Mammalian Prohibitin Proteins Respond to Mitochondrial Stress and Decrease during Cellular Senescence

Experimental Cell Research 265, 262–273 (2001) doi:10.1006/excr.2001.5166, available online at http://www.idealibrary.com on

Mammalian Prohibitin Proteins Respond to Mitochondrial Stress and Decrease during Cellular Senescence P. J. Coates,* ,† ,1 R. Nenutil,‡ A. McGregor,§ S. M. Picksley, ¶ D. H. Crouch,㛳 P. A. Hall,* and E. G. Wright* ,† *Department of Molecular and Cellular Pathology and 㛳Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom; ‡Department of Pathology, Faculty Hospital Brno, Obilni trh 11, 656 17 Brno, Czech Republic; §Department of Neurology, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom; ¶Biomedical Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, United Kingdom; and †Radiation and Genome Stability Unit, Medical Research Council, Harwell, Didcot, Oxfordshire OX11 ORD, United Kingdom

The two prohibitin proteins, Phb1p and Phb2p BAP37, have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses. The gene promoter sequences contain binding sites for the Myc oncoprotein and overexpression of Myc induces expression of the prohibitins. The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging. © 2001 Academic Press Key Words: prohibitin; BAP37; mitochondria; metabolism; aging.

INTRODUCTION

Expression of the two structurally related prohibitin proteins, Phb1p and Phb2p BAP37, influences the aging process in budding yeast, with deletion of both genes, PHB1 and PHB2, accelerating the rate of aging [1, 2]. The prohibitin genes have been highly conserved, and one or other or both of the protein products have been localized to the mitochondria in mammals, plants, and 1 To whom reprint requests should be addressed. Fax: ⫹44 (0)1382 633952. E-mail: [email protected].

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yeast [1– 6]. In mammals and yeast, the two proteins have also been shown to physically associate with each other [1, 5, 6]. Despite such information, the function of the prohibitins has remained unclear, particularly in view of the number of different roles that have been proposed. PHB1 was originally identified in mammals as a putative negative regulator of the cell cycle, based on decreased levels of mRNA in rat liver cells during cell cycle entry induced by partial hepatectomy [7]. The ability of microinjected PHB1 mRNA to inhibit cell cycle progression strongly supported the idea that Phb1p acts as a negative regulator of cell cycle entry [8]. Human Phb1p has also been identified in a yeast two-hybrid screen as a retinoblastoma-binding protein which inhibits E2F transcriptional activation [9]. Further studies indicated that prohibitin can also directly bind to E2F and the Raf-1 kinase and that there are differences in the responses of prohibitin and Rb to various cell cycle stimuli [10]. Human Phb2p BAP37 has been isolated from a yeast two-hybrid screen as an estrogen receptor-binding protein that can repress transcriptional activation [11]. Taken together, these data suggest that the prohibitins act as independent nuclear cell cycle regulatory proteins. In other studies using coimmunoprecipitation, both Phb1p and Phb2p BAP37 have been identified as proteins associated with cell-surface IgM on murine B lymphocytes, implying a role in receptor-mediated signaling at the cell surface [12]. However, none of these observations are in accord with the demonstration that the mammalian prohibitins exist in the mitochondria [1, 3]. Furthermore, it has recently been demonstrated that the growth-suppressive activity of microinjected PHB1 mRNA is located in the 3⬘ untranslated region, with microinjection of the protein-coding sequence having no effect on cell cycle entry [13, 14]. Consequently, the idea that the prohibitin proteins act as negative regulators of the cell cycle is brought into question. Observations made in lower organisms have indicated widely different roles for the highly conserved

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prohibitin proteins. A function in programmed cell death pathways has been suggested because the levels of PHB1 mRNA are seen to rise during apoptosis of trypanosomes [15]. In yeast, genetic interactions suggested roles for PHB1 and PHB2 in mitochondrial inheritance [2]. The prohibitins have also been implicated in regulating the assembly of respiratory enzyme complexes in this organism. The two Saccharomyces cerevisiae prohibitins form a high-molecular-weight complex that copurifies with the m-AAA protease in the mitochondrial inner membrane [5]. Deletion of either of the prohibitin genes in combination with deletion of either of the protease subunits causes a severe growth defect. Furthermore, newly synthesized mitochondrial proteins are degraded more rapidly in the absence of the prohibitins, suggesting that they function as inhibitors of the protease. The prohibitins additionally act as a direct chaperone in the inner mitochondrial membrane of yeast, reinforcing the view that their primary role lies in the assembly of respiratory chain enzymes in this organism [6]. Their expression patterns in yeast imply a role as a “holdase” in situations of altered mitochondrial metabolism [6]. These observations suggest that the prohibitins act in cooperation with each other to modulate mitochondrial activity, particularly in situations of mitochondrial stress. In view of the link between expression of the prohibitin proteins and the aging process, and the confusion surrounding the cellular role of the proteins from experiments in different species, we have investigated the localization, structure, and expression patterns of the two proteins in mammals. Our results provide evidence for a conserved role for the prohibitins in regulating mitochondrial metabolism and in cellular aging, but are not compatible with functions relating to the regulation of cell cycle or apoptosis. MATERIALS AND METHODS All experiments involving animal tissues were performed following the relevant governmental guidelines and regulations. Immunocytochemistry. Mouse embryos were collected from day 8.5 of gestation through to birth. Tissues were also collected from young adult mice and rats (3– 6 months). A series of 70 primary human neoplasms were obtained from surgery, comprising tumors of the breast, prostate, colon, testis, and skin. Tissues were fixed in buffered 10% formalin or in Methacarn solution (60% methanol, 30% chloroform, 10% acetic acid) before being processed to paraffin wax. Tissue sections were incubated overnight at 4°C with primary rabbit serum to Phb1p (APP-2 serum) or Phb2p BAP37 (supplied by BioVation, Aberdeen, Scotland, UK) diluted in 5% normal swine serum in phosphate-buffered saline, pH 7.4 (PBS). For tissues fixed in formalin, sections were pretreated by microwave antigen retrieval in 0.01 M citric acid, pH 6.0, as described [16] prior to staining for Phb1p (the Phb2p BAP37 serum is not effective following formalin fixation). Following washes in PBS, peroxidase-conjugated swine anti-rabbit immunoglobulins (Dako Ltd., UK) diluted 1/100 in PBS containing 0.1% bovine serum albumin (BSA) were applied for 30 min at room tem-

