Down-regulation of the Mitochondrial Translation System during Terminal Differentiation of HL-60 cells by 12- O -Tetradecanoyl-1-phorbol-13-acetate COMPARISON WITH THE CYTOPLASMIC TRANSLATION SYSTEM*

Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell func-tioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12- O -tetradecanoyl-1-phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly down-regulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPA-induced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory


Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell functioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12-O-tetradecanoyl-1phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly downregulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors
showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPAinduced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory mechanisms of mt translational activity upon terminal differentiation (in response to the growth arrest) is different to that of the cytoplasmic system, where the control of mRNA translational efficiency of major translation factors is the central mechanism for their down-regulation.
Mitochondrial (mt) biogenesis depends on both the nuclear and mitochondrial genomes (1,2). Mitochondrial DNA (mtDNA) 1 is present in 10 3 -10 4 copies per cell and encodes 13 proteins, which are critical subunits of the respiratory chain complexes I, III, IV, and V, as well as 2 ribosomal RNAs and 22 transfer RNAs, which are necessary for mitochondrial protein synthesis (3). Nuclear genes encode the majority of the respiratory chain subunits and all protein components necessary for maintenance and expression of mtDNA. Mitochondria play pivotal roles in eukaryotic cells in producing cellular energy and essential metabolites as well as in controlling apoptosis by integrating numerous death signals (4). Recently, evidence has emerged that mitochondria are also implicated in the regulation of cell growth and differentiation. Inhibition of mitochondrial activity either by deleting mtDNA (rho°cells) or by blocking translation in the organelle has been shown to arrest or decrease proliferation in various cell lines (5)(6)(7)(8)(9). Mitochondrial protein synthesis inhibition is associated with the impairment of differentiation in different cell types, including mouse erythroleukemia (10) and mastocytoma cells (11), neurons (12), and human (13), avian (14), or murine myoblasts (15). The coordination of mitochondrial and nuclear genetic systems in the cell is necessary for proper mitochondrial biogenesis and cellular functioning. However, little is known either about the control of mitochondrial translation activity or about the regulatory mechanisms governing the expression of mammalian mitochondrial translation factors.
As a first step toward understanding the integrated processes that regulate mitochondrial translation we studied the expression of mt translation factors during TPA-induced differentiation of HL-60 cells. The promyelocytic leukemia cell line, HL-60, is one of the best studied models of cell differentiation (16,17). This line comprises 90 -95% of cells with myeloblastic/ promyelocytic morphology. A variety of agents can induce differentiation of these cells either to granulocyte-like (dimethyl sulfoxide) or to monocyte/macrophage-like (TPA or 1,25-dihydroxyvitamin D3) cells (16). The effects of TPA are thought to be due in part to the activation of protein kinase C␤ (18,19). Induction of differentiation by TPA in HL-60 cells is associated with activation of the stress-activated protein kinase, mitochondrial swelling, permeability transition, release of cytochrome c, activation of caspases, and ultimately the induction of apoptosis (20 -22).
In this report we demonstrate down-regulation of the mt translation system during terminal differentiation of HL-60 cells by TPA. In contrast to cytoplasmic translation factors, which are down-regulated at the level of mRNA translation during terminal differentiation (in response to the growth arrest), the regulation of stability and transcription rather than translational efficiency of mRNA is crucial for gene expression of mt translation factors. The biological significance of down-regulating the mt translation system during terminal differentiation is also discussed.

