©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Changing Patterns of Transcriptional and Post-transcriptional Control of -F-ATPase Gene Expression during Mitochondrial Biogenesis in Liver (*)

José M. Izquierdo (1), Javier Ricart (1)(§), Luciana K. Ostronoff (1)(¶), Gustavo Egea (1) (2), José M. Cuezva (1)(**)

From the (1) Departamento de Biologa Molecular, Centro de Biologa Molecular ``Severo Ochoa'' (Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Cientficas), Universidad Autónoma de Madrid, 28049 Madrid, Spain and the (2) Departamento de Biologa Celular y Anatoma Patológica, Facultad de Medicina, Universidad de Barcelona, 08036 Barcelona, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To elucidate the mechanisms that regulate the expression of nuclear genes during biogenesis of mammalian mitochondria, the expression pattern of the -subunit of the ATP synthase gene has been characterized in rat liver between day 20 in utero and 12 weeks postnatal. The parallelism existing between transcriptional activity of the gene and the amount of -F-ATPase protein in liver indicates that proliferation of mitochondria is controlled at the transcriptional level. On the other hand, an increased stability (4-5-fold) of -F-ATPase mRNA during early neonatal life as well as a rapid postnatal activation of translation rates affecting mitochondrial proteins appear to control mitochondrial differentiation. Immunoelectron microscopy of the F-ATPase complex during liver development revealed that the rapid postnatal increase in the in vivo rate of F-ATPase synthesis was mostly used for functional differentiation of pre-existing organelles (Valcarce, C., Navarrete, R. M., Encabo, P., Loeches, E., Satrstegui, J., and Cuezva, J. M. (1988) J. Biol Chem. 263, 7767-7775). The findings support that -F-ATPase mRNA decay is developmentally regulated in liver, indicating that gene expression is also controlled at this level during physiological transitions that affect biogenesis of mitochondria.


INTRODUCTION

Complex physiological responses require the simultaneous and coordinated regulation of multiple genes. This is most evident in the case of the regulation of biogenesis of mitochondria, where molecular components of the organelle are encoded in two physically separated genomes. Although it is assumed that nuclear genome activity controls the program of organelle biogenesis, other events in the cytoplasm, on the mitochondrial membranes and in the organelle interior, are likely to play prominent roles in biogenesis of mitochondria (Attardi and Schatz, 1988; Clayton, 1991; Nagley, 1991; Wallace, 1992).

The regulation of the expression of nuclear encoded proteins involved in mitochondrial energy metabolism has been described mostly at the transcriptional level (Scarpulla et al., 1986; Williams et al., 1987; Izquierdo et al., 1990; Torroni et al., 1990; Izquierdo and Cuezva, 1993b). Specific cis-acting elements and transcription factors for these genes have been characterized (Li et al., 1990; Suzuki et al., 1991; Chau et al., 1992; Chung et al., 1992; Virbasius et al., 1993; Haraguchi et al., 1994; Vander Zee et al., 1994; Gopalakrishnan and Scarpulla, 1994). Recently, it has been suggested that some of these trans-acting proteins may be directly involved in the coordinated transcriptional expression of both genomes (Suzuki et al., 1991; Virbasius and Scarpulla, 1994; Haraguchi et al., 1994). However, the changes in the expression pattern of a given protein during development could be the result of a combination of regulatory mechanisms operating at multiple levels of gene expression, i.e. gene transcription, mRNA stability, translational efficiency, protein stability, etc. In this regard, it is possible that other mechanisms operating at the post-transcriptional level could share the control of biogenesis of mitochondria at different stages of development. In fact, recent findings have provided evidence that mitochondrial biogenesis in mammals is regulated at the post-transcriptional level (Attardi et al., 1990; Polosa and Attardi, 1991; Izquierdo and Cuezva, 1993a; Luis et al., 1993).

Biogenesis of mitochondria during liver development results both in an increase in the number of mitochondria per cell, organelle proliferation (Rohr et al., 1971; Hommes, 1975), and in an increase in the functional capabilities of mitochondria, organelle differentiation (Pollack and Sutton, 1980; Valcarce et al., 1988; Cuezva et al., 1990, 1993). During the last years, we have characterized mechanisms that regulate the onset of mitochondrial function and differentiation in liver (Valcarce et al., 1988, 1990; Izquierdo et al., 1990; Valcarce and Cuezva, 1991; Izquierdo and Cuezva, 1993b; Luis et al., 1993) and in other rat tissues (Luis and Cuezva, 1989; Cuezva et al., 1990; Izquierdo and Cuezva, 1993a). Recently, we have found that the increase in -F-ATPase protein experienced in liver during the first postnatal hour is due to the activation of translation initiation as well as to a rapid increase in the translational efficiency of the template as determined in vitro (Luis et al., 1993).

In this work, we have addressed the study of the mechanisms that control biogenesis of mitochondria during both differentiation and proliferation of the organelle. The study of the mechanisms controlling differentiation is important because it has been found that -F-ATPase mRNA is accumulated in fetal liver (Luis et al., 1993). As in previous studies from our group (Izquierdo et al., 1990; Luis et al., 1993; Izquierdo and Cuezva, 1993a), the nuclear encoded -catalytic subunit of the mitochondrial ATP synthase has been used as a reporter protein in mitochondrial biogenesis. Since previous documentation of the differentiation process of mitochondria has always been inferred from data provided in the isolated organelle (Valcarce et al., 1988; Izquierdo et al., 1990), it is necessary to ascertain, by immunoelectron microscopy techniques, the occurrence of this process in situ. An additional important question raised in this study is whether mitochondrial proliferation and differentiation share the same molecular mechanisms of expression. The findings reported reveal that whereas proliferation of the organelle is controlled at the transcriptional level, differentiation of mitochondria is controlled at the post-transcriptional level, both by stabilization and translational activation of the transcript.


