help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2008-0400
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lenglet, S.
Right arrow Articles by Rossier, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lenglet, S.
Right arrow Articles by Rossier, M. F.
Endocrinology Vol. 149, No. 11 5461-5469
Copyright © 2008 by The Endocrine Society

Hint2 Is Expressed in the Mitochondria of H295R Cells and Is Involved in Steroidogenesis

Sébastien Lenglet, Fabrice Antigny, Laurène Vetterli, Jean-François Dufour and Michel F. Rossier

Services of Endocrinology & Diabetology (S.L., F.A., L.V., M.F.R.) and Laboratory Medicine (M.F.R.), University Hospital of Geneva, CH-1211 Geneva 14, Switzerland; and Institute for Clinical Pharmacology (J.-F.D.), University of Bern, CH-3012 Bern, Switzerland

Address all correspondence and requests for reprints to: Dr. Michel F. Rossier, Service of Endocrinology & Diabetology, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: michel.rossier{at}hcuge.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROIDOGENESIS is a continuous process involving several biosynthetic steps, the most important of them, in terms of regulation, occurring within the mitochondria. The transfer of cholesterol, the common precursor for all steroids, from the cytosol into the mitochondrial matrix is generally considered as the rate-limiting step. For example, in the case of aldosterone synthesis, this step appears to be sensitive to the presence of the steroidogenic acute regulatory protein but is also regulated by Ca2+-dependent and Ca2+-independent processes (1, 2, 3). After cholesterol entry into the mitochondria, the precursor is rapidly converted, by cleavage of its side chain, into pregnenolone, the first of a series of intermediates to the final steroid products. Therefore, the rate of pregnenolone production can be considered as an index of the steroidogenic activity of the cells, and more specifically reflects the early and rate-limiting mitochondrial steps of steroidogenesis.

Calcium entry into the mitochondria of adrenocortical cells has been sufficient for stimulating steroidogenesis (4, 5), and particular pathways for conveying Ca2+ to these organelles upon stimulation by agonists like angiotensin II (AngII) or extracellular K+ have been proposed (6, 7, 8). Additional agonists, such as ACTH for example, act through Ca2+-independent mechanisms, involving principally cAMP and protein kinase A, although cross talk between Ca2+ and cAMP signaling pathways has been described in steroidogenic cells (9, 10). However, in any case the mitochondria remain central to the regulation of the cell steroidogenic activity.

Hint proteins belong to the histidine triad AMP-lysine hydrolase superfamily, characterized by the presence of a conserved histidine motif (His-X-His-X-His) conferring to its members nucleotide binding properties and catalytic activity (11, 12). Hint2, one of the five members of the Hint subfamily, is a 17-kDa dimeric protein originally misidentified as a protein kinase C inhibitor, and is highly expressed in mitochondria of hepatic and pancreatic cells. It has been recently shown that this protein sensitizes these cells to mitochondria-dependent apoptosis and appears down-regulated in hepatocellular carcinoma (13). However, Hint2 physiological function in other living cells still remains to be determined.

Because of its mitochondrial location, Hint2 appeared to us as a putative candidate for regulating steroidogenesis. Therefore, we initiated this study by determining whether Hint2 is expressed in H295R cells, a classical model largely used for investigating adrenal steroid biosynthesis (14). We found that the protein is highly expressed in H295R cells, and, therefore, we decided to characterize its role in steroidogenesis by experimentally manipulating its expression and measuring the consequence of this manipulation on steroid production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
AngII, ionomycin, 25-hydroxycholesterol (25-OHChol), forskolin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), BSA, and HEPES were purchased from Sigma-Aldrich Corp. (St. Louis, MO). [3H]Pregnenolone was obtained from New England Nuclear Life Science Products (Switzerland) and antipregnenolone antiserum from Biogenesis Ltd. (Poole, UK). Trilostane (WIN 19798) was purchased from Farillon (London, UK). Plasmids (pcDNA3) containing DNA coding for the mitochondrial ratiometric pericam were kindly provided by Dr. T. Pozzan (University of Padova, Padova, Italy), and that coding for the Hint2 protein was prepared as previously described (13). Normal human adrenocortical tissues were obtained from Dr. H. Lefebvre (Institut National de la Santé et de la Recherche Médicale, Unité 413, University of Rouen, Rouen, France) and bovine adrenals from a local slaughterhouse.