EXPERIMENTAL PROCEDURES
Cells and Culture Conditions-The HL-60-rapid growth (RG) derivative of the HL-60 cell line was used throughout the study (a gift from Dr. T. Yamaguchi, National Institute of Health Sciences). Because its DNA profiling and sensitivity to TPA-induced differentiation are almost the same as that of the parental HL-60 cell line, we chose HL-60RG to take advantage of its rapid growth rate (normal doubling time, 24 h). Cells were routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Intergen) in the presence of penicillin (100 units/ml) and streptomycin (100 unit/ml). Cells were routinely passaged twice weekly to a density of 0.2-1.0 ϫ 10 6 cells/ml and grown at 37°C in 5% CO 2 . Induction of differentiation (20 ml of 4 ϫ 10 5 cells/ml for 150-mm plate) into monocyte/macrophage-like cells was achieved by the addition of 50 nM TPA (Sigma). After a 48-h exposure to TPA, proliferation was arrested, and almost all the cells adhered to the plastic dishes in the form of aggregates; they subsequently spread out and acquired a spindle-shaped morphology and prominent pseudopodia. This series of events was confirmed microscopically in all experiments.
RNA Isolation and Northern Analysis-Total RNA was isolated from cells using ISOGEN (Nippongene). RNA (10 -20 g/sample) was sizefractionated on a 1% agarose-formaldehyde gel, transferred to a Hybond-Nϩ membrane (Amersham Biosciences), and then UV-crosslinked to the membrane. The membrane was prehybridized for 1 h at 68°C in ExpressHyb (Clontech) and then hybridized in fresh Ex-pressHyb with a 32 P-labeled cDNA probe at 1-2 ϫ 10 6 cpm/ml and salmon sperm DNA at 20 g/ml overnight (or for 2 nights for rare RNA species) at 68°C. The membrane was washed with 2ϫ SSC (1ϫ SSC ϭ 0.15 M NaCl and 0.015 M sodium citrate), 0.05% SDS at 68°C and then with 0.1ϫ SSC -0.1% SDS at 55°C. Membranes were analyzed by autoradiography and quantified using a BAS5000 analyzer (Fuji Film). The radiolabeled cDNA probes for Northern blots were prepared by random priming with a BcaBEST labeling kit (TAKARA). For analysis of mt transcripts (Fig. 1, 16 S, cytochrome c oxidase II (COII) and COIII), DNA templates were obtained by PCR of isolated HL-60RG total DNA. For analysis of nuclear transcripts ( Fig. 1 remaining samples), template DNAs were amplified either by RT-PCR using HL-60RG total RNA and oligo-dT primers or by PCR of plasmids containing the objective sequences. Primers used for PCR are summarized in Table I. mRNA Turnover Studies-Cells were grown to 4 ϫ 10 5 cells/ml, and the transcription inhibitor actinomycin D (ActD) (Sigma) was added to the medium at the concentration of 5 g/ml. For differentiated cells, the ActD chase was initiated as follows. After 48 h of exposure to TPA, the culture medium was aspirated off to remove unattached cells and replaced with a new medium containing ActD (5 g/ml). Total RNA was isolated from cells at the indicated time points and subjected to Northern analysis.