EXPERIMENTAL PROCEDURES

Animals

Timed pregnant albino Wistar rats weighing 200 g were fed standard laboratory chow and water ad libitum. The fetuses were delivered by rapid hysterectomy from cervical dislocated pregnant rats on days 20 and 22 of gestation, respectively. The newborns were maintained without feeding as described previously (Valcarce et al., 1988) and killed by decapitation at 1 and 6 h postnatal. Animals for other time points were taken at 5 days postnatal and 12 weeks (adult) of development.

In Vivo Determination of the Relative Rates of Synthesis of the F -ATPase Complex

[S]Methionine incorporation into liver proteins of newborn rats were measured taking into consideration the methods used for determining rates of protein synthesis (Schimke, 1975). To determine the relative rate of synthesis of the F-ATPase complex, neonates were intraperitoneally injected with 250 µCi of L-[S]methionine (1000 Ci/mmol) either at the time of birth (0 h) or 1 h later (1 h old) (Izquierdo et al., 1990). 10 min after receiving the tracer, newborns were killed by decapitation, their livers (0.3 g) were homogenized, and mitochondria were isolated. Washed mitochondrial pellets were solubilized in 1 ml of buffer containing 20 mM potassium phosphate, 1% Triton X-100, 0.9% NaCl, pH 7.0; frozen and thawed three times; and centrifuged at 40,000 rpm for 30 min at 4 °C. To measure the rate of [S]methionine incorporated into cytosolic liver proteins, 2-µl aliquots of liver homogenates were precipitated with trichloroacetate (Izquierdo et al., 1990).

The F-ATPase complex was immunoprecipitated from solubilized mitochondrial proteins using rabbit anti-rat liver mitochondrial F-ATPase serum (Valcarce et al., 1988) as described previously (Izquierdo et al., 1990). The immunoprecipitates were washed as follows: (i) 2% Triton X-100 + 300 mM NaCl; (ii) 0.5 M NaCl + 0.1% Triton X-100 + 10 mM Tris-HCl, pH 8; and (iii) 0.05% Triton X-100 + 10 mM Tris-HCl, pH 8. The washed precipitates were dissociated in loading buffer, resolved by SDS-12.5% polyacrylamide gel electrophoresis, and further processed for fluorography as described previously (Izquierdo et al., 1990; Luis et al., 1993).

DNA Isolation and Southern Blots

Rat genomic DNA was isolated and digested with three restriction endonucleases ( EcoRI, BamHI, and PstI). DNA fragments were resolved on 0.8% agarose gels and transferred to nylon membranes (GeneScreen, DuPont NEN). An EcoRI- EcoRI fragment (a-cDNA) and an EcoRI- HindIII fragment (b-cDNA) from rat liver -F-ATPase cDNA (Garboczi et al., 1988) were used as probes for hybridization experiments. Probes were labeled with [P]dCTP by nick translation. A synthetic 41-mer oligonucleotide (Garboczi et al., 1988; Izquierdo et al., 1990) contained in the sequence of the EcoRI- EcoRI cDNA fragment was also used as a probe after labeling with polynucleotide kinase (data not shown). After hybridization, membranes were washed once using the following protocol: (i) 6 SSC at room temperature for 10 min, (ii) 2 SSC containing 0.1% SDS at 65 °C for 10 min, and (iii) 0.1 SSC containing 0.1% SDS at 65 °C for 10 min. Membranes were further exposed to x-ray films at 70 °C with intensifying screens.

Northern Blot Analysis

Total liver RNA was extracted from freeze-clamped rat livers and separated by electrophoresis on formaldehyde-1.4% agarose gels (Izquierdo et al., 1990; Luis et al., 1993). Denatured RNAs were either stained with ethidium bromide or vacuum-blotted onto GeneScreenmembranes for hybridization procedures. Membranes were incubated with [P]dCTP-labeled cDNA probes. The rat liver DNA probes used in this study were -F-ATPase (Garboczi et al., 1988) and mitochondrial encoded subunits 6 and 8 of the ATP synthase complex and subunit III of the cytochrome c oxidase and 12 S rRNA (Gadaleta et al., 1990), generously provided by Drs. P. L. Pedersen (The Johns Hopkins University, Baltimore) and P. Cantatore (University of Bari, Bari, Italy), respectively. Rat liver -microglobulin and human -actin cDNAs were also used. Hybridization conditions have been previously reported (Izquierdo et al., 1990). After hybridization, membranes were washed according to the protocol described above for Southern blots. For stripping labeled DNA probes, membranes were incubated in sterile water at 90-100 °C for 20 min. Membranes were exposed to x-ray films. Autoradiograms falling within the linear range of the films were analyzed by laser densitometric scanning.

In Vivo RNA Half-life Determination

Animals used for the determination of RNA half-lives were intraperitoneally injected with 100 mg of actinomycin D (Sigma)/100 g of body weight. In adult rats, a second dose of transcriptional inhibitor was administered 1 h after the first dose. In neonatal rats, additional doses of actinomycin D were administered 3 and 6 h after the first dose. To inhibit protein synthesis, animals received an intraperitoneal injection of 10 mg of cycloheximide (Sigma)/100 g of body weight. A second dose of cycloheximide was administered 1 h later. The animals were killed at various times post-injection, and the livers were processed as described above.

