells infected with Ad-PHB1-eGFP were applied to each lane and analyzed for overexpressed prohibitin. C, Granulosa cells were infected with sense adenovirus-PHB1-eGFP (MOI = 10) or an adenovirus-eGFP vector control (MOI = 10) for 2 h and maintained in culture for 24 h. Thereafter, granulosa cells were treated with STS (1 μm) for indicated time periods (0, 30, 60, 120, and 180 min) followed by Western blot analysis. Equal amounts of protein (15 μg) from STS-treated granulosa cells were applied to each lane, and the blots were analyzed for prohibitin (30-kDa) and cleaved caspase-3 protein levels. Cyclophilin A and α-tubulin levels were measured as an internal control. A representative of three individual experiments (n = 3) were performed for B and C. Fig. 3. Open in new tabDownload slide Effects of recombinant adenovirus-directed overexpression of prohibitin on procaspase-3 cleavages in granulosa cells. A, Overexpression of prohibitin (30-kDa) in granulosa cells by transient infection with recombinant adenovirus constructs containing prohibitin sense cDNA and eGFP (Ad-PHB1-eGFP). Granulosa cells were infected with the adenoviral prohibitin constructs for 2 h and cultured for 24 h. Live cell photographs were taken under an inverted epifluorescence microscope showing green fluorescence for the overexpressed eGFP-prohibitin for the different concentrations of adenovirus-PHB1-eGFP, respectively (MOI = 1, 5, and 10 pfu/cell). B, Western blot analysis of overexpressed prohibitin protein in granulosa cells. Equal amounts of protein (15 μg) from granulosa cells infected with Ad-PHB1-eGFP were applied to each lane and analyzed for overexpressed prohibitin. C, Granulosa cells were infected with sense adenovirus-PHB1-eGFP (MOI = 10) or an adenovirus-eGFP vector control (MOI = 10) for 2 h and maintained in culture for 24 h. Thereafter, granulosa cells were treated with STS (1 μm) for indicated time periods (0, 30, 60, 120, and 180 min) followed by Western blot analysis. Equal amounts of protein (15 μg) from STS-treated granulosa cells were applied to each lane, and the blots were analyzed for prohibitin (30-kDa) and cleaved caspase-3 protein levels. Cyclophilin A and α-tubulin levels were measured as an internal control. A representative of three individual experiments (n = 3) were performed for B and C. In further studies, we examined whether overexpression of Phb1 resulted in inhibition of the processing of procaspase-3 to the active enzyme. As shown in Fig. 3C, Western blot analyses revealed that cleaved caspase-3 protein content in control (adenovirus-eGFP) infected granulosa cells was initially detectable at 2 h, and the levels of cleaved caspase-3 protein increased significantly (P < 0.05) with time up to 3 h after transfection. In marked contrast, adenovirus PHB1 eGFP-infected granulosa cells show the first appearance of cleaved caspase-3 at 3 h, suggesting that virally expressed exogenous Phb1 delayed the activation of cleaved caspase-3 expression in granulosa cells. At the 3-h time point, there was an increase in caspase-3 cleavage with a corresponding decrease in exogenous Phb1 levels. In this study, we investigated whether the inhibition of the processing of procaspase-3 to the active enzyme in STS-treated granulosa cells was due, in part, to the inhibition in cytochrome c release from the mitochondria to the cytosol. As shown in Fig. 4, A and D, a decrease in cytochrome c protein content was observed from isolated mitochondrial extract at 0.5 h after treatment with 1 μm STS. This decrease continued at subsequent times confirming that STS induces the release of cytochrome c from the mitochondria of granulosa cells. Fig. 4. Open in new tabDownload slide Effects of recombinant adenovirus-directed overexpression of prohibitin on cytochrome c release from mitochondria in granulosa cells. A, Cells were cultured in serum-free media in the presence of STS for 0, 0.5, 1, 1.5, and 2 h and collected for Western blotting analysis of cytochrome c release from mitochondria in granulosa cells. B, Adenovirus-infected (MOI = 10) granulosa cells were treated with STS (1 μm) for the indicated times and lysed, and aliquots of the extracts were analyzed by Western blotting as indicated above. C, Western blot analysis of expression of cytochrome c in mitochondria of granulosa cells infected with increasing amounts of recombinant adenoviral lysates (MOI = 10, 20, 40, and 80). Equal amounts of mitochondrial protein (30 μg) from STS-treated granulosa cells were applied to each lane in A–C. Representative blots were scanned using the NIH Image software program computer-assisted analysis system for quantitative assessment of changes in protein levels. The bar graphs represent the mean ± sem