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perature and immunoreactive sites were revealed by incubation with diaminobenzidine/H 2O 2. For sections of skin, aminoethylcarbazole was used as the chromogen to give a red product, more easily distinguished from endogenous melanin pigment. Cell nuclei were stained with hematoxylin. Control sections were immunostained with preimmune serum or with serum which had been incubated with the corresponding immunizing peptide prior to use for immunocytochemistry. For immunofluorescence of cultured cells grown on glass slides, fixation was performed by immersion in cold acetone/methanol for 10 min. Primary rabbit sera to Phb1p or Phb2p BAP37 were diluted in PBS containing 3% BSA. After washes in PBS, rabbit primary antibodies were detected with FITC-conjugated swine anti-rabbit immunoglobulins (Dako) and nuclei were counterstained with propidium iodide. For double-labeling studies, mouse monoclonal antibodies to either cytochrome c oxidase subunit IV or subunit VIc (Molecular Probes Europe, Leiden, The Netherlands) were mixed with the rabbit serum and the mouse monoclonal antibody was detected with Texas red-conjugated horse anti-mouse IgG (Vector Laboratories, Peterborough, UK) diluted in PBS containing 5% normal rabbit serum and 5% normal swine serum. Control sections were stained with both fluorescent markers in the absence of either the polyclonal or the monoclonal antibodies. Cell cycle entry, cellular senescence, and MYC transformation. Human lymphocytes were isolated from whole blood using density centrifugation through Ficoll (Pharmacia, UK) according to the manufacturer’s instructions. Cells were resuspended in DMEM containing 20% fetal calf serum (FCS) (Life Technologies, UK) and monocytes removed by adhesion to plastic for 30 min. The purified lymphocytes were exposed to phytohemaglutinin (PHA; Life Technologies) to induce cell cycle entry. Equal numbers of cells were collected after various times, washed in PBS, and lysed for SDS– PAGE and Western blotting. Primary human fibroblasts (HF19) were maintained in MEM-␣ with 10% FCS. Cells were subcultured until they reached senescence, judged by a flattened and enlarged morphology, a lack of continued proliferation following subculture, and the appearance of senescence-associated ␤-galactosidase activity [17]. Mitochondrial membrane potential of young and senescent cells was measured by FACS analysis after staining with the lipophilic cationic carbocyanine dye DiOC 6(3) at a final concentration of 0.1 ␮M [1]. Chick embryo fibroblasts (CEF) were prepared from 10-day-old White Leghorn embryos as previously described [18]. Cells were subcultured by splitting 1 to 5 until they reached senescence, judged as for human cells. To generate Myc-transformed CEF, early passage cells were infected with the appropriate retroviral SFCV-Myc constructs (10 ␮g) together with 4 ␮g RCAN(A) helper and selected with G418 at 1 mg/ml [18, 19]. Cells were infected with an empty viral vector or with viruses containing c-MYC or v-MYC. Overexpression of the Myc protein was verified by Western blotting of whole-cell lysates using the anti-Myc antibody sc-764 (Santa Cruz) at a dilution of 1:1000. The effects of mitochondrial and cellular stresses. Human HepG2 liver cells were grown in Eagle’s minimal essential medium and mitochondrial protein synthesis was inhibited by the addition of 50 ␮g/ml thiamphenicol as previously described [20]. Cells were removed at various times up to 10 days and analyzed by Western blotting. In a separate experiment, cells were exposed to thiamphenicol continuously for 11 days, after which the mitochondrial protein synthesis inhibitor was removed. Samples were analyzed by Western blotting at various times in the presence of thiamphenicol and at 3 days after its removal. Genotoxic stress was induced in adult mice exposed to 3 Gy of X ray, and tissues were removed after various times and manually homogenized into sample buffer. For each homogenate, 25 ␮g of total protein was analyzed by Western blotting. The responses of the prohibitins to a variety of stresses were measured in HF19 human fibroblasts. Cells were exposed to a range of doses of ultraviolet light