Nuclear Run-on Transcription Assay-Isolation of nuclei, run-on transcription, and hybridization were performed essentially as described in Greenberg and Bender (23), with minor modifications. Ap-proximately 1 ϫ 10 8 growing (4 ϫ 10 5 cells/ml) and differentiated (50 nM TPA, 48 h) HL-60RG cells were harvested and washed using ice-cold PBS. Before harvesting differentiated HL-60RG cells, unattached cells were carefully removed by washing twice with PBS. Cell pellets were lysed in 3.6 ml of ice-cold sucrose buffer I (0.32 M sucrose, 3 mM CaCl 2 , 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40), and 3.6 ml of ice-cold sucrose buffer II (2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol) was added to the nuclei before they were collected in 4 ml of sucrose buffer II in a SW41Ti tube (Beckman Instruments). After centrifugation (45 min at 30,000 ϫ g, 4°C), the nuclear pellet was resuspended in glycerol storage buffer (50 mM Tris-HCl (pH 8. and incubated at 30°C for 30 min. The mixture was treated with 100 units of RNase-free DNase I for 10 min at 30°C, then further incubated with 400 l of proteinase K solution (20 mM Tris-HCl (pH 7.5), 2% SDS, 10 mM EDTA, 200 g/ml proteinase K) for 30 min at 42°C. After phenol/CHCl 3 extraction, transcripts were precipitated with isopropanol. The RNA was again treated with DNase I, proteinase K, phenol/ CHCl 3 extraction, and ethanol precipitation. Labeled RNA was dissolved in 50 l TES (10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.2% SDS). Approximately 2 ϫ 10 7 cpm of labeled RNA was reproducibly obtained from 1 ϫ 10 8 growing cells and 1.5 ϫ 10 7 cpm from the same amount of differentiated cells. Linearized plasmid constructs (10 g) containing the relevant cDNA were alkali-denatured and immobilized onto a Hybond Nϩ membrane (Amersham Biosciences). Hybridization was carried out in 2 ml of ULTRA Hyb (Ambion) with 2 ϫ 10 7 cpm of radiolabeled RNA and salmon sperm DNA at 100 g/ml over 2 nights at 50°C with rotation. The membrane was washed with 2ϫ SSC, 0.1% SDS at 50°C, and unhybridized RNA was finally removed with 10 g/ml RNase A in 2ϫ SSC (37°C, 30 min). Membranes were analyzed using a BAS5000 analyzer (Fuji Film). We confirmed that the intensity of the actin signal was linearly obtained using 4 ϫ 10 6 -2 ϫ 10 7 cpm of radiolabeled transcripts.
The plasmids containing ␤-actin and the myeloperoxidase (MPO) gene were prepared as follows. Part of the ␤-actin and MPO cDNA sequences were amplified by RT-PCR of HL-60RG total RNA with the oligo-dT primer and the primers described in Table I. DNA fragments were cloned into pCR 2.1 TOPO vector (Invitrogen). pcDNA3.1/Zeoϩ (Invitrogen) and hEF-Tumt/pcDNA3.1/Zeoϩ were used for the detection of human EF-Tumt transcripts.
Immunoblot Assay for Mitochondrial Protein-Growing (4 ϫ 10 5 cells/ml) and differentiated HL-60RG cells were harvested and lysed in ice-cold lysis buffer (PBS containing 1% (v/v) Triton X-100 and protease inhibitor mixture (Roche Applied Science)). After 30 min on ice, the lysate was centrifuged at 14,000 ϫ g for 20 min at 4°C, after which the supernatant was recovered and stored at Ϫ80°C. Total protein concen- Primers used for the construction of plasmids for nuclear run-on assays.
MPO 1068 Primers used for RT-PCR in polysome analysis. L19 324 mt Translation Down-regulation during HL-60 Differentiation tration of the lysate was determined using the Bio-Rad protein assay; the proteins (10 -50 g/lane) were separated by SDS-PAGE and subsequently transferred to nitrocellulose. The membrane was blocked with 5% nonfat dried milk in PBS containing 0.1% Tween 20 at 22°C for 1 h before incubation with a human EF-Tu/Ts mt polyclonal antibody (1: 1000; anti-rabbit) or human COII monoclonal antibody (1:1000; antimouse, Molecular Probes) and horseradish peroxidase-conjugated antirabbit (or mouse) immunoglobulin G (1:4000; Amersham Biosciences); the protein was visualized by ECL (Amersham Biosciences) and quantified by LAS-plus (Fuji Film). The blots were also probed with a human monoclonal ␤-actin antibody (1:5000; Sigma) as a control for loading. Pulse Labeling of Mitochondrial Translation Products-Labeling of mt translation products was performed as described in Hayashi et al. (24). Briefly, cells were labeled for 60 min at 37°C in methionine-free Dulbecco's modified Eagle's medium containing 100 Ci/ml [ 35 S]methionine and 100 g/ml emetine. Cell pellets were resuspended in sonication buffer (2% SDS, 10 mM Tris-HCl (pH 6.7)) and sonicated, and total cellular protein (100 g) was loaded and separated on Tricine-SDS-PAGE (16.5% (w/v); acrylamide/bis-acrylamide ϭ 32:1) gels. Radiolabeled mitochondrial proteins were analyzed by autoradiography and quantified using a BAS5000 analyzer (Fuji Film).