of results from three replicate experiments after normalization of data against porin protein (D, E, and F). An asterisk indicates a significant difference (P < 0.05) when compared with control. Fig. 4. Open in new tabDownload slide Effects of recombinant adenovirus-directed overexpression of prohibitin on cytochrome c release from mitochondria in granulosa cells. A, Cells were cultured in serum-free media in the presence of STS for 0, 0.5, 1, 1.5, and 2 h and collected for Western blotting analysis of cytochrome c release from mitochondria in granulosa cells. B, Adenovirus-infected (MOI = 10) granulosa cells were treated with STS (1 μm) for the indicated times and lysed, and aliquots of the extracts were analyzed by Western blotting as indicated above. C, Western blot analysis of expression of cytochrome c in mitochondria of granulosa cells infected with increasing amounts of recombinant adenoviral lysates (MOI = 10, 20, 40, and 80). Equal amounts of mitochondrial protein (30 μg) from STS-treated granulosa cells were applied to each lane in A–C. Representative blots were scanned using the NIH Image software program computer-assisted analysis system for quantitative assessment of changes in protein levels. The bar graphs represent the mean ± sem of results from three replicate experiments after normalization of data against porin protein (D, E, and F). An asterisk indicates a significant difference (P < 0.05) when compared with control. As shown in Fig. 4, B and E, when granulosa cells were infected with 10 MOI of adenoviral PHB1 eGFP, there was an initial delay in the release of cytochrome c content from mitochondrial fraction at time 0.5–1.0 h followed by a significant decrease at 1.5 and 2.0 h. As illustrated in Fig. 4, C and F, increasing MOI of adenovirus-directed overexpression of prohibitin in granulosa cells significantly inhibited the release of cytochrome c protein content in a dose-related manner. Prohibitin levels increased proportionally with higher MOI (data not shown). The release of cytochrome c coincided with activation of caspase-3 as previously demonstrated in Fig. 3. Subcellular localization of prohibitin during STS-induced apoptosis To assess whether prohibitin is associated with the mitochondria in primary rat granulosa cells, immunocytochemical and Western blotting studies were performed. As shown in Fig. 5A, we confirmed mitochondrial localization of prohibitin by demonstrating colocalization (yellow) of prohibitin and cytochrome c. We also observed a faint immunoreactive signal within the nucleus. In subsequent studies, the granulosa cells were lysed and fractionated into mitochondrial, cytosolic, and nuclear fractions and equal amounts of protein extracts were analyzed by two-dimensional gel electrophoresis and Western blot. As shown in Fig. 5B, two-dimensional gel analyses delineated three polypeptide species of the prohibitin 30-kDa protein from the mitochondrial fractions and one spot in the nuclear fraction but not in the cytosol (data not shown). The polypeptide species in the mitochondrial fractions had isoelectric points (pI) of 5.6, 5.8, and 5.9, whereas the polypeptide spot in the nuclear fraction had a pI of 5.8. Because two apparent locations of prohibitin were detectable with the antibody, studies were performed to ascertain whether Phb1 isoforms were primarily associated with the mitochondria of granulosa cells. To facilitate these studies, an eGFP-full-length prohibitin fusion construct was made that allowed us to monitor the distribution of exogenous Phb1 in vitro. Granulosa cells were transiently transfected with prohibitin-eGFP fusion construct or an eGFP vector control (without PHB1). Western blotting analysis carried out using the transfected cells showed that the exogenously expressed prohibitin protein levels were more than twice that of the endogenous prohibitin expressed in cultured granulosa cells (Fig. 5C). Furthermore, Western blot analysis of cells that were transfected for 24 h with the Phb1 fusion construct and subsequently separated into subcellular compartments (mitochondria and nuclear), confirmed the association of the exogenous Phb1 with the mitochondria of granulosa cells (Fig. 5D) but not in the nucleus. Porin and lamin were used as specific markers for mitochondrial and nuclear fractions, respectively. Fig. 5. Open in new tabDownload slide Localization of endogenous and exogenous prohibitin in primary granulosa cells. A, Immunocolocalization of mitochondrial marker cytochrome c detected with Alexa fluor 488 (a, green) labeled secondary antibody, and endogenous prohibitin detected with Alexa fluor 594 (b, red) labeled secondary antibody. The DNA was counterstained with 4′,6-diamidino-2-phenylindole (c, blue). The area of colocalization is seen as yellow (d) punctated spots within the cytoplasm. B, Eighty micrograms of protein purified from mitochondria and nuclear fractions isolated from cultured granulosa cells were focused in the first dimension on IPG pH gradient 4–7 strips for 60 kilovolt-hours using a Bio-Rad Protean IEF Cell and second dimension followed by the Western blotting procedure and prohibitin antibody to detect protein spots corresponding to prohibitin. C, Western blot analysis of protein expression for endogenous and exogenous prohibitin in granulosa cells transfected with a full-length PBH1-eGFP fusion construct (lane 1) or vector without PHB1 fusion (as a control, lane 2). D, Western blot analysis of exogenous prohibitin localization in granulosa cells. Granulosa cells were transfected with full-length prohibitin GFP fusion construct followed by cell fractionation and localization of PBH1-eGFP fusion protein in mitochondrial (M), and nuclear (N) fractions detect by analysis with GFP antibody. All blots were probed with porin and lamin A/C antibodies. Western blots are representative of three individual experiments (n = 3). Bar, 20 μm. Fig. 5. Open in new tabDownload slide Localization of endogenous and exogenous prohibitin in primary granulosa cells. A, Immunocolocalization of mitochondrial marker cytochrome c detected with Alexa fluor 488 (a, green) labeled secondary antibody, and endogenous prohibitin detected with Alexa fluor 594 (b, red) labeled secondary antibody. The DNA was counterstained with 4′,6-diamidino-2-phenylindole (c, blue). The area of colocalization is seen as yellow (d) punctated spots within the cytoplasm. B, Eighty micrograms of protein purified from mitochondria and nuclear fractions isolated from cultured granulosa cells were focused in the first dimension on IPG pH gradient 4–7 strips for 60 kilovolt-hours using a Bio-Rad Protean IEF Cell and second dimension followed by the Western blotting procedure and prohibitin antibody to detect protein spots corresponding to prohibitin. C, Western blot analysis of protein expression for endogenous and exogenous prohibitin in granulosa cells transfected with a full-length PBH1-eGFP fusion construct (lane 1) or vector without PHB1 fusion (as a control, lane 2). D, Western blot analysis of exogenous prohibitin localization in granulosa cells. Granulosa cells were transfected with full-length prohibitin GFP fusion construct followed by cell fractionation and localization of PBH1-eGFP fusion protein in mitochondrial (M), and nuclear (N) fractions detect by analysis with GFP antibody. All blots were probed with porin and lamin A/C antibodies. Western blots are representative of three individual experiments (n = 3). Bar, 20 μm. Next, we examined whether prohibitin expression patterns changed with the insult of a cell stressor such as STS using a prohibitin GFP fusion construct. STS-induced apoptosis resulted in changes in the cellular distribution of Phb1 in live granulosa cells. In Fig. 6A, immunofluorescence microscopy of cells transfected with prohibitin-eGFP vector revealed an initial diffuse cytoplasmic localization of the prohibitin-eGFP fusion protein (see inset). By 2 h, mitochondrial sequestration of the fusion protein was evident (Fig. 6A, 2 h). Increased mitochondrial sequestration of the fusion protein was observed within 4–6 h (Fig. 6A, 4 h and 6 h). This expression pattern and localization was confirmed by Western blot analysis of isolated mitochondrial fraction that revealed a marked increase in prohibitin-eGFP fusion protein levels (Fig. 6, B and C). This effect occurred in a time-dependent manner when compared with transfected controls. In Fig. 6D, overexpression of Pbh1-eGFP fusion protein delayed the onset of apoptosis in STS-treated granulosa cells. Fig. 6. Open in new tabDownload slide Overexpression and translocation of prohibitin into the mitochondria upon apoptotic stimulation supporting its role in apoptosis. Granulosa cells were transfected with full-length PBH1-eGFP fusion construct or vector without PBH1 followed by apoptotic stimulation with STS (1 μm) for the indicated time periods. A, Expression and translocation of Pbh1-eGFP fusion protein upon apoptotic stimulation was studied by fluorescence imaging for GFP time-lapse photography under an inverted fluorescence microscope at 10-min intervals. Photographs are representative of three individual experiments that were arranged using Adobe Photoshop; bar, 20 μm. B, Western blot analysis of Pbh1-eGFP fusion protein (15 μg) in isolated mitochondria from granulosa cells using GFP antibody. Porin was used as a loading control. C, Representative blots were scanned using the NIH Image software program computer-assisted