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using a commercial cross-linking apparatus (Stratagene Europe, The Netherlands), or to a range of doses of H 2O 2, or were heat-shocked at 42°C for 30 min. In these experiments, duplicate plates were used to count cell numbers, and cells were collected at various times by direct lysis into sample loading buffer for Western blotting. Models of apoptosis. Apoptosis of prostatic epithelial cells was induced by castration of adult male Wistar rats. Prostates were removed at various time points ranging from 6 h to 7 days afterward, fixed in Methacarn, and processed to paraffin wax for immunoperoxidase studies. Apoptosis of T lymphocytes was induced by treatment of thymocytes from neonatal male Wistar rats with 10 ⫺5 M prednisolone succinate (Solu-Medrone, Upjohn) in RPMI 1640 medium, as previously described [21]. Cells were collected at hourly time points and stained with acridine orange to assess the proportion of cells undergoing apoptosis or were lysed for Western blotting. Subcellular localization. Mitochondria were purified from rodent tissues after homogenization into 250 mM sucrose, 10 mM Tris–HCl, 1 mM EDTA, pH 7.5. The cell suspension was centrifuged for 10 min at 800g, and the pellet was collected as a crude nuclear fraction. Mitochondria were recovered by centrifugation at 8500g for 10 min and the pellet was resuspended in fresh buffer. After a second spin at 800g for 10 min to remove further debris, the mitochondria were again recovered by centrifugation at 8500g for 10 min. Submitochondrial localization used the standard methods of bicarbonate treatment and accessibility to protease digestion after the preparation of mitoplasts by osmotic shock [2, 5]. Immunodepletion. Immunoprecipitation procedures were performed using ice-cold solutions and centrifugations at 4°C. Murine liver or spleen was homogenized manually in PBS to produce a single-cell suspension and lysed in PBS containing 1% Triton X-100 (PBST) and protease inhibitors for 20 min. Human fibroblasts in culture were rinsed twice in PBS before lysis in PBST. The lysates were centrifuged for 10 min at 14,000g and the supernatants retained. The detergent lysates from 2 ⫻ 10 6 HF19 cells in 800 ␮l of buffer were incubated with 100 ␮l of antiserum to Phb2p BAP37 (which had been preloaded onto 100 ␮l of protein A–Sepharose beads (Sigma) and washed three times with PBS to remove serum proteins). As controls, either preimmune serum or protein A–Sepharose alone was used for the immunoprecipitation. After 2 h at 4°C, beads were recovered by centrifugation, and a 50-␮l aliquot of the lysate was removed for Western blot analysis. The remaining lysate was subjected to further rounds of precipitation by adding fresh antibody/ protein A–Sepharose. The protein A beads were also collected after four washes in lysis buffer. SDS–PAGE and Western blotting. All protein extracts and immunoprecipitates were separated by SDS–PAGE using 10 or 12% polyacrylamide resolving gels and blotted onto nitrocellulose membranes, which were blocked in 5% nonfat milk powder in PBS. The primary antibodies to Phb1p or Phb2p BAP37 were diluted in PBS containing 5% swine serum and, following washes in PBS containing 0.05% Tween 20, peroxidase-labeled swine anti-rabbit serum (Dako) was applied diluted 1/1000 for 1 h. After further washes in PBS/ Tween and PBS, immunoreactive sites were revealed by chemiluminescence (Amersham) and exposure to X-ray film. Quantitation of results was carried out using densitometric measurements and the AlphaEase image analysis software package. Analysis of promoter sequences. Computer algorithms were employed to identify potential transcription factor sites in the promoters of human PHB1 (362 nucleotides upstream of the initiating ATG; GenBank L14272 [22]) and rat PHB1 (291 nucleotides; GenBank U17178 [23]), and the 1 kb upstream sequences of human and mouse PHB2 BAP37 [24] (GenBank U47924 and AC002397, respectively). Results are given for the MatInspector program [25].