Isolation of Nuclei and Run-on Transcription Assays

Nuclei were isolated from 1 g of liver as described previously (Izquierdo and Cuezva, 1993b). Briefly, the tissue was minced and homogenized in 10 mM Tris-HCl, pH 8.0, containing 0.32 M sucrose, 3 mM CaCl, 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride. Nuclei were prepared by centrifugation through a 2 M sucrose cushion (Babiss et al., 1986) with slight modifications (Kikuchi et al., 1992). Isolated nuclei were resuspended in 25% glycerol, 50 mM Tris-HCl, pH 8, 5 mM magnesium acetate, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA, and 5 mM dithiothreitol. The nuclei were quickly frozen in liquid nitrogen and stored at 70 °C for a few months.

Run-on experiments were carried out according to Marzluff and Huang (1984) with the modifications reported by Izquierdo and Cuezva (1993b). Nuclei (2-3 10) were used in run-on assays. The transcription reaction was incubated at 25 °C for 60 min and terminated by the addition of ribonuclease-free DNase I (23 units/ml; Boehringer Mannheim) and 20 mM CaCl. For specific detection of radioactive nascent RNA transcript, 10-20 10cpm of the isolated RNA was hybridized for 60 min at 60 °C and for 72 h at 42 °C with the following plasmid DNAs immobilized on nylon filters: cytosolic phosphoenolpyruvate carboxykinase (Yoo-Warren et al., 1981), -F-ATPase (Garboczi et al., 1988), -actin (Gunning et al., 1983), and pUC18 (Yanisch-Perron et al., 1985). Undigested plasmid DNAs (10 µg) were denatured with 3 M NaOH at 65 °C for 60 min and neutralized with 2 M NHAc, pH 7. Denatured DNAs were applied to nylon membranes using a slot blot apparatus (Schleicher & Schuell). After hybridization, membranes were washed according to the following protocol: 1 SSC containing 0.1% SDS at 65 °C for 15 min (two times) and 0.1 SSC containing 0.1% SDS at 65 °C for 15 min (two times). Membranes were exposed to x-ray films and analyzed by densitometric scanning.

Tissue Processing for Electron Microscopy

Small pieces of livers from 0-h, 1-h-old, and adult rats were fixed by immersion in freshly prepared 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M Sörensen phosphate buffer, pH 7.2, for 2 h at 4 °C. Other tissue samples were fixed for 1 h with 1% glutaraldehyde in phosphate-buffered saline (PBS; () 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.2) and 2% tannic acid. Tissue samples were rinsed in buffer, and the free aldehyde groups were quenched by immersion in 50 mM ammonium chloride in PBS for 60 min at room temperature (two changes of 30 min each), rinsed in PBS, and finally processed for embedding in Lowicryl K4M (Chemische Wercke Lowi, Waldkraiburg, Germany) according to the manufacturer's instructions. Gold interferential color ultrathin sections were collected in Formvar/carbon-coated nickel grids and stored until use.

Immunocytochemical Localization of the Mitochondrial F -ATPase Complex

Grids containing thin sections of fetal, neonatal, and adult rat liver were equilibrated for 5 min with PBS and then incubated in parallel with a 1:50-1:75 dilution in PBS of anti-rat liver mitochondrial F-ATPase serum (Valcarce et al., 1988). After brief rinses in PBS, grids were incubated with protein A complexed to 15-nm gold particles (BioCell Research Laboratories, Cardiff, United Kingdom), which were diluted to A= 0.4 in PBS containing 1% bovine serum albumin, 0.01% Triton X-100, and 0.1% Tween 20. After 45 min of incubation with the protein A-gold complexes, grids were washed twice in PBS and distilled water and air-dried. Counterstaining was performed with 2% aqueous uranyl acetate (6 min) and 1% lead citrate (45 s). Several series of standard controls for immunocytochemical techniques were conducted in parallel to assess the specificity of the immunoreactive signal. Quantitation of F-ATPase protein density in mitochondria (number of gold particles/square micrometer of mitochondrial section) was performed according to Griffiths and Hoppeler (1986).

Other Methods

Preparation of liver homogenates (Izquierdo et al., 1990), isolation of liver mitochondria (Valcarce et al., 1988), and one-dimensional (Luis et al., 1993) and two-dimensional (Santarén et al., 1993) Western blot analyses of the -subunit of the F-ATPase complex have been previously described in detail.


RESULTS

One Gene and One Protein for -F -ATPase

The regulation of the expression of the nuclear encoded genes involved in energy metabolism in mammalian cells has been mostly described at two levels: (i) differential expression of tissue-specific isoforms and (ii) variable expression of functional single copy genes (for review, see Wallace (1992)). The existence of more than one gene (Gay and Walker, 1985; Breen, 1988; Walker et al., 1989; Pierce et al., 1992) and the differential expression of isoforms, originated by alternative splicing of the same transcript (Matsuda et al., 1993a, 1993b; Endo et al., 1994), have been described for other subunits of the mitochondrial ATP synthase.

To determine whether rat -F-ATPase is encoded by a single gene or whether there are additional genomic sequences related to the -subunit, we analyzed rat genomic DNA by Southern blot hybridization analysis with 5`- and 3`-fragments of the rat liver -F-ATPase cDNA (Fig. 1). The 5`-most cDNA (a-cDNA) hybridized with single DNA fragments (4.7-kb EcoRI, 2.0-kb BamHI, and 3.5-kb PstI) (Fig. 1 B, left panel). Also, an additional faint band appeared in each track (Fig. 1 B, left panel). The 3`-most cDNA (b-cDNA) hybridized with 23-kb EcoRI, 2.3-kb BamHI, and two PstI (0.7 and 1.8 kb) DNA fragments (Fig. 1 B, right panel). Likewise, with this probe, one or two additional faint bands (25 kb in EcoRI, 5 and 3 kb in BamHI, and 2.2 and 4.5 kb in PstI) could be seen (Fig. 1 B, right panel). These hybridization studies suggest that rat -F-ATPase is encoded by a single copy gene. The hybridization of the probes with a -F-ATPase pseudogene (Ohta et al., 1988; Neckelmann et al., 1989; Walker et al., 1989) could account for the appearance of faint hybridization signals.