analysis system for quantitative assessment of changes in protein levels. The bar graph represents the mean ± sem of results from three replicate experiments after normalization of data against porin protein. An asterisk indicates a significant difference at P < 0.05. D, Overexpression of Pbh1-eGFP fusion protein mediated delayed in percentage of apoptotic cells observed after STS treatment. Apoptosis was assessed by change in nuclear morphology after staining with Hoechst 33248. Nuclei were considered apoptotic if the nucleus became increasingly bright and decreased in size or fragmented into apoptotic bodies (see Fig. 1, inset B). The bar graph represents the mean ± sem of results from three replicate experiments. An asterisk and plus indicate a significant difference (P < 0.05) when compared with control. Fig. 6. Open in new tabDownload slide Overexpression and translocation of prohibitin into the mitochondria upon apoptotic stimulation supporting its role in apoptosis. Granulosa cells were transfected with full-length PBH1-eGFP fusion construct or vector without PBH1 followed by apoptotic stimulation with STS (1 μm) for the indicated time periods. A, Expression and translocation of Pbh1-eGFP fusion protein upon apoptotic stimulation was studied by fluorescence imaging for GFP time-lapse photography under an inverted fluorescence microscope at 10-min intervals. Photographs are representative of three individual experiments that were arranged using Adobe Photoshop; bar, 20 μm. B, Western blot analysis of Pbh1-eGFP fusion protein (15 μg) in isolated mitochondria from granulosa cells using GFP antibody. Porin was used as a loading control. C, Representative blots were scanned using the NIH Image software program computer-assisted analysis system for quantitative assessment of changes in protein levels. The bar graph represents the mean ± sem of results from three replicate experiments after normalization of data against porin protein. An asterisk indicates a significant difference at P < 0.05. D, Overexpression of Pbh1-eGFP fusion protein mediated delayed in percentage of apoptotic cells observed after STS treatment. Apoptosis was assessed by change in nuclear morphology after staining with Hoechst 33248. Nuclei were considered apoptotic if the nucleus became increasingly bright and decreased in size or fragmented into apoptotic bodies (see Fig. 1, inset B). The bar graph represents the mean ± sem of results from three replicate experiments. An asterisk and plus indicate a significant difference (P < 0.05) when compared with control. Overexpression of prohibitin inhibits serum withdrawal-induced apoptosis in granulosa cells To further investigate the role of Phb1 in serum withdrawal-induced apoptosis of granulosa cells, experiments were performed in which granulosa cells were infected with adenovirus, adenovirus PHB1, and controls (absence of adenovirus) with or without serum. Primary granulosa cells were infected with adenovirus at a MOI of 10 pfu/cell for 24 h. Immunofluorescence microscopy was used to evaluate percentage of apoptotic cells by annexin V staining. As shown in Fig. 7A, rat granulosa cells cultured in the absence of serum showed significant increase (50%, 24 h) in numbers of annexin V-positive cells beyond 3 h after serum removal when compared with controls (5%, 24 h; with serum or serum plus adenovirus-infected cells). The observed increase in annexin V-positive cells corresponded with increased caspase-3 activity (5-fold, 24 h) when compared with serum controls (1-fold, 24 h; Fig. 7, A and C). Cells infected with adenovirus PHB1 for 24 h and deprived of serum and subsequently labeled with annexin V at 0, 3, 6, 12, and 24 h, displayed only sporadic labeling. A representative image of granulosa cells infected with adenovirus without PHB1 (Fig. 7B, a–c) or with PHB1 (Fig. 7B, d and e) and subsequently cultured in the absence of serum for 12 h is presented. A higher magnification image (Fig. 7B, c) allows better resolution of annexin V labeling in granulosa cells. Fig. 7. Open in new tabDownload slide Effects of recombinant adenovirus-directed overexpression of prohibitin on serum withdrawal-induced cell death and on mitochondrial membrane potential in rat granulosa cells. Granulosa cells were plated overnight in 4F medium containing 10% fetal calf serum. The next morning, media and unattached cells were removed and monolayers were exposed to the present and absence of adenovirus constructs (adenovirus without PHB1 or adenovirus with PHB1 at MOI = 10) for 2 h; subsequently, virus-containing media were removed and replaced with fresh 4F medium plus 10% fetal calf serum. After 24 h, medium was replaced with DMEM-F12 with or without fetal calf