RESULTS

Phb1p and Phb2p BAP37 Exist as Heteromeric Proteins in the Mitochondrial Inner Membrane The two prohibitin proteins have each been shown to exist in the mitochondria in yeast, amphibians, and mammals [1, 2, 5, 6]. Previous biochemical and fractionation studies have further localized yeast Phb1p and Phb2p BAP37 to the inner mitochondrial membrane [2, 5]. Mammalian Phb1p has also been localized to the inner mitochondrial membrane [26]. Although no similar data exist for mammalian Phb2p BAP37, an interaction with Phb1p has been demonstrated [1], suggesting that it too resides in the inner mitochondrial membrane. To confirm this location, we isolated mitochondria from rodent liver and spleen cells. The two proteins are found in the mitochondrial fraction after differential centrifugation. Neither is solubilized by treatment with carbonate buffer, indicating that they are both integral membrane proteins. The two mammalian proteins are also resistant to proteolytic digestion with proteinase K after the outer membrane has been disrupted by osmotic shock, compatible with results from yeast and indicating their location in the inner mitochondrial membrane. We have previously demonstrated a physical interaction between the two mammalian prohibitin proteins by coimmunoprecipitation and nonreducing electrophoresis [1, 6]. Unresolved issues therefore include whether any free, uncomplexed proteins exist and whether the two prohibitins must bind to each other or can bind to themselves. To answer these questions, we depleted a detergent extract of human fibroblasts by successive immunoprecipitations with an antiserum to Phb2p BAP37 and searched for the presence of Phb1p and Phb2p BAP37 in both the immunoprecipitate and the remaining lysate. We found that immunodepletion of Phb2p BAP37 resulted in a corresponding depletion of Phb1p (Fig. 1). There was no detectable loss of either protein when mock immunoprecipitations were performed using either preimmune serum (not shown) or protein A beads only (Fig. 1), showing that the loss of Phb1p was specific and not caused by protein degradation or nonspecific adsorption. The experiment was also performed using a separate antiserum to Phb1p for immunoprecipitation, and again complete removal of Phb1p from the lysate was accompanied by a full depletion of Phb2p BAP37 . The same results have also been obtained using primary rodent splenocytes and hepatocytes. These data show that all of the Phb1p present in a cell is bound to Phb2p BAP37 , and vice versa.

PROHIBITIN PROTEINS RESPOND TO MITOCHONDRIAL STRESS

FIG. 1. Immunodepletion of Phb2p BAP37 causes depletion of Phb1p. Human fibroblasts were repeatedly immunoprecipitated with anti-Phb2p BAP37 serum bound to protein A beads (lanes 1– 4) or were mock immunoprecipitated with protein A beads only (lanes 5– 8). The original cell lysates and the supernatants after one, two, or three rounds of immunoprecipitation were separated on the same gel and simultaneously assessed for the presence of Phb1p (32 kDa, lower band) and Phb2p BAP37 (37 kDa, upper band) by Western blotting. Lanes 1 and 5 show the original lysates. Lanes 2 and 6 show the proteins present after one addition, lanes 3 and 7 after two additions, and lanes 4 and 8 after three additions of anti-Phb2p BAP37 serum or protein A–Sepharose beads, respectively. Note that both Phb1p and Phb2p BAP37 have been completely depleted in lane 4. In contrast, the prohibitins are maintained in the control precipitations shown in lanes 6 – 8. Lane M contained molecular weight standards.

Phb1p and Phb2p BAP37 Are Widely Expressed Cytoplasmic Proteins in Normal Tissues Murine embryos, fetuses, and adult tissues were immunostained using polyclonal sera specific for either of the two prohibitins. Replacing each of the primary antisera with preimmune serum, or with antiserum which had been preincubated with the corresponding immunizing peptide, resulted in an absence of staining, confirming the specificity of the reagents. The staining patterns of the prohibitin proteins are identical to each other during development and in adult tissues (Fig. 2). Both proteins appear to be expressed in all tissues and cell types, although there are distinct differences in the levels of the proteins in different cell populations. Staining was always cytoplasmic with a distinct granular pattern, in keeping with their location in mitochondria. In adult tissues, relatively high level staining was seen for both Phb1p and Phb2p BAP37 in cardiomyocytes, smooth muscle, and individual skeletal muscle fibers, in renal tubule epithelial cells, in liver hepatocytes, in neural cells in the brain and in ganglia in the intestinal wall, in pancreatic islets, in ovarian oocytes, and in scattered phagocytic cells in the spleen and epithelial tissues such as the lung and skin. Moderate levels were seen in epithelial cells of the skin, gastrointestinal tract, adrenal cortex, bladder, and prostate and in large lymphoblastic cells in the spleen and thymus. Low level staining was seen in connective tissue fibroblasts, in supporting cells in the brain, in alveolar lining cells of the lung, in the exocrine pancreas, in the endothelial cells of blood vessels and kidney glomeruli, in the adrenal medulla, in mature spermatids in the testis, and in small lymphocytes in the spleen and thymus. Representative sections are shown in Fig. 2. These data are in broad agreement with more limited immunohistochemical studies of the expression of