Figure 1: Southern blot analysis of rat genomic DNA. A, shown is a schematic representation of DNA fragments derived from rat liver -F-ATPase cDNA with three restriction endonucleases. a- and b-cDNA represent the DNA fragments of -F-ATPase cDNA used to hybridize digested genomic DNA. B, EcoRI-, BamHI- and PstI-digested DNA fragments were resolved on 0.8% agarose gels, transferred to nylon membranes, and hybridized with P-labeled a- and b-cDNA probes. Molecular size markers are shown on the left in kilobases.



Two-dimensional Western blots of mitochondrial proteins revealed that -F-ATPase protein had the same apparent molecular mass and isoelectric point (49 kDa and 5.3 pH units, respectively) at different stages of liver development (data not shown) as previously reported for the adult liver protein (Santarén et al., 1993), i.e. no evidence for isoforms of the protein could be detected during development. Altogether, the results herein reported indicate the existence of a single copy -F-ATPase gene in rat genome. This finding is in agreement with similar findings in humans (Neckelmann et al., 1989) and in bovines (Breen et al., 1988).

Expression of -F -ATPase during Liver Development Reveals Two Different Processes of Mitochondrial Biogenesis

The existence of one gene and one protein product for -F-ATPase in liver provided a good marker to monitor mitochondrial biogenesis during development. Accumulation of -F-ATPase protein in liver showed almost a continuous increase (Fig. 2 A). However, if the rate of accumulation of -F-ATPase protein is taken into consideration, two distinct periods of mitochondrial biogenesis can be distinguished (Fig. 2 A). The first, defined by the highest rate, occurs immediately after birth and results in a 2-fold increase in the relative amount of -F-ATPase in just 1 h postnatal (Fig. 2 A). The second, defined by a much lower rate, results in a similar 2-fold increase between 6 h postnatal and the adult stage (Fig. 2 A).


Figure 2: Developmental changes in the relative amount of -F-ATPase protein in rat liver and in isolated organelles. A, liver homogenates from fetal (days 20 and 22 of gestation ( lanes 1 and 2, respectively)), neonatal (1, 6, and 5 days old ( lanes 3-5, respectively)), and adult ( lane 6) rats. Left panel, Coomassie Blue-stained gel of 50 µg of liver proteins fractionated by SDS-polyacrylamide gel electrophoresis. Molecular mass markers (200, 97, 69, 46, 30, 21, and 14 kDa) are shown on the left. Middle panel, representative blot obtained from a parallel gel transferred to polyvinylidene difluoride membranes and probed with affinity-purified anti--F-ATPase antibodies. Right panel, quantitation of immunoreactive -F-ATPase protein performed by laser densitometric scanning of the resulting bands. Data are expressed in arbitrary units ( a.u.)/gram of liver and are the means ± S.E. p < 0.05 (*), p < 0.025 (**), and p < 0.01 (***) when compared with the previous value of the time course by Student's t test. B, postnatal increase in -F-ATPase protein in isolated liver mitochondria. 10 µg of day 22 fetal (0 h) ( lane 1), 1-h-old neonatal ( lane 2), and adult ( lane 3) liver mitochondrial proteins were resolved by SDS-polyacrylamide gel electrophoresis. The left panel shows a Coomassie Blue-stained gel ( upper) and a parallel Western blot ( lower) processed with affinity-purified anti--F-ATPase antibodies. Note the rapid postnatal increase ( right panel) in the steady-state amounts of -F-ATPase protein in isolated organelles. The values are expressed as a percentage of -F-ATPase protein detected in adult mitochondria and are the means ± S.E. *, p < 0.0025 when compared with the 0-h stage by Student's t test. C, synthesis in vivo of the mitochondrial F-ATPase complex in fetal ( open bars) and neonatal ( hatched bars) rat liver. F-ATPase was immunoprecipitated from solubilized mitochondrial proteins. Relative rates of F-ATPase synthesis are expressed as the ratio of the counts/minute in the F-ATPase complex to the counts/minute in cytosolic proteins. The results are the means ± S.E. for three to four experiments. p < 0.01 (*) and p < 0.0005 (**) when compared with the 0-h stage by Student's t test.



Visualization of the two different processes affecting organelle biogenesis during development was illustrated when the steady-state amounts of -F-ATPase protein were expressed relative to the amount of isolated mitochondrial proteins (Fig. 2 B). It is shown that the relative -F-ATPase amount increases 2-fold between the fetal (0 h) and neonatal (1 h old) stages of development. Interestingly, the relative content of this protein in isolated mitochondria from adult liver is the same as that detected in 1-h-old samples (Fig. 2 B). These findings suggest that the increase in liver -F-ATPase between 1 h postnatal and the adult stage (Fig. 2 A) is due to an increase in the number of differentiated mitochondria in liver. On the other hand, the major period of organelle differentiation appears to occur during the first hour after birth because the surge in liver -F-ATPase protein (Fig. 2 A) is accounted for by the increase of the protein in isolated organelles (Fig. 2 B).