serum and apoptosis was determined at 0, 3, 6, 12, and 24 h after serum withdrawal. Cells were stained for phosphatidylserine with Alexa 488-conjugated annexin V and propidium iodide (PI) as a vital stain and viewed under epifluorescent illumination and by phase contrast microscopy. A, Percent apoptosis was determined as indicated in Materials and Methods. B, Representative images of granulosa cells infected with viruses without PHB1 (a–c) or with PHB1 (d and e). Image C is at a higher magnification, demonstrating staining specificity for annexin V. Arrowheads indicate examples of cells in early stages of apoptosis based on positive staining for annexin V but no uptake of PI at 12 h (B). C, Caspase-3 activity in cytosolic protein extracts from granulosa cells cultured in the presence or absence of serum or infected with viruses with or without PHB1 were measured using the spectrophotometric substrate DEVD-pNA. D, Mitochondrial membrane potential was assessed in cells infected with viruses without PHB1 (a–c) or with PHB1 (e–g). Phase contrast (a and d) and epifluorescence (b, c, e, and f) of cells stained with JC-1 to demonstrate a reduction in mitochondrial membrane potential in the absence of overexpressed prohibitin (b and c, red to green transition). The line graph represents the mean ± sem of results from three replicate experiments. Significant difference is at P < 0.05. Bar, 20 μm. Fig. 7. Open in new tabDownload slide Effects of recombinant adenovirus-directed overexpression of prohibitin on serum withdrawal-induced cell death and on mitochondrial membrane potential in rat granulosa cells. Granulosa cells were plated overnight in 4F medium containing 10% fetal calf serum. The next morning, media and unattached cells were removed and monolayers were exposed to the present and absence of adenovirus constructs (adenovirus without PHB1 or adenovirus with PHB1 at MOI = 10) for 2 h; subsequently, virus-containing media were removed and replaced with fresh 4F medium plus 10% fetal calf serum. After 24 h, medium was replaced with DMEM-F12 with or without fetal calf serum and apoptosis was determined at 0, 3, 6, 12, and 24 h after serum withdrawal. Cells were stained for phosphatidylserine with Alexa 488-conjugated annexin V and propidium iodide (PI) as a vital stain and viewed under epifluorescent illumination and by phase contrast microscopy. A, Percent apoptosis was determined as indicated in Materials and Methods. B, Representative images of granulosa cells infected with viruses without PHB1 (a–c) or with PHB1 (d and e). Image C is at a higher magnification, demonstrating staining specificity for annexin V. Arrowheads indicate examples of cells in early stages of apoptosis based on positive staining for annexin V but no uptake of PI at 12 h (B). C, Caspase-3 activity in cytosolic protein extracts from granulosa cells cultured in the presence or absence of serum or infected with viruses with or without PHB1 were measured using the spectrophotometric substrate DEVD-pNA. D, Mitochondrial membrane potential was assessed in cells infected with viruses without PHB1 (a–c) or with PHB1 (e–g). Phase contrast (a and d) and epifluorescence (b, c, e, and f) of cells stained with JC-1 to demonstrate a reduction in mitochondrial membrane potential in the absence of overexpressed prohibitin (b and c, red to green transition). The line graph represents the mean ± sem of results from three replicate experiments. Significant difference is at P < 0.05. Bar, 20 μm. Analysis of mitochondrial membrane potential during serum withdrawal-induced apoptosis in granulosa cells JC-1 binds to the polarized mitochondrial membrane as aggregates. The JC-1 aggregates (Fig. 7D, b–e), upon excitation, have an emitted fluorescence signal at approximately 590 nm (orange). Upon depolarization of the mitochondrial membrane, the JC-1 forms monomers (Fig. 7D, c) that emit fluorescence at approximately 530 nm (green fluorescence). As shown in Fig. 7D, when cells are infected with adenovirus (without PHB1; Fig. 7D, a–c) for 24 h and subsequently deprived of serum for 12 h, the monomeric form of JC-1 dye in cells at early stages of apoptosis is evident (Fig. 7D, c). When cells are infected with adenovirus PHB1 (Fig. 7D, d–f) for 24 h and subsequently deprived of serum for 12 h, there is an absence of the monomeric form of JC-1 dye (Fig. 7D, f). Thus, overexpression of prohibitin inhibits the collapse of the electrochemical gradient across the mitochondrial membrane during serum withdrawal-induced apoptosis. Discussion In the present study, two-apoptotic model systems, STS, and serum-withdrawal activation of the intrinsic apoptotic pathway were used to study the role of Phb1 in suppressing granulosa cell