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Phb1p [27, 28]. The data additionally reveal the very close association of expression of the two individual members of the prohibitin family in mammals. When considering the proposed role of the prohibitins as negative regulators of the cell cycle, we found a lack of correlation between protein expression and proliferative state. For instance, some nonproliferative cells such as neurons, hepatocytes, and heart muscle express high levels, while other postmitotic cells such as sperm, endothelium, and neural-derived cells of the adrenal medulla express low levels of the proteins (Fig. 2). Examination of the expression of Phb1p and Phb2p BAP37 during murine development showed similarly complex patterns of staining between different tissues and at different stages of development, with an overlap in the expression patterns between embryonic and adult tissues. Thus, throughout gestation, high level expression was seen in brown fat, heart, liver, developing renal tubules, and neurons, while tissues such as lung and exocrine pancreas showed relatively lower levels of expression. We also found that the expression of both proteins was higher in undifferentiated cells of newly forming tissues, such as in the developing lung and pancreas, compared to their fully differentiated counterparts. Immunocytochemistry performed on 70 surgically removed human neoplasms from a variety of anatomical sites revealed high level expression of both Phb1p and Phb2p BAP37 in all tumors (Fig. 3). When there were areas of tumor surrounded by nonneoplastic tissue in the same section, we noticed a clear overexpression of both prohibitins in the neoplastic areas, compared to the surrounding normal tissue (Fig. 3A). In view of the nonquantitative nature of the immunoperoxidase assay, combined with variations of size and length of fixation of these clinical samples, it is not possible to quantify the apparent overexpression of the prohibitins in neoplastic versus normal cells. However, studies of experimental tumors and cell lines show that elevated Phb1p expression is a general feature of neoplastic cells [13, 14, 29, 30]. Phb1p and Phb2p BAP37 Levels Decrease Heterogeneously during Cellular Aging In yeast, deletion of the PHB1 and PHB2 genes accelerates the aging process. To investigate a role for the prohibitins in aging of higher species, we studied whether the levels of the prohibitins are altered during cellular senescence of human fibroblasts. We used HF19 human diploid fibroblasts, which cease to divide after approximately 60 population doublings (PD) but remain viable for a prolonged period of time. The onset of senescence was accompanied by an increase in population doubling time, by the characteristic increase in

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FIG. 2. Immunocytochemical distribution of the prohibitins in rodent tissues. Immunoreactivity is seen as brown staining, and cell nuclei are stained blue. The left shows the distribution of Phb1p, while the right shows the distribution of Phb2p BAP37 in the same tissues. In the brain (A and B), strong staining for Phb1p and Phb2p BAP37 is seen in neurons, while glia and other supporting cells are relatively unstained (scale bar, 50 ␮m). In the kidney (C and D), strong staining of renal tubular cells is apparent, while glomeruli are only weakly positive (scale bar, 200 ␮m). In the prostate (E and F), Phb1p and Phb2p BAP37 are most prominent in the lining epithelial cells (scale bar, 50 ␮m). In the adrenal gland (G and H), moderate staining for both Phb1p and Phb2p BAP37 is seen in the cortex (c), with the adrenal medulla (m) shows only very weak expression (scale bar, 200 ␮m).

cell size and flattened morphology of senescent fibroblasts, and by the accumulation of cells exhibiting senescence-associated ␤-galactosidase activity [17]. Immunofluorescence of young cells (PD 20 or less) showed a uniform pattern of expression of both Phb1p and Phb2p BAP37 in mitochondria, identified by the colocal-

ization of each antiserum with monoclonal antibodies against mitochondrial cytochrome c oxidase subunits (Figs. 4A and 4C). In contrast, senescent cells showed variability in their expression of both the prohibitins, with approximately 10% of cells exhibiting a marked reduction in levels. Figures 4B and 4D present exam-

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FIG. 3. Immunocytochemical demonstration of Phb1p and Phb2p BAP37 in human tumors. Sections of melanoma with surrounding normal skin were stained for Phb1p (A) and for Phb2p BAP37 (B), using aminoethyl carbazole to give a red color, and cell nuclei were counterstained with hematoxylin (blue). The tissue is oriented such that the keratinized external surface of the skin runs across the left-hand side of the photographs. Note the increased intensity of immunoperoxidase staining in a tumor nodule, compared to the surrounding normal epithelium (scale bar, 50 ␮m). The sections in C and D are of a breast carcinoma, stained for Phb1p and Phb2p BAP37, respectively, using DAB as chromogen (brown). Islands of tumor cells show strong staining and are surrounded by more weakly stained nonneoplastic stromal cells (bar, 25 ␮m). E and F show sections of a testicular seminoma. Strong staining is seen in tumor cells, identified by their enlarged nuclei.

ples of senescent cells showing diminished fluorescence for the prohibitins but an unchanged level of staining for cytochrome c oxidase subunit IV, indicating that the cells are viable but have a specific decreased expression of the prohibitins. Measurement of mitochondrial membrane potential in these cells showed a uniform peak in young cells, compared to markedly heterogeneous levels in senescent cells (Fig. 5). To demonstrate that decreased expression of the prohibitins is a conserved feature of cellular senescence, we also examined the expression patterns of the prohibitins in primary chick fibroblasts, which senesce after approximately 40 PD. Here again, immunocytochemical analysis revealed a marked heterogeneity in the staining of late passage cells, which was not apparent in early passage cells (Fig. 6).

Expression during Cell Cycle, during Apoptosis, or in Response to Stress Western blotting of proteins extracted from primary human lymphocytes revealed temporally coordinated increases in the levels of Phb1p and Phb2p BAP37 in cells stimulated to enter the cell cycle by exposure to phytohemaglutinin. Densitometry indicated a 3-fold increase in both proteins when the cells have entered the G 1 phase of the cell cycle and expression was maintained during subsequent progression of the cells through S phase and mitosis (Fig. 7A). As a marker protein known to be induced during cell cycle entry, these samples were also probed for proliferating cell nuclear antigen (PCNA) using the monoclonal antibody PC10. As expected, the levels of PCNA were in-