Polysome Analysis-Fractionation of HL-60RG polysomes and isolation of RNA contained in the fractions was carried out using a modification of a published protocol (25). Approximately 3 ϫ 10 7 cells were used for each gradient (growing cells, 4 ϫ 10 5 cells/ml; differentiated cells, 50 nM TPA, 48 h). Before harvesting, cells were incubated with the medium containing 100 g/ml cycloheximide for 5 min and washed twice with PBS (containing 100 g/ml cycloheximide). Cell pellets was resuspended in 1 ml of lysis buffer (20 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl 2 , 0.04 M sucrose, 0.5% Nonidet P40, 1 mM dithiothreitol) containing 100 units of RNase inhibitor and lysed by incubation on ice for 10 min with occasional shaking. Nuclei and cell debris were removed by centrifugation at 1,000 ϫ g for 10 min. The lysate was layered on top of a 11-ml 15-50% (w/v) sucrose gradient and centrifuged at 36,000 rpm in a Beckman SW41Ti rotor for 2 h 15 min at 4°C. Gradients were separated into 12 equal fractions using a density gradient fractionator (Towa Labo, Model 152-001) while monitoring absorbance at 260 nm. Each fraction was treated with proteinase K, and RNA was extracted by phenol/CHCl 3 , precipitated with ethanol, and analyzed for each mRNA species. L19 and ␤-actin mRNAs were detected by semi-quantitative RT-PCR using oligo-dT primers. The number of amplification cycles in PCR was optimized to maintain the PCR reaction within the linear range. Primers used for PCR are summarized in Table I. EF-Tumt, L7/L12mt, and S12mt mRNAs were detected by Northern blotting.

Mitochondrial Translation Factor mRNAs Are Markedly Down-regulated in a Coordinate and Specific Manner during TPA-induced Differentiation of HL-60 Cells-Despite a myriad
of recent studies on gene expression during TPA-induced differentiation of HL-60 cells using both DNA microarray (26,27) and differential display methods (28), little is known about the expression of mt translation factors. It is possible that the results obtained to date might be ambiguous due to the low levels of these mRNAs or because some objective genes were absent from the DNA arrays.
In contrast we observed only a slight reduction of mRNA expression for the cytoplasmic (cyt) translation factor, ribosomal protein L19 (Fig. 1B). Similarly, others also observed, using DNA microarray technology, that mRNA levels of some cyt translation factors (such as elongation factor 1␣, ribosomal proteins S27 and L27a) are not down-regulated during this differentiation process (27). Therefore, mt translation factor mRNAs appear to be specifically down-regulated in a stringent manner during TPA-induced differentiation of HL-60 cells.
Nuclear respiratory factors (NRF-1 and NRF-2) govern the transcription of many, but not all, nuclear-coded mt genes (29,30). mtDNA-coded mRNAs are also downstream of NRFs, since their transcription is governed by Tfam (mt transcription factor a), whose transcription itself is governed by NRF-1 (31). The mRNA levels of both NRF-1 and NRF-2␣ were significantly decreased by TPA-induced differentiation, and thus, the mt mt Translation Down-regulation during HL-60 Differentiation oxidative phosphorylation-related genes COII, cytochrome c oxidase III (COIII), and F1-ATP synthetase ␤ subunit (F1-ATPase-␤) also declined, as was expected (Fig. 1, C and D). Furthermore, previous studies reported that the DNA binding activity of NRF1 is growth-regulated (32) and that expression of L7/L12mt mRNA is regulated in a growth-dependent manner (33). These results suggest that most mt genes are coordinately down-regulated, at least at a transcriptional level, through NRFs either in a direct or indirect manner in response to growth arrest that is associated with terminal differentiation. In agreement with this, another nuclear-coded mt mRNA, mt heat shock protein 70 (mt Hsp70), is also down-regulated after TPA-induced differentiation of HL-60 cells (34).