H295R cell culture
H295R cells were obtained from Dr. W. E. Rainey (University of Texas, Dallas, TX). Cells were grown in DMEM:F12 (1:1) with 15 mM HEPES, supplemented with 120 IU/ml penicillin, 120 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD), 1% insulin, transferrin, selenium (ITS Plus; BD Biosciences, Bedford, MA), and 2% Ultroser SF (BioSepra SA, Villeneuve-la-Garenne, France).

Cells were routinely grown in 75 cm2 flasks in a humidified atmosphere at 37 C (95% air, 5% CO2). The medium was changed every 3 d.

Before experiments, cells were detached with 0.25% trypsin-EDTA and plated into culture petri dishes or on glass coverslips. A few days after trypsinization, the medium was replaced by DMEM:F12 without insulin/transferrin/selenium and Ultroser during the 24 h preceding the start of the various treatments.

Isolation of mitochondria and preparation of submitochondrial fractions
H295R cells were homogenized with a Potter-Elvehjem homogenizer (1200 rpm, 35 strokes) in a 5 mM Tris-HCl buffer (pH 7.4) containing 275 mM sucrose. The homogenate was centrifuged at 200 x g for 15 min to remove large debris and nuclei. Further centrifugation of the supernatant at 10,000 x g for 10 min yielded the mitochondria. The mitochondrial pellet was then washed twice at 8000 x g with the same buffer.

Submitochondrial particles were prepared as described elsewhere (15). Briefly, washed mitochondrial pellets were exposed to a swelling procedure by incubation for 20 min in 10 mM sodium phosphate buffer (pH 7.4), at a final protein concentration of approximately 1 mg/ml. Incubation was terminated by addition of sucrose to obtain a 0.45 M concentration. Aliquots (10 ml) were mildly sonicated (3 x 30 sec), using a probe sonicator (Branson Sonifier 250; Branson Ultrasonics Corporation, Danbury, CT). The suspension was centrifuged at 8000 x g to remove unbroken mitochondria. The supernatant was collected and centrifuged again at 150,000 x g for 90 min. The pellet, containing the submitochondrial membrane fractions, was mixed in 10 mM sodium phosphate buffer (pH 7.4) containing 0.45 M sucrose (final protein concentration, 5 mg/ml) using a Teflon homogenizer (DuPont Co., Wilmington, DE). Membranes (1–2 mg protein) were loaded onto a linear (15–50%) sucrose density gradient and centrifuged for 20 h at 100,000 x g. Subsequently, the gradient was divided into 500-µl fractions that were assayed for marker enzyme activities. Protein was quantified using the Bio-Rad protein microassay (Bio-Rad Laboratories, Inc., Hercules, CA) and BSA as a standard. Cytochrome C oxidase (COX) (Enzyme Commission of the International Union of Biochemistry 1.9.3.1) activity was determined according to Appelmans et al. (16).

Proteinase K digestion of extramitochondrial proteins
The basic experimental procedure was previously described (6). Briefly, freshly isolated membranes (100 µg) were incubated in homogenizing buffer (without protease inhibitors) alone or in the presence of proteinase K (0.1 µg/ml) only or proteinase K plus Triton X-100 (1% final concentration). After 10 min incubation on ice, 2 µl phenylmethylsulfonyl fluoride (100 mM) was added to terminate proteolysis. Samples were then boiled in loading buffer for 5 min and analyzed by immunoblotting.

Hint2 immunoblot analysis
Submitochondrial distribution of Hint2 was analyzed on a commercially available membrane (Biochain, Lugano, Switzerland). Briefly, fractionated mitochondrial membranes were loaded on a 4–20% denaturating polyacrylamide gel. After electrophoresis, proteins were transferred, and the blot was tested with a homemade rabbit anti-Hint2 antibody. After blocking for 1 h at 37 C with 5% nonfat milk, membranes were incubated with the primary antibody overnight at 4 C. The membranes were then washed and incubated for 1 h with peroxidase-conjugated secondary antibody, antirabbit donkey IgG diluted 1:65,000 (Pierce, Rockford, IL). Immunoblots were revealed using an enhanced chemiluminescence detection system (SuperSignal West Pico Chemiluminescent Substrate; Pierce), and exposure was performed with a Fujifilm LAS-1000 CCD camera (Dielsdorf, Switzerland) coupled to a computer.