A Selective Increase in the Relative Rate of Synthesis of Mitochondrial Proteins Triggers Differentiation of the Organelle

To analyze the mechanisms responsible for the rapid postnatal increase in the amount of -F-ATPase protein in the liver, the in vivo rates of F-ATPase synthesis were determined in fetal (0 h) and neonatal (1 h old) rats (Fig. 2 C). The rates of incorporation of L-[S]methionine into liver cytosolic proteins increased 5-fold during the first postnatal hour (Fig. 2 C). In contrast, the rates of incorporation of L-[S]methionine into the immunoprecipitated F-ATPase complex (Fig. 2 C; see also Fig. 3in the work of Izquierdo et al. (1990)) showed a 12-fold increase during the same time period. The relative rates of synthesis of the F-ATPase complex were calculated in order to correct the possible differences in (i) the dose and site of administration of the tracer, (ii) the rates of uptake of L-[S]methionine, and (iii) the liver methionine pool size. The relative rate of synthesis was expressed as the ratio of tracer incorporation into F-ATPase to that into liver cytosolic proteins. The relative rate of F-ATPase synthesis showed a 2-fold stimulation during the first postnatal hour (Fig. 2 C). These results illustrate the activation of translation initiation that occurs in liver immediately after birth (Luis et al., 1993). Even more important, they illustrate in vivo that the synthesis of mitochondrial proteins is preferential over other non-mitochondrial cellular proteins (Luis et al., 1993), suggesting that new synthesis of the mitochondrial F-ATPase complex (Fig. 2 C) is used for differentiation of pre-existing fetal mitochondria.


Figure 3: Immunoelectron microscopy of the F-ATPase complex evidences liver mitochondrial differentiation. Shown is F-ATPase immunogold labeling in day 22 fetal ( a), 1-h-old neonatal ( b), and adult ( c) rat liver ultrathin sections from paraformaldehyde-fixed samples. Gold particles decorate the inner mitochondrial membrane. In d, quantitation of the gold particle density/square micrometer of mitochondrial section evidences a major presence of the F-ATPase complex in neonatal ( 1h) and adult ( Ad) rat liver. *, p < 0.01 when compared with the 0-h stage by Student's t test.



Quantitative Immunoelectron Microscopy Evidences Mitochondrial Differentiation after Birth

Immunoelectron microscopy of the mitochondrial F-ATPase complex in rat liver ultrathin sections showed that gold particles decorated the inner mitochondrial membrane (Fig. 3). Quantitation of F-ATPase gold particle density/square micrometer of mitochondrial section in fetal, neonatal, and adult liver revealed a significant increase at the 1-h time point (Fig. 3). The percentage of the increase in F-ATPase immunolabeling at 1 h ranged from 50 to 250%, depending upon whether tissue samples were fixed in paraformaldehyde (Fig. 3) or glutaraldehyde (data not shown), respectively. Interestingly, the mitochondrial gold particle density in 1-h-old and adult hepatocytes was the same (Fig. 3), in agreement with the results of Western blotting experiments (Fig. 2 B). The differences observed in the increase in the mitochondrial F-ATPase complex between fetal and neonatal samples estimated by electron microscopy of in situ organelles (Fig. 3) and Western blotting of the isolated organelles (Fig. 2 B) may be explained by the differences in the conservation of antigenic determinants between the two fixation procedures of the electron microscopic technique. In fact, when liver samples were fixed in glutaraldehyde, the results obtained by both techniques were comparable (data not shown).

Proliferation of Liver Mitochondria Occurs when Steady-state mRNA Levels Show a Sharp Decline

Fig. 4 summarizes the developmental changes in liver steady-state levels of various mitochondrial (nuclear (-F-ATPase) and mitochondrial (cytochrome c oxidase subunit III + ATPase 6 and 8 and 12 S rRNA) encoded) and non-mitochondrial (-microglobulin and -actin) transcripts. Interestingly, the developmental profile for the two mRNAs of the ATP synthase complex was similar although they are encoded in different genomes. They steadily accumulated in liver, reaching maximum levels at 6 h postnatal. It should be noted that when differentiation of the organelle occurred (during the first postnatal hour), there were no changes in steady-state levels for these two transcripts, as reported earlier (Luis et al., 1993) (Fig. 4). Surprisingly, at the time when most of the -F-ATPase protein accumulated in liver (between the 5-day neonatal and adult stages; see Fig. 2A), steady-state transcript levels were at a minimum (Fig. 4). The specific developmental profile for these two mitochondrial mRNAs was further supported by the expression pattern of the other three transcripts (Fig. 4). While mitochondrial 12 S rRNA showed no changes during development, steady-state mRNA levels of -microglobulin and -actin showed the opposite developmental profile (Fig. 4).


Figure 4: Developmental changes in the relative amount of -F-ATPase mRNA in rat liver. 30 µg of total RNA extracted from fetal livers at days 20 and 22 of gestation ( 20d-F and 22d-F, respectively), from neonatal livers at 1 h, 6 h, and 5 days postnatal ( 1h-N, 6h-N, and 5d-N, respectively), and from adult livers were analyzed by Northern blot hybridization procedures with the indicated probes. A parallel ethidium bromide-stained gel shows 28 S and 18 S rRNAs. The relative abundance of the RNAs (arbitrary units ( a.u.)/microgram of RNA) was determined by laser densitometric scanning of the autoradiograms. The values shown are the mean of two different total liver RNA preparations. COIII, cytochrome c oxidase subunit III; -microg., -microglobulin.