apoptosis. We focused on the intrinsic pathway because prohibitin is predominantly associated with mitochondria. The classical intrinsic apoptotic pathway is activated by a death signal, which subsequently leads to the release of cytochrome c from the mitochondrial intermembrane space into the cytosol and to changes in the mitochondrial membrane potential. The mitochondrial and down-stream events in the intrinsic pathway are well described (34, 37–49). Our results demonstrate that the intrinsic apoptotic pathway in primary granulosa cells isolated from immature rats are directly activated by STS and serum withdrawal treatments. Under these conditions, cytochrome c release was observed from mitochondrial intermembrane space into the cytosol. Also observed were procaspase 3 cleavage (34), positive staining of phosphatidyl serine on the outer cell membrane by annexin V (50, 51), and alteration in the mitochondrial membrane potential (52). Cytochrome c has been shown previously to play an integral role in initiating the activation of a cascade of caspases as it is released into the cytosol (43–45). Caspase-3 is a key executioner component of the apoptotic pathway because it catalyzes the proteolytic degradation of several crucial target proteins such as poly (ADP-ribose) polymerase (45, 46), protein kinase C δ (47), and sterol-regulatory element-binding protein (48). Our results confirm that cytochrome c release, procaspase-3 processing, and alteration in mitochondrial membrane potential are key components mediated by STS and serum withdrawal in immature rat granulosa cells. When apoptosis was induced with the protein kinase C inhibitor STS, a corresponding increase in prohibitin expression was observed in granulosa cells. This increase in prohibitin expression corresponded with an increased processing of procaspase-3 to the active enzyme. These results are in agreement with previous findings indicating that the processing of procaspase-3 is an early marker for the demise of granulosa cells (41, 42, 51–53). Interestingly, a decline in Phb1 levels was observed at higher STS concentration (4 μm). It is not clear at this point whether this observation is due to cellular responses to stress or other mechanisms. There are two possible explanations for these findings. One is that prohibitin expression is induced by an apoptotic stimulus as a cellular response to prevent apoptosis, but if the apoptotic signal is too great, the extent of prohibitin expression is not sufficient to prevent apoptosis. The second, from a physiological perspective, is that the FSH induction of prohibitin (18) would further increase prohibitin levels and, therefore, would provide additional protection to the cell if it is subjected to an apoptotic signal. In fact, it has been report by Vander Heiden et al. (54), that overexpression of prohibitin protected the murine prolymphocytic cell line from growth factor withdrawal-induced apoptosis. Our finding that modulation in the level of prohibitin expression is in agreement with previous observations made in yeast, Caenorhabditis elegans, Drosophila melanogaster, and humans that verify conservation of Phb1 in mitochondrial function (16, 18, 20, 27, 54). To evaluate whether Phb1 is involved in modulating apoptosis, we used a recombinant adenovirus vector to constitutively overexpress prohibitin in granulosa cells. Greater than 90% of granulosa cells in primary culture exhibited adenovirus expression of GFP reporter gene without adverse effects on cellular morphology and apoptosis (Fig. 3A). Because the viruses infect virtually all cells in culture, direct analysis of virally mediated overexpression of specific proteins on granulosa cell function can be assessed. In the current study, adenovirus vectors that direct overexpression of the PHB1 gene, tilted the balance in favor of survival by diminishing cytochrome c release, modulating the activation of caspase-3, stabilizing mitochondrial membrane potential and inhibiting the translocation of phosphatidyl serine from the inside to the outside of the cell membrane. Infection of granulosa cells with adenovirus vectors lacking the prohibitin gene resulted in apoptosis after STS and serum withdrawal treatments. Although these results provide evidence for a functional role of prohibitin in suppressing apoptosis in immature rat granulosa cells, the molecular mechanism(s) involved are currently unknown. We further extended our studies to examine the intracellular expression patterns of the PHB1 gene product in relation to apoptotic stimulation. Previously, using the commercially available antibody for immunohistochemical analysis of atretic follicle cells and aberrant embryos, we reported that prohibitin was localized