Cell transfection with small interfering RNAs (siRNAs) and plasmids
siRNA fragments and plasmids were introduced in H295R cells by electroporation, using a Nucleofector (Amaxa Biosystems, Köln, Germany). Batches of 5 x 106 dissociated cells were used in each transfection experiment. Cells were washed with PBS and centrifuged at 1000 rpm for 5 min. They were then resuspended in 100 µl Nucleofector solution R (Amaxa Biosystems) at room temperature, and 4 µg nonsilencing siRNA was added, or a mixture of two siRNAs directed against Hint2 (Table 1Go). The cells were transferred into a 2 mm-wide electroporation cuvette (Amaxa Biosystems), and electroporation was performed according to the indications of the Nucleofector manufacturer, using the T-20 program. Immediately after transfection, H295R cells were diluted with 500 µl DMEM/Ham’s F-12 (1:1) medium. Cells were then seeded in 24-well chamber plates or 35-mm Petri dishes. In separate experiments a plasmid (pcDNA3) coding the Hint2 gene (QIAGEN, Germantown, MD) was used in place of siRNAs.


View this table:
[in this window]
[in a new window]

  		TABLE 1. siRNA sequences

 
RNA extraction and mRNA analysis
Total RNA was extracted from H295R cells or adrenal tissues with the Nucleospin kit and on-column deoxyribonuclease digested (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. The concentration and purity of RNA were determined spectrophotometrically, and cDNA was prepared from 250–500 ng total RNA using the iScript cDNA biosynthesis kit (Bio-Rad Laboratories, Reinach, Switzerland), as suggested by the manufacturer.

Amplification by PCR was performed in a DNA Thermal Cycler 9700 (PerkinElmer, Courtaboeuf, France), using 10 pmol Hint or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers (Table 2Go). After 30 PCR cycles (94 C for 30 sec, 58 C for 30 sec, and 72 C for 30 sec), amplicons were analyzed by electrophoresis on a 2% agarose gel, and their sequence was confirmed on a Li-Cor 4000L DNA sequencer (Science Tec, Les Ulis, France).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Primers and probes used for PCR

 
Hint2 mRNA was quantified by real-time RT-PCR, using TaqMan probes (Microsynth, Balgach, Switzerland) on an iCycler iQ (Bio-Rad Laboratories, Inc., Hercules, CA). Probes were designed using the Primer3 program, and specificity was confirmed by Basic Local Alignment Search Tool analysis (Table 2Go). Briefly, real-time PCR was performed in a 25-µl reaction medium containing 12.5 µl master mix (ABgene, Epsom, UK), 500 nM primers, 100 nM TaqMan probe, and previously reverse-transcribed cDNA. The PCR thermal protocol consisted of 45 cycles (denaturating at 95 C for 15 sec, and annealing/extension at 60 C for 1 min). Hint2 mRNA levels were normalized to those of GAPDH, using the comparative 2-{Delta}{Delta}Ct method (17), and expressed as a function of levels obtained under control conditions.

Steroid measurement
Pregnenolone and aldosterone production was determined by direct RIA of the steroids in the extracellular medium (Diagnostic Systems Laboratories, Inc., Webster, TX). After washing with a Krebs buffer [136 mM NaCl, 1.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 1.2 mM CaCl2, and 5.5 mM D-glucose (buffered to pH 7.4) with 20 mM HEPES], cells were incubated for 2 h in the same buffer, supplemented or not with AngII or KCl. For pregnenolone assessment, trilostane (5 µM) was present in wells throughout the stimulation period to prevent the conversion of pregnenolone to progesterone. At the end of the incubation, the medium was collected for pregnenolone or aldosterone assays, as previously described (18). The cell protein content was determined by the Coomassie blue method (Bio-Rad Laboratories GmbH, Munich, Germany). Each condition was tested in duplicate in each experiment, and steroid assay was performed in duplicate.