Nuclear Transcription Rates of the -F -ATPase Gene during Mitochondrial Biogenesis

Nuclear run-on assays in isolated liver nuclei from different stages of development revealed that transcription rates of the -F-ATPase gene (Fig. 5) did not parallel the sharp reduction in steady-state -F-ATPase mRNA levels (Fig. 4). In fact, -F-ATPase transcription rates in adult nuclei (Fig. 5) were found to be at least 3-fold higher than at any other stage of liver development ( p < 0.025 and p < 0.005 when compared with the 5-day neonatal and day 20 fetal stages, respectively). The transcription rates of the -F-ATPase gene during development closely resembled those of another housekeeping gene, -actin (Fig. 5). Comparison of the relative -F-ATPase/-actin transcription rate during development (data not shown) revealed that -F-ATPase gene transcription increased (3-5-fold) toward the adult stage. Determination of transcription rates of the inducible phosphoenolpyruvate carboxykinase gene further supported the results obtained from nuclear run-on assays (Fig. 5). Transcription rates of the phosphoenolpyruvate carboxykinase gene, in agreement with previous reports (Garcia Ruiz et al., 1978; Lyonnet et al., 1988), were switched on immediately after birth to allow development of gluconeogenesis and the supply of glucose to neonatal tissues (Patel et al., 1982; Mayor and Cuezva, 1985).


Figure 5: Developmental changes in transcription rates of the -F-ATPase gene. Liver nuclei were isolated from fetal (days 20 and 22), neonatal (1 h, 6 h, and 5 days old), and adult rat liver. Nuclear run-on assays were carried out for determining transcription rates of the phosphoenolpyruvate carboxykinase, -F-ATPase, and -actin genes. A, representative autoradiograms hybridized with radioactive nascent RNA transcripts at the indicated time periods during liver development. Note the rapid postnatal activation of phosphoenolpyruvate carboxykinase ( PEPCK) gene transcription. B, quantitation of transcription rates (arbitrary units ( a.u.)/10cpm) of the genes after densitometric scanning of the resulting hybridization signals. The results shown are the means ± S.E. for three to four independent experiments.



Changing Patterns of -F -ATPase mRNA Half-life during Development

The lack of correlation between steady-state mRNA levels (Fig. 4) and transcription rates of the -F-ATPase gene (Fig. 5) prompted us to estimate in vivo the half-life of the messenger during development. This approach is possible if transcription rates are blocked after administration of an inhibitor (Belasco and Brawerman, 1993). Administration of actinomycin D to rat neonates prevented the postnatal accumulation of phosphoenolpyruvate carboxykinase mRNA in their livers (Fig. 6 A). Similarly, administration of actinomycin D to adult rats promoted the disappearance of this short-lived mRNA (Fig. 6 A). These findings indicate that mRNA synthesis is blocked after actinomycin D administration. Thus, quantitation of steady-state amounts of the transcripts at various times after administration of the inhibitor (Fig. 6 A) and calculation of RNA decay assuming first-order rate kinetics (Fig. 6 B) (Belasco and Brawerman, 1993) allowed the estimation of -F-ATPase mRNA half-life.


Figure 6:In vivo determination of -F-ATPase mRNA half-life in neonatal and adult liver. Transcription rates were inhibited by intraperitoneal administration of actinomycin D to neonatal and adult rats (+). Untreated age-matched neonates were processed to illustrate the inhibitory effect of actinomycin D on transcription rates of the phosphoenolpyruvate carboxykinase ( PEPCK) gene (). A, total RNA was prepared from livers at various times during neonatal development and after administration of the inhibitor and analyzed by Northern blot hybridization procedures with the probes indicated on the left. Quantitation of mRNA content was carried out by laser densitometric scanning of the resulting hybridization signals. B, calculation of RNA half-lives was done assuming first-order kinetics. The log of steady-state -F-ATPase mRNA levels (arbitrary units ( a.u.)/microgram of total RNA) is represented at various times after administration of actinomycin D to neonatal ( closed circles) and adult ( closed triangles) rats. The log of steady-state -F-ATPase mRNA levels in untreated neonates is also shown ( open circles). B represents the experiment shown in A. Note that the 6-h time point is not represented because of artifactual RNA loading in the gel. C, shown are the calculated RNA half-lives. The results shown are the means ± S.E. for three to four independent experiments. p < 0.0025 (*) and p < 0.0005 (**) when compared with the neonatal stage by Student's t test.



Interestingly, the disappearance of the hybridization signals for the nuclear encoded -F-ATPase transcript after administration of the inhibitor was more pronounced in adult than in neonatal liver (Fig. 6 A; please note differences in the time scale). In fact, calculation of the apparent mRNA half-life (Fig. 6 B) revealed that the neonatal -F-ATPase mRNA was 4-5 times more stable than the adult transcript (Fig. 6 C).

It has been described that actinomycin D is also effective in inhibiting yeast mitochondrial transcription in vitro (Barat-Gueride et al., 1987) and in cultured liver cells (Chrzanowska-Lightowlers et al., 1994). The results shown in Fig. 6 A revealed a significant decline in the 12 S rRNA hybridization signal after actinomycin D administration to the neonates, thus suggesting that the drug is also effective as an inhibitor of mammalian mitochondrial transcription in vivo. Although inhibition of mitochondrial transcription could be directly or indirectly mediated, the decline in 12 S rRNA hybridization signals allowed a rough estimation of the half-life of a transcript encoded in the mitochondrial genome. The results obtained also revealed that 12 S rRNA has a higher stability in neonatal than in adult liver (Fig. 6, A and C). It should be mentioned that 12 S rRNA half-life could be subestimated due to accumulation of this transcript in liver during the first 12 postnatal hours (Fig. 6 A).

A Role for Labile Protein(s) in Stabilizing -F -ATPase mRNA during Development?