within the mitochondrial and nuclear compartments (18). Although it has also been reported that prohibitin is primarily in mitochondria (15, 16, 20, 27–33, 55), others have reported that prohibitin is located in the nucleus (11–13, 56). In an attempt to understand trafficking of prohibitin under our experimental conditions, we initially used immunolocalization strategies to colocalize prohibitin with cytochrome c expression in untreated granulosa cells. The results of these colocalization studies strongly suggest that Phb1 is primarily associated with mitochondria. However, a small percentage of immunoreactive staining of a prohibitin-like molecule was recognized by the prohibitin antibody in nuclei of rat granulosa cells. To verify the cellular distribution pattern of Phb1, we fractionated cytosolic, mitochondrial and nuclear fractions of granulosa cells and performed two-dimensional electrophoresis and Western blot analyses. The prohibitin antibody identified three 30-kDa isoforms of pIs 5.6, 5.8, and 5.9 in the mitochondrial fraction; however, a single 30-kDa protein spot with a pI of 5.8 was also detected in the nuclear fraction of granulosa cells. We are currently investigating the significance of the nuclear prohibitin protein in granulosa cells. It is likely that the specific role of individual members of the prohibitin family may depend on cell type examined, physiological status, and cellular concentrations (57). Moreover, the data in this study do not exclude the possibility that processing of the 30-kDa prohibitin protein occurs before, or during, targeting to the mitochondria and/or nucleus in mammalian cells. We have previously demonstrated that the more acidic isoform of prohibitin (pI, 5.6) is due in part to posttranslational modification by phosphorylation. Furthermore, the subcellular localization of the 30-kDa prohibitin, the major isoform associated with mitochondria, and its relationship to apoptotic stimulation is not well understood and has not been addressed by previous studies. We addressed this issue by transfecting granulosa cells with a prohibitin-eGFP fusion protein followed by induction of apoptosis with STS and time-lapse immunofluorescence microscopy. These studies showed the sequestration of the fusion protein from the cytosol to the mitochondria in a time-dependent manner. No nuclear localization signal was evident. However, it is possible that the GFP tag at the carboxyl terminus may inhibit translocation to the nucleus. Additionally, we observed a delay in the onset of apoptosis in STS-treated granulosa cells. In summary, this study reveals a potential role of prohibitin in suppressing apoptosis in undifferentiated granulosa cells of the rat. The mechanism by which overexpression of prohibitin reduced apoptosis appears to be targeting of the mitochondria. Whether the cellular response to prohibitin in granulosa cells from immature follicles vs. preovulatory follicles are different, awaits further investigation. The protective nature of prohibitin in granulosa cells and the cellular response to two cell stressors are in agreement with previous studies using camptothecin-induced apoptosis as well as growth factor withdrawal-induced apoptosis (54, 56). It is important to understand the details of the signaling pathway(s) mediating Phb1 expression in granulosa cells during apoptosis. The ability to use adenoviral vectors to selectively overexpress or block prohibitin in granulosa cells will provide additional clues in the elucidation of the signaling pathways that govern proliferation, differentiation, and apoptosis in granulosa cells. We thank Dr. Dong Liu (Morehouse School of Medicine, Cardiovascular Research Institute) for his valuable contributions in the design of the adenovirus vectors. We also thank Patrick Abramson from the Department of Information Technology for photographic and computer imaging assistance. This work was supported by National Institutes of Health Grants U54 HD41749 and RR03034 and the Department of Defense Ovarian Cancer Program OC020258. This work was presented, in part, at the 36th Annual Meeting of the Society for the Study of Reproduction in Vancouver, Canada, July 31–August 5, 2004. Disclosure Statement: I.C., W.X., J.K.S., A.Z., X.Y., R.M., K.T., and W.E.T. have nothing to declare. 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TI - Apoptosis of Rat Granulosa Cells after Staurosporine and Serum Withdrawal Is Suppressed by Adenovirus-Directed Overexpression of Prohibitin
JO - Endocrinology
DO - 10.1210/en.2006-0187
DA - 2007-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/apoptosis-of-rat-granulosa-cells-after-staurosporine-and-serum-IVoSfBfSuU
SP - 206
VL - 148
IS - 1
DP - DeepDyve
ER -