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duced 30-fold after exposure to phytohemaglutinin in this experiment (data not shown). These results therefore indicate a moderate induction of the two prohibitin proteins during cell cycle entry. Examination of the possible relationship between the prohibitin proteins and programmed cell death pathways was studied in two independent models. Apoptosis of immature thymocytes was induced 2 h after steroid treatment and apoptotic nuclei increased steadily up to 8 h. Western blotting of lysates from equal numbers of cells showed no changes in the levels of either Phb1p or Phb2p BAP37. For the prostate model, apoptotic nuclei were first seen in epithelial cells after 24 h, and the numbers peaked at 72 h and declined thereafter. By immunoperoxidase staining, we could find no evidence of any alteration in the expression of either Phb1p or Phb2p BAP37 in apoptotic or surviving cells. Consequently, it is doubtful that the prohibitins play a role in regulating apoptotic pathways in mammals, despite the observation that trypanosome prohibitin is up-regulated during programmed cell death of the parasites [15]. In view of the links between aging and stress responses, particularly oxidative stress [31], we investigated a possible role for the prohibitins in the cellular response to oxidative and other stresses. Western blotting and immunocytochemistry were used to assess the levels of Phb1p and Phb2p BAP37 in primary fibroblasts exposed to hydrogen peroxide, UV light, or heat shock. Proteins extracted from rodent tissues following wholebody X-irradiation were also examined. We could find no changes in the levels of either Phb1p or Phb2p BAP37 in any of these experiments (data not shown), and yeast cells deleted for the prohibitins do not show different sensitivities to a variety of stresses [1]. Thus, it is unlikely that the prohibitins influence aging by modulating these types of stress responses. To investigate a role for the mammalian prohibitins in the regulation of mitochondrial enzyme assembly during times of altered mitochondrial metabolism, we created an imbalance in respiratory enzyme subunits by inhibiting mitochondrial protein synthesis in human liver cells with thiamphenicol [20]. Levels of Phb1p and Phb2p BAP37 were measured by Western blotting and quantitated by densitometry. The levels of GDH were similarly measured and used to normalize any changes in prohibitin expression. Both Phb1p and Phb2p BAP37 showed a relative twofold increase after 11 days of continuous thiamphenicol treatment. Interestingly, withdrawal of drug at this time caused a further elevation of prohibitin levels 3 days later, to threefold the original levels. In a separate experiment, cells were continuously exposed to thiamphenicol for up to 10 days, and the prohibitins and the mitochondrial chaperone cpn60 were analyzed by Western blotting at intervals. The levels of the prohibitins were unchanged

or slightly lower for the first 5 days of treatment and showed a twofold increase after 10 days of continuous exposure. In contrast, the levels of cpn60 remained relatively constant throughout this period (Fig. 7B). The PHB1 and PHB2 Genes Are Regulated by Myc A previous analysis of the promoter regions of the rat and human PHB1 genes revealed the presence of conserved CAAT boxes and Sp1 sites [23], which are present in many housekeeping genes. We have reexamined the promoter region of PHB1 from human and rat and compared these with 1 kb of the promoter regions from the human and mouse PHB2 BAP37 genes. Similar to PHB1, there are conserved CAAT and Sp1 sites in these PHB2 BAP37 promoter regions. We also discovered that both of the PHB2 BAP37 promoters contain consensus sites for Myc at position ⫺95, and a Myc site is present in the human PHB1 promoter at nucleotide ⫺355 (the corresponding sequence for the rat promoter is not available). To investigate whether the PHB1 and the PHB2 BAP37 genes are directly regulated by Myc, we infected primary chick fibroblasts with avian retroviruses coding for the Myc oncoprotein. Western blotting of proteins extracted from equal numbers of cells infected with retroviruses containing cMYC or v-MYC showed a clear induction of both Phb1p and Phb2p BAP37 compared with cells infected with an empty virus control (Fig. 7C). DISCUSSION

The prohibitins comprise a pair of highly conserved proteins whose expression influences mitochondrial function and the life span of budding yeast [1, 2, 5, 6]. The two proteins bind to each other in mammalian and yeast cells and exist in a high-molecular-weight complex in the inner mitochondrial membrane of these species [1, 5, 6]. The prohibitins are probably the only components of this complex, implying that they exist in an oligomeric state [6]. Deletion of the PHB1 gene from yeast results in an apparent absence of Phb2p, while deletion of PHB2 gene causes a similar lack of Phb1p [2], suggesting that each prohibitin protein is unstable in the absence of its partner and that they rely on each other for their function(s). Our depletion experiments clearly show that the mammalian proteins are always bound to each other and, in combination with our immunochemical demonstrations of coexpression, support the idea of mutual interaction being a critical feature of the two proteins. The function of the prohibitins has recently become clear in yeast from studies indicating a role in the assembly of mitochondrial respiratory chain enzymes [5, 6]. They appear to be especially important in situations in which there is an imbalance in the synthesis