Mitochondrial calcium measurements
Mitochondrial calcium fluctuations were recorded using the mitochondrial ratiometric pericam probe as previously described (7). Plasmid (pcDNA3) containing DNA coding for ratiometric pericam fused in-frame on the 5' extremity to the first 36 nucleotides of the mitochondrial COX subunit IV was introduced in H295R cells by transient transfection with the Nucleofector technique. After transfection (plasmid at 340 ng/ml for 9–16 h), cells were washed and left in culture medium supplemented with serum for 2–3 additional days before being used in the experiments.

Variations in mitochondrial calcium concentrations were recorded by imaging pericam fluorescence on an Axiovert S100TV microscope, using a x40, 1.3NA oil immersion objective (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Excitation was alternatively performed at 480 ± 10 and 410 ± 10 nm with the use of a DeltaRam monochromator (Photon Technology International, Inc., Monmouth Junction, NJ) through a 505 DCXR dichroic mirror (Chroma Technology Corp., Rockingham, VT) and emission filtered at 535 nm (535DF25; Omega Optical, Brattleboro, VT). Image acquisition (every 2 sec at each excitation wavelength) and analysis were performed with the Metamorph/Metafluor 4.1.2 software (Universal Imaging, West. Chester, PA). The fluorescence intensity ratio (at 480/410 nm) was calculated pixel by pixel and averaged within each manually delimited entire cell. Changes in fluorescence ratio, reflecting mitochondrial calcium variations, were then expressed as a function of time.

Statistical analysis
All results are shown as mean ± SEM of data from at least three separate experiments. The statistical significance of the changes induced by treatments was analyzed by paired or unpaired t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intramitochondrial expression of Hint2 in H295R cells
Hint2 has been shown to be involved in the mitochondrial process of apoptosis. Because of similarities, in terms of mitochondrial calcium requirements, for steroidogenesis and apoptosis, we hypothesized that this protein could also play a role in steroid biosynthesis. Therefore, the expression of Hint2 within adrenocortical H295R cells was investigated at the levels of both mRNA and protein. As shown in Fig. 1AGo (left panel), RT-PCR analysis using specific primers revealed the presence in these cells of mRNA coding for three Hint protein isoforms, namely Hint1, Hint2, and, in a less pronounced manner, Hint3. The absence of amplification when the reverse transcription (RT) was omitted before PCR (–RT lanes) excluded any contamination by genomic DNA.


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 1. Hint2 expression in H295R cells. The presence of Hint2 in H295R cells and adrenal tissues was investigated by RT-PCR (A) and immunoblot analysis (B). A, Total RNA was extracted from H295R cells, reverse transcribed (+RT) or not (–RT), and amplified with primers specific for Hint1, Hint2, and Hint3 (Table 2Go). Amplicons were then analyzed by gel electrophoresis (left panel). Similar experiments were performed (for Hint2) with normal adrenocortical tissues isolated from two patients (h1 and h2) and with freshly isolated bovine adrenal cortex (b1, right panel). B, Proteins from various cellular fractions were analyzed by sodium dodecyl sulfate gel electrophoresis and immunoblotting. Left panel, Cell homogenate (Hom) and mitochondrial fractions (Mito) were prepared by differential centrifugation as described in Materials and Methods. Right panel, Isolated mitochondria were exposed for 10 min to various treatments: control (Mito1), proteinase K alone (Mito2), proteinase K, and Triton X-100 (Mito3). Arrow indicates the size of intact Hint2. Neg, Negative control.

 
We then focused our attention only on Hint2 for the rest of the study. Because H295R cells were originally derived from a human adrenal tumor, we verified that Hint2 was also expressed in normal human adrenal as well as in adrenals from other species. For this purpose, additional RT-PCR experiments were performed with adrenals removed from two patients (h1 and h2), as well as with freshly prepared bovine adrenal cortex (b1). As shown in Fig. 1AGo (right panel), Hint2 was also clearly expressed in these tissues, supporting a general role for this protein in adrenal function.