TPA-induced Decrease in EF-Tumt mRNA Results
Mainly from the Decreased mRNA Stability-It is noteworthy that the extent of mRNA reduction is much more profound for mt translation factors than for other mt-related transcripts (Fig. 1, A  and D). This indicates that decreased stability as well as the decreased transcription rate of mt translation factor mRNA contributes to their marked down-regulation during TPA-induced differentiation. To verify this possibility, nuclear run-on and ActD chase assays were employed to assess the effect of TPA on transcription rate and mRNA stability, respectively. The EF-Tumt transcript, which is one of the most abundant transcripts among mt translation factors, was analyzed.
MPO mRNA level is also down-regulated after the TPAinduced differentiation of HL-60 cells, mainly due to its decreased transcription rate (35,36). A nuclear run-on assay indicated that EF-Tumt transcription is apparently not downregulated after exposure of HL-60RG cells to TPA (50 nM, 48 h), whereas MPO gene transcription is reduced to less than 20% basal level ( Fig. 2A). On the contrary, ActD chase studies demonstrated that EF-Tumt mRNA was significantly destabilized after the exposure to TPA (50 nM, 48 h) (Fig. 2B). It is difficult to precisely evaluate mRNA stability for each condition by simply comparing the mRNA half-life estimated by linear regression analysis. Because ActD induces the differentiation of HL-60 cells into granulocytes and promotes apoptosis (37,38), it may modulate the expression of various cytoplasmic proteins, possibly including the trans-acting proteins that govern the stability of EF-Tumt mRNA. Indeed, the apparent increase of EF-Tumt mRNA level during the first 1.5 h of ActD chase study may reflect such an effect. However, taken together the results of the nuclear run-on and ActD chase assays indicate that the TPA-induced decrease in EF-Tumt mRNA mainly reflects decreased mRNA stability.
In our preliminary observations we also found that TPAinduced decreases in mRNA levels of other mt translation factors, MTFmt, MetRSmt, and IF-2mt, result from a combination of decreased mRNA stability and transcription, the for- mt Translation Down-regulation during HL-60 Differentiation mer appearing dominant. 2 Thus, we suggest that down-regulation of mRNA stability is a general and crucial mechanism for the marked down-regulation of mt translation factor mRNA during the differentiation of HL-60 cells by TPA and probably in response to growth arrest in general.
It is ambiguous how the transcriptional regulation for mt translation factors contributes to the change in their mRNA levels. Genomic sequence data base searching indicates that the promoter regions of EF-Tumt, MTFmt, MetRSmt, and IF-2mt carry putative NRF-binding sites; however, it remains to be elucidated whether these are functional. It is still possible that the transcription factors other than NRFs are important for the transcriptional regulation of mt translation factors, and that would explain why we did not observe the significant transcriptional reduction for EF-Tumt mRNA during differentiation ( Fig. 2A). Functional analyses of their promoter regions are currently under investigation. It is interesting to note here that transcription of the mitochondrial adenine nucleotide translocator 2 (ANT2) gene is growth-regulated, but that NRF is not involved in its control (39,40). In this case the NRFbinding sites are apparently absent in its promoter region.
So far any common cis elements that may regulate mRNA stability of mt translation factors have not been identified, although we compared their cDNA sequences. Thus, it is likely that each gene employs its own cis and probably trans elements. Searches for such cis-and trans-acting elements in each gene are also under way.