The experiments described above open up the possibility that the stability of the -F-ATPase transcript in fetal and early neonatal liver depends on mechanisms such as translation rates of the template itself (Greenberg et al., 1986; Greenberg and Belasco, 1993) or on the presence of a specific trans-acting factor(s) that could modulate its stability at that stage of development. As a preliminary approach to address this question, an inhibitor of cytosolic protein synthesis was administered to newborn and adult rats, and the steady-state mRNA levels for -F-ATPase and ATPase 6 and 8 and for 12 S rRNA were determined in liver at various times after cycloheximide administration (Fig. 7).


Figure 7: Inhibition of protein synthesis increases -F-ATPase mRNA decay in neonatal liver. 10 mg of cycloheximide/100 g of body weight was intraperitoneally administered to newborns at the time of birth. At 0, 30, 60, and 120 min ( lanes 1-4, respectively) after administration of the first dose of inhibitor, livers were removed, and RNA was extracted. 30 µg of total RNA were processed for Northern blotting and hybridized with the probes indicated on the left. Note the sharp reduction in the -F-ATPase mRNA hybridization signal at 30 min after cycloheximide administration, whereas subtle changes occur for the other two transcripts, indicating a specific effect of the inhibitor on -F-ATPase mRNA levels. Two different experiments ( A and B) are shown.



12 S rRNA and the mitochondrial encoded ATPase 6 and 8 mRNA showed no major changes after administration of the inhibitor. However, the nuclear encoded -F-ATPase mRNA showed a sharp decline in the neonatal liver 30 min after cycloheximide administration (Fig. 7). By 1 h post-injection, the mRNA levels again reached normal levels (Fig. 7). Interestingly, administration of the cytosolic protein synthesis inhibitor to adult rats did not promote such rapid (30 min) changes in the steady-state levels for any of the three transcripts studied (data not shown). These results, obtained in vivo, should be interpreted with caution because of possible side effects of cycloheximide in other cellular functions. However, they suggest that the stability of the transcript during early stages of development is more dependent on ongoing protein synthesis than at later stages (adult). On the other hand, since determination of protein synthesis in liver (Valcarce et al., 1988) after 2 h of cycloheximide administration to neonates indicates a significant reduction in the amount of radioactivity incorporated into protein (4026 ± 267 and 303 ± 49 dpm/mg of protein for saline- and cycloheximide-injected age-matched neonates, respectively), we suggest that the transient nature of the -F-ATPase mRNA response (Fig. 7) may represent an adaptation of liver cells to inhibition of protein synthesis.


DISCUSSION

The -subunit of the ATP synthase plays a key role in mitochondrial function. It is the catalytic subunit of the complex and thus a bottleneck for oxidative phosphorylation. Here, we show the result of mitochondrial differentiation in rat liver at the subcellular level (Fig. 3). This finding illustrates in situ that new synthesis of -F-ATPase protein (Fig. 2) is mostly used for increasing the relative amount of this protein in pre-existing organelles, confirming previous results obtained at the molecular and functional levels with the isolated organelles (Valcarce et al., 1988; Izquierdo et al., 1990). Furthermore, the two processes that affect biogenesis of mitochondria during development, proliferation and differentiation, differ in time and intensity scales and do not share the same molecular mechanisms for their expression, most likely reflecting the different physiological states and energy demands of the liver during development. Differentiation is controlled at the post-transcriptional level by increasing the stability (Fig. 6) and translational efficiency (Fig. 2; see Luis et al. (1993)) of mitochondrial transcripts. Proliferation is controlled at the transcriptional level (Fig. 5). In both differentiation and proliferation processes, the participation of additional levels of regulation at the post-translational level cannot be excluded, i.e. differential regulation of protein stability during development.

Recent findings indicate that the -F-ATPase gene is under complex transcriptional regulation (Webster et al., 1990; Torroni et al., 1990; Stepien et al., 1992). It is interesting to note that the -F-ATPase gene (Ohta et al., 1988; Neckelmann et al., 1989) contains a putative nuclear respiratory factor-2-responsive element (Virbasius and Scarpulla, 1991), in common with other genes involved in energy metabolism. It also contains an enhancer element, in common with human cytochrome cand subunit E1 of the pyruvate dehydrogenase complex (Tomura et al., 1990). Similarly, other cis-acting elements have been found in common between the ATP synthase -subunit and other oxidative phosphorylation genes (Li et al., 1990; Suzuki et al., 1991; Chau et al., 1992). On the other hand, thyroid hormones have been shown to effectively regulate the transcriptional expression of the -F-ATPase gene (Izquierdo et al., 1990; Izquierdo and Cuezva, 1993b). Ubiquitous and tissue-specific nuclear factors that interact with overlapping promoter elements (OXBOX/REBOX) of the gene have been described (Li et al., 1990; Chung et al., 1992; Haraguchi et al., 1994). Interestingly, these factors interact differentially with cis-acting elements depending on cellular conditions (Chung et al., 1992). Thus, it seems reasonable to suggest that differential transcription of the -F-ATPase gene during liver development (Fig. 6) could result, among other factors, from the differences in thyroid hormone availability and functional maturation of the thyroid transduction pathway that accompany mammalian development.

The opposite developmental profile between steady-state -F-ATPase mRNA levels (Fig. 4) and transcription rates of the gene (Fig. 5) indicated the existence of a mechanism for controlling mRNA decay that is differentially expressed during development of the mammalian liver. The post-transcriptional regulation of -F-ATPase mRNA availability in fetal and neonatal liver is further supported by the higher half-life of the transcript at this stage of development (Fig. 6). Little, if anything, is known about the existence of specific cis-acting elements, endonucleolytic pathways, mechanisms of mRNA decay, and trans-acting factors involved in determining the stability of nuclear encoded transcripts for mitochondrial proteins.