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of enzyme subunits [6]. To directly test whether the prohibitins have a similar function in mammals, we inhibited mitochondrial protein synthesis to create an imbalance in the ratios of nuclear- and mitochondrialencoded gene products. In these experiments, both of the prohibitins were induced by prolonged treatment with thiamphenicol, a specific inhibitor of mitochondrial protein synthesis. The results are comparable to those seen in yeast with reduced mitochondrial protein synthesis due to genetic mutation, which causes a similar up-regulation of expression of the prohibitins [6]. These experiments clearly show that the mammalian prohibitin proteins respond to situations in which there is an imbalance between cytoplasmic and mitochondrially translated proteins, indicating their role during situations of metabolic stress. Our proposal that the prohibitins function to regulate mitochondrial respiration, particularly in situations of mitochondrial stress, is supported by analyses of the expression patterns of the two proteins. In normal tissues, there is no correlation between expression of the prohibitins and proliferative status. However, the prohibitins are most highly expressed in cells that show a particular reliance on mitochondrial metabolism, including neurons, muscle, renal tubules, adrenal cortex, brown adipocytes, and pancreatic islet cells. These cell types are especially susceptible to mitochondrial dysfunction, and mitochondrial diseases affect one or more of these tissue types [32]. Previous data relating an increased expression of Phb1p during cell cycle entry in vitro have been interpreted to indicate a role in cell proliferation [26]. In our studies, we found an increase in expression of both of the prohibitins during cell cycle entry of primary human lymphocytes, similar to the previous observations of Phb1p induction in primary fibroblasts. However, since there is no relationship between prohibitin expression levels and proliferation in vivo, and because it is known that primary lymphocytes undergo a switch from aerobic to anaerobic metabolism during cell cycle entry [33], these results are more easily explained by a role for the prohibitins during metabolic change. Furthermore, tumor cells characteristically rely on glycolysis for energy production even when oxygen is present, the so-called “Warburg effect” [34], and our data show that tumors consistently express high levels of both of the prohibitins. Thus, high expression of the prohibitin proteins in tumors is also compatible with their proposed role during situations of altered metabolism. Similarly, during embryogenesis the high level expression seen in relatively undifferentiated tissues again indicates that the high metabolic activity of these rapidly changing cells is accompanied by high level expression of the two prohibitins. We are particularly interested in the connection between the prohibitins and the aging process. In S.

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cerevisiae, artificially reducing the levels of Phb1p and/or Phb2p BAP37 by gene deletion causes a reduction in life span [1, 2]. To determine whether the relationship between expression of the prohibitins and aging extends into higher species, we used Western blotting and immunofluorescence to measure protein levels during cellular senescence. In senescent human and avian fibroblasts, immunofluorescence revealed a clear decrease in the levels of Phb1p and Phb2p BAP37 in some cells. In agreement with a previous report that studied only Phb1p [35], we could not detect a fall in protein levels using Western blotting. The lack of changes in protein levels using Western blotting is not surprising, since this method indicates the average amount of protein per cell, and only a small proportion of the population of senescent cells shows a decrease in expression of the prohibitins by immunofluorescence. This variable decrease in prohibitin expression ties in with the marked heterogeneity of mitochondrial membrane potential seen in senescent cells by FACS analysis. These observations highlight the usefulness of single-cell assays to identify changes in individual cells, and such alterations in mitochondrial function have previously been reported during in vitro aging of human fibroblasts, as well as in rodent and human hepatocytes and human muscle in vivo [36 –39]. Analysis of mitochondrial DNA abnormalities has also revealed heterogeneity in the accumulation of mutations in individual fibers in aged human heart [40]. Consequently, there are extensive data showing that a variety of mitochondrial parameters change heterogeneously during aging both in vitro and in vivo, and our studies indicate that the mitochondrial prohibitin proteins are a part of these processes. A simple explanation for the fall in levels of the prohibitins in individual cells during aging would be that a loss of mitochondrial function causes a concomitant loss of the prohibitins. However, levels of the prohibitins are maintained in rho 0 mammalian cells, which have no mitochondrial respiratory activity due to a lack of mitochondrial DNA [6], indicating that the observed aging-associated mitochondrial dysfunction does not by itself cause any loss of prohibitin expression. On the other hand, yeast deleted for the prohibitins show increased mitochondrial abnormalities [1, 2], suggesting that lower levels of the prohibitins during cellular senescence of human cells contribute to the age-related loss of mitochondrial function. In yeast, absence of the PHB genes causes an accelerated rate of aging [1, 2] and recent data have linked this effect to increased levels of reactive oxygen species in PHB-null cells [6]. Therefore, given the similarities between the yeast and the human proteins, the decreased expression of the prohibitins that we have found during aging of mammalian cells would be predicted to cause a similar increase in the production of

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FIG. 4. Cellular senescence of fibroblasts is associated with heterogeneity of the content of Phb1p and Phb2p BAP37. HF19 human diploid fibroblasts were stained with rabbit antiserum against Phb1p or Phb2p BAP37 (green, left) and simultaneously stained with a mouse monoclonal antibody to cytochrome c oxidase subunit IV as a mitochondrial marker (red, middle). The right side represents a merging of the two images. (A) Early passage fibroblasts show colocalization of Phb1p and COX IV, resulting in a yellow color in the merged image. (B) The late passage fibroblasts pictured show a comparative reduction in Phb1p staining, resulting in a redder appearance in the merged image. (C) There is uniform expression of Phbp2 BAP37 and COX IV in the mitochondria of young cells, resulting in a yellow coloration in the merged image. (D) In senescent cells, both of the senescent cells pictured are equally positive for COX IV, while the cell to the right of the image shows decreased Phb2p BAP37. In consequence, the merged image shows one cell with an overall yellow appearance and the other appears predominantly red. Scale bar, 25 ␮m for all images. FIG. 6. Expression of the prohibitins in chick fibroblasts. Cells were stained with Phb2p BAP37 antiserum shown in green and nuclei were counterstained with propidium iodide shown in red. (A) Young cells show a uniform expression of Phb2p BAP37. (B) Senescent cells show heterogeneity in the level of Phb2p BAP37, with the cell pictured on the right showing reduced expression.