Down-regulation of mt Translation Factor mRNA Is Associated with the Decreased Protein Level and mt Translational
Activity-We confirmed by immunoblot analysis that EF-Tumt protein expression is also down-regulated in a manner that mirrors its mRNA levels (Fig. 3A, EF-Tumt). The band indicated with the arrow is the putative proteolytic product. A phosphatase treatment diminishes the lower band, indicating that the phosphorylated form of EF-Tumt described elsewhere (41) is preferentially in the lower band). 2 The temporal modulation of the protein level followed that of mRNA by ϳ50 h, probably because the protein half-lives of the mt translation factors are much longer than those of mRNAs. Fig 3B shows the analysis of mt translation products by pulse labeling, where only de novo-synthesized mt translation products are radiolabeled. Mitochondrial translation activity drops to a level less than 50% that of basal within 24 h and reaches a minimal level (ϳ10% of basal) within 48 h. Downregulated mt translational activity is actually associated with decreased levels of mt translation factors. Because mt translation activity drops off much more quickly than the levels of EF-Tumt protein (Fig. 3, A and B), cells may down-regulate the mt genetic system in several ways in addition to down-regulating the levels of mt translation factors. For example, the proteolysis of EF-Tumt may be involved. We reproducibly observe that the fraction of the lower band of EF-Tumt increases and the amount of the intact EF-Tumt decreases after TPA treatment (Fig. 3A). Other factors, such as mitochondrial polynucleotide phosphorylase, may be also involved (see ''Discussion'').
It is interesting to note that the expression of COII protein, an mtDNA-coded protein, remained constant up to 96 h after TPA treatment (Fig. 3A, COII) irrespective of mt translational activity down-regulated. This is probably because COII is very stable or, rather, because it is stabilized in response to mt translation dysfunction, as is also observed in mice with a moderate reduction of mt DNA copy number (42)(43)(44). In any case, this observation suggests that the biological significance of down-regulating mt translational activity may be independent of the regulation of respiratory activity, as is further discussed under ''Discussion.'' Synthesis of mt Translation Factors Is Not Regulated at the Translational Level during Cellular Differentiation-The synthesis of many cytoplasmic translation factors is selectively regulated in a growth-dependent manner at the translational level. A structural hallmark common to the mRNAs encoding much cyt translational machinery is the presence of a 5Ј-terminal oligopyrimidine tract (5Ј-TOP), referred to as TOP mRNAs. The TOP motif comprises the core of the translational cis regulatory element of these mRNAs (45). Terminal differentiation is associated with growth arrest, and indeed, recent studies showed that TOP mRNAs are translationally downregulated during TPA-induced differentiation of HL-60 cells (28). We wondered whether decreased protein expression of mt translation factors during TPA-induced differentiation (Fig.  3A, EF-Tumt, for example) simply reflects decreased mRNA levels. According to a dbEST search, it is unlikely that most mt translation factor mRNA species are TOP mRNAs. However, it is reported that the translation of S12mt mRNA is downregulated in response to serum starvation, and its regulation is not mediated through the TOP sequence (46).
To examine whether mt translation factor mRNA was translationally regulated in a manner independent of the TOP se-quence, sucrose density gradient centrifugation was used to separate cell lysates into polysomal and subpolysomal (messenger ribonucleoprotein particles) fractions. Such gradients were prepared from HL-60RG cells before (ϪTPA) and after differentiation (ϩ50 nM TPA, 48 h). RNA was extracted from successive fractions across the gradients and analyzed for EF-Tumt, L7/L12mt, and S12mt mRNA expression by Northern hybridization. These mRNAs were selected because their counterparts in the cyt translation system, i.e. mRNAs of EF-1␣, and ribosomal proteins, are TOP mRNAs. Two control mRNAs, ␤-actin, which is efficiently translated in both conditions, and L19, a cytosolic ribosomal protein encoded by a typical, translationally regulated TOP mRNA, were also analyzed by RT-PCR. As seen in Fig. 4, L19 mRNA shows typical TOP mRNA behavior, mainly polysomal in growing cells (ϪTPA) and mainly non-polysomal in resting cells (ϩTPA). mRNA species of mt translation factors (EF-Tumt, L7/L12mt, and S12mt) are efficiently translated in both conditions. These mitochondrial mRNAs were still associated with polysomal fractions even after 72 h of TPA treatment (data not shown). These results suggest that there is no translational control of mt translation factor mRNA expression during differentiation and in response to growth arrest. Thus, the decreasing protein levels of mt translation factors during differentiation would directly mirror the decreasing mRNA levels, although their temporal modulation vary depending on each protein stability. Behavior of S12mt mRNA, akin to TOP mRNA in response to the serum starvation described in Mariottini et al. (46), might not reflect the cessation of cellular proliferation.