The degradation process of short-lived mammalian mRNAs, those encoded by the early response gene family (Herschman, 1991), is dependent on protein synthesis (Greenberg et al., 1986; Greenberg and Belasco, 1993). When cellular protein synthesis is inhibited, the stability of these mRNAs increases. Contrary to this situation and as a further interesting contrast between different stages of development, inhibition of protein synthesis in neonatal liver promotes a rapid and profound reduction in steady-state -F-ATPase mRNA levels (less than half of the original values in <30 min; Fig. 7 ). This finding could argue against the idea that nuclear templates encoding for mitochondrial proteins are degraded as a result of their translational activation after birth (Fig. 2 C; see Luis et al. (1993)). On the contrary, it may suggest that -F-ATPase mRNA stability is dependent on ongoing protein synthesis, a mechanism that seems to be differentially regulated during liver development.

It is interesting to note that the relative changes in the amount of -F-ATPase/gram of liver (Fig. 2 A) parallel the changes in the number of mitochondria/hepatocyte (Rohr et al., 1971; Hommes, 1975) and almost parallel the changes in the amount of mtDNA/cellular DNA during rat liver development (Cantatore et al., 1986). On the other hand, it appears that representation of the mitochondrial translational machinery, when expressed relative to the cellular one (assessed by 12 S rRNA) (Fig. 4), is kept constant throughout development. Therefore, we suggest that maintenance of the constancy of the level of 12 S rRNA results from the concerted operation of constitutive transcription of mtDNA (Cantatore et al., 1986), which will increase as development proceeds, and mechanisms of regulation controlling mitochondrial rRNA decay, which are also expected to increase during development. This is consistent with the observed difference in 12 S rRNA half-life estimated at two stages of liver development (Fig. 6).

On the other hand, the parallelism observed during liver development between steady-state levels of the nuclear and mitochondrial encoded mRNAs (Fig. 4) further reinforces the hypothesis, reported recently within different mammalian cellular types (Izquierdo and Cuezva, 1993a), of the existence of a conserved mechanism for coordination of the expression of the two genomes. The parallel changes observed in RNA decay rates during development for transcripts encoded in both genomes (Fig. 6) seem to add additional support to this hypothesis.

Several experimental systems have been described in which mRNAs are being stored ``masked'' until required to fulfill a predetermined morphogenetic or differentiation program of the organism (Wickens, 1990; Jackson and Standart, 1990). The activation of these mRNAs is usually mediated by specific covalent modification and/or trans-activation of the stored mRNAs in response to given stimuli (Bachvarova, 1992; Bouvet and Wolffe, 1994). This situation mimics the differentiation process of mitochondria in rat liver. The -F-ATPase mRNA accumulated in liver during fetal stages of development changes its sedimentation behavior (Luis et al., 1993) and translational efficiency immediately after birth, as assessed both in vivo (Fig. 2 C) and in vitro (Luis et al., 1993). At the present time, we have no explicit finding that could explain such differences in the physical (Luis et al., 1993) and functional (Fig. 2 C; Luis et al., 1993) behavior of -F-ATPase mRNA.

It is obvious that future investigations aimed at identifying putative cis- and trans-acting factors of -F-ATPase mRNA sorting, stabilization, and translational activation will contribute to a better picture of the mechanisms that control the post-transcriptional regulation of mitochondrial biogenesis in mammals. In this regard, it is worth mentioning that some mitochondrial proteins are bifunctional, i.e. they are catalysts of metabolic pathways, and they also present mRNA binding activities (Preiss et al., 1993; Preiss and Lightowlers, 1993; Klausner et al., 1993; Beinert and Kennedy, 1993). This situation opens up new trends for positive and negative feedback autoregulation of the post-transcriptional expression of the genes involved in mitochondrial biogenesis. We believe that the developing rat liver provides an excellent experimental system in which to address future studies at this level.

In conclusion, our findings indicate that the expression of nuclear encoded genes required for mitochondrial biogenesis during liver development is exerted at various levels. We hypothesize that mitochondrial biogenesis in mammals is regulated primarily by the variable transcriptional expression of certain genes that control key cellular functions (the catalytic subunit of the ATP synthase complex is one example). To encompass or finely tune the cellular outfit of mitochondria with energy demands, other mechanisms exert a complex regulation of these genes at various post-transcriptional levels during certain stages of development. These findings are of utmost importance for understanding the mitochondrial pathophysiology of mammals during adaptation to extrauterine life (Valcarce et al., 1994).


FOOTNOTES

*
This work was supported in part by Comunidad de Madrid Grant CAM 92/039 and Dirección General de Investigación Cientfica y Técnica Grant PB91/0032 and by an institutional grant from the Fundación Ramón Areces, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a predoctoral fellowship from the Universidad Autónoma de Madrid.

Recipient of a predoctoral fellowship from the Instituto de Cooperación Iberoamericana-Ministerio de Asuntos Exteriores.

**
To whom correspondence should be addressed. Tel.: 34-1-397-4866; Fax: 34-1-397-4799.

The abbreviations used are: PBS, phosphate-buffered saline; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Prof. J. Satrstegui for critical reading of the manuscript. Drs. A. M. Luis and J. F. Santarén are acknowledged for help in measuring rates of protein synthesis and running two-dimensional gels, respectively. We are grateful to M. Chamorro and C. San Martn and to D. Jelenic for expert technical and secretarial assistance, respectively.


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