reactive oxygen species. Indeed, senescence of human fibroblasts is accompanied by increased oxidative stress [41], and hepatocytes from old rats contain subpopulations of cells with reduced mitochondrial metabolism and higher levels of reactive oxygen species [37]. Increased accumulation of reactive oxygen species is clearly important for the aging process, since protection from oxidative damage extends the life span of human fibroblasts in vitro and of intact organisms [41– 43]. Furthermore, oxidative stress causes telomere shortening [44], linking nuclear changes that

occur during cellular aging with the mitochondrial changes that also take place. In this respect, it is interesting to note that Myc expression can extend life span in vitro, an effect which has been related to direct transcriptional activation of telomerase [45], but which from our data might also be mediated by an enhanced expression of the prohibitins. These observations emphasize the importance of metabolic factors in the overall aging process and suggest the existence of interrelationships between the nuclear and the cytoplasmic changes that are seen during aging.

PROHIBITIN PROTEINS RESPOND TO MITOCHONDRIAL STRESS

In conclusion, we have provided evidence to indicate a conserved role for the mammalian prohibitin proteins in mitochondrial metabolism and have shown that the levels of the proteins decrease during aging. Because deletion of the prohibitin genes accelerates aging in yeast, in which they act as chaperone/holdase-like molecules for assembly of respiratory enzymes, the data indicate that the ability of cells to regulate mitochondrial enzyme assembly might be a factor that influences the rate of aging. This is the first time that the regulation of assembly of mitochondrial respiratory enzyme complexes has been implicated in aging, even though it is well known that metabolic activity and stress responses have profound effects on longevity [31]. It is also of interest that mutations in a human member of the m-AAA protease family (with which the prohibitins interact in yeast [5]) are responsible for some forms of the neuromuscular degenerative disease heredi-

FIG. 5. Heterogeneity of mitochondrial membrane potential during cellular aging. HF19 cells were stained with the mitochondrial membrane-potential-sensitive dye DiOC 6(3) and analyzed by FACS. Fluorescence values are plotted on a logarithmic scale on the x axis and the number of cells with a given fluorescence value are given on the y axis. (A) Young fibroblasts show a uniform peak of fluorescence. (B) Senescent cells show a wide variation in mitochondrial membrane potential, with additional peaks visible both above and below the value seen for young fibroblasts.

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FIG. 7. (A) Induction of Phb1p and Phb2p BAP37 following PHA stimulation of lymphocytes. Human peripheral blood lymphocytes were exposed to PHA and collected at time 0 and at the indicated hours afterward. Proteins extracted from 1.5 ⫻ 10 5 cells were Western blotted for Phb1p and the blot was stripped and probed again for Phb2p BAP37. (B) Inhibiting mitochondrial protein synthesis induces expression of the prohibitins. Human HepG2 hepatocytes were treated with thiamphenicol for the number of days indicated. Levels of the prohibitins and of the mitochondrial chaperone cpn60 were analyzed by Western blot analysis. (C) Overexpression of Myc induces expression of the prohibitins. Chick fibroblasts were infected with an empty vector (lanes 2 and 6) or with retroviruses coding for c-Myc (lanes 3 and 7) or for v-Myc (lanes 4 and 8). Lanes 1 and 5 contain control uninfected cells. Duplicate samples of proteins from 5 ⫻ 10 4 cells were subjected to Western blotting for Phb1p in lanes 1– 4 or for Phb2p BAP37 in lanes 5– 8.

tary spastic paraplegia [46]. Thus, defects in the processing of mitochondrial proteins can directly cause progressive cellular degeneration in humans. From this, deficiencies in any of the components of the quality control system for mitochondrial respiratory enzyme assembly, including other members of the protease family, the prohibitins, and other mitochondrial chaperones, are candidates for accelerated aging and age-related degenerative diseases. Factors which are able to maintain or reactivate these pathways may therefore help to delay the onset of these processes. This approach would represent an alternative to manipulating mitochondrial metabolism by reducing calorie intake, or the administration of mitochondrial coenzymes or antioxidants, each of which can be at least partially effective in delaying aging and/or cellular senescence [31, 41, 42, 47]. We are grateful to K. Townsend for help with confocal microscopy, to P. Bonner for irradiations, and to T. Hacker for histology. The work was supported by the Medical Research Council and the Ministry of Health in the Czech Republic (Grant IGA 4783-3).

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COATES ET AL. against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalinfixed paraffin sections. J. Pathol. 168, 357–363.

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