The regulatory mechanism of mt translational activity upon terminal differentiation (in response to the growth arrest) shows a remarkable contrast with that of the cytoplasmic system. The regulation of stability and transcription rather than translational efficiency of mRNA species is crucial for gene expression of mt translation factors, whereas the control of mRNA translational efficiency is more important for the expression of cyt translation factors.
Biological Significance of Down-regulating Mitochondrial Translational Activity after TPA-induced Cell Differentiation of HL-60 Cells-It should be emphasized that the down-regulation of mt translation described in this paper is a phenomenon associated with terminal differentiation rather than a requirement for the onset of cellular differentiation, apoptosis, and FIG. 4. Synthesis of mt translation factors is not regulated at the translational level during cellular differentiation. Polysome association in normal (ϪTPA, left) and differentiated (ϩTPA, 50 nM 48 h, right) HL-60RG cells of mRNAs were analyzed. Cellular extracts were fractionated by centrifugation through 15-50% sucrose density gradients. Fractions were collected from the top of the gradient with continuous monitoring at 260 nm (upper). Each fraction was analyzed by RT-PCR (␤-actin and L19, EtBr staining) or by Northern blotting (EF-Tumt, L7/L12mt, and S12mt) (lower). For polysome analysis of differentiated HL-60RG cells, RNA was hybridized with the indicated probes over 2 nights.
mt Translation Down-regulation during HL-60 Differentiation differential gene expression for the new phenotype. It is reported that high mt activity appears to be associated with the preliminary steps of avian myoblast differentiation, and its induction just before the onset of terminal differentiation could characterize an irreversible engagement in terminal differentiation (47,48). In line with this, we observed that HL-60RG cells treated with thiamphenicol, a specific inhibitor of mt translation, were unable to differentiate to monocyte/macrophage-like cells in response to TPA. 2 This indicates that downregulated mt translation is not a prerequisite for terminal differentiation and apoptosis. Mitochondrial translation activity in the early stages of HL-60RG differentiation is currently under investigation.
It is unclear whether down-regulation of mt translation is just a result of growth arrest or is also of biological significance after the cellular commitment to terminal differentiation. It is unlikely that cells down-regulate mt translation activity to inhibit the mt respiration activity and to stop cell proliferation because rho°cells, which are devoid of mtDNA and deficient in respiratory activity, are able to proliferate. One fascinating possibility is that down-regulation of mt translation activity may promote spontaneous apoptosis during terminal differentiation. In this respect, it is interesting that polynucleotide phosphorylase, a recently identified mt protein probably involved in the degradation of mt transcripts (49), is up-regulated before growth arrest and terminal differentiation (50). The decreased mRNA levels of mt transcripts (Fig. 1C, COII and COIII) and decreased mt translation activity (Fig. 3B) described in this report might be caused in part by enhancement of polynucleotide phosphorylase activity. A change in mt translational activity would effect the assembly of the respiratory complex, production of reactive oxygen species, maintenance of mt membrane permeability, formation of the voltage-dependent anion channel, opening of the permeability transition pore, release of cytochrome c, and so on. Thus, cells may downregulate the mt genetic system to execute apoptosis during terminal differentiation.