The presence of the protein in H295R cells was then confirmed by Western blot analysis. As expected, the specific antibody raised against Hint2 revealed a 17-kDa band in cell homogenate (Fig. 1BGo), and the protein was clearly recovered in the mitochondrial fraction (Mito), prepared by differential centrifugation. Hint2 submitochondrial localization was then investigated according to two different approaches. We first tested the susceptibility of mitochondria-associated Hint2 to degradation by proteinase K (Fig. 1BGo, right). For this purpose, freshly prepared mitochondria were exposed to proteinase K in the presence (Mito3) or absence (Mito2) of Triton X-100. Control mitochondria (Mito1) were incubated without proteinase K. The fact that Hint2 was relatively resistant to proteinase treatment in the absence of detergent suggests that the protein was not directly accessible from the cytosol and, therefore, mostly expressed inside the organelle.

We also fractionated mitochondrial membranes on a sucrose density gradient as previously described (2). The distribution of Hint2 among the various fractions, as determined by Western blot analysis (Fig. 2Go), and of COX, an enzyme marker of inner mitochondrial membranes, did not exactly overlap. In fact, intact Hint2 was maximal within fractions F11 to F13, at densities intermediate between those of outer and inner mitochondrial membranes, and corresponding the best to that of contact sites in adrenal glomerulosa cells, the putative site of cholesterol transport into the mitochondria (2, 15).


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 2. Submitochondrial localization of Hint2. Mitochondrial membranes were fractionated on a sucrose density gradient as described in Materials and Methods, and the presence of Hint2 in each fraction was determined by immunoblot analysis (only fractions 8–16 are shown). Mito corresponds to the mitochondrial membrane before fractionation. Protein content and COX activity were also measured in the same fractions and expressed as a percentage of the maximal measurement. The approximate distribution of outer (OMM) and inner mitochondrial membranes (IMM), and of contact sites (CS) is indicated on the top of the figure.

 
Role of Hint2 in the control of steroidogenesis
Mitochondria being the intracellular site of the main steps of steroid biosynthesis, it was suggested that Hint2 could participate in the control of steroid production. To determine whether Hint2 was involved in the steroidogenic function of H295R cells, we measured the production of pregnenolone and aldosterone by these cells either after overexpressing Hint2 or after its silencing with specific siRNA.

As shown in Fig. 3Go, 48 h after cell transfection with a pcDNA plasmid coding for Hint2, one could observe a marked increase of both the mRNA levels (Fig. 3AGo) and the protein (Fig. 3BGo). In contrast, combining two different siRNAs against Hint2 led to a 90% reduction of mRNA after 48 h (Fig. 3CGo) and a 60% decrease of the protein levels (Fig. 3DGo).


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 3. Measurement of Hint2 overexpression or silencing. Hint2 expression was increased by transfecting H295R cells with a pcDNA plasmid coding for the protein (A and B), or reduced (C and D) by introducing in the cells a combination of two specific siRNAs directed against Hint2 (Table 1Go). Control (Ctrl) conditions were performed with the empty vector or a nonsilencing siRNA. The effect of treatments on Hint2 mRNA and protein levels was then assessed by real-time RT-PCR (TaqMan assay) (A and C) and immunoblot analysis (B and D), respectively. Results were normalized with GAPDH (mRNA) or actin (protein) and expressed as a function of the levels found in control conditions. Insets of panels B and D show representative results with the arrow indicating the size of Hint2 protein. If not otherwise mentioned, a combination of both siRNA a and b was used. Data are the mean values from three independent experiments.

 
Hint2 overexpression had no influence on the steroidogenic responses to either AngII (Fig. 4AGo) or K+ (Fig. 4BGo). Indeed, pregnenolone formation was not significantly different in control cells and in cells transfected with the Hint2 plasmid. Because this lack of effect could be due to the fact that, under normal conditions, Hint2 content in H295R cells was already optimal in the steroidogenic pathway and, therefore, was not rate limiting, the opposite approach, consisting of decreasing its expression with siRNA, was used.


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 4. Effect of Hint2 on the steroidogenic response of H295R cells. H295R cells overexpressing Hint2 (A and B, closed circles) or with reduced Hint2 expression (C–F, closed circles) were stimulated with increasing concentrations of AngII or K+. After 2 h incubation, pregnenolone (A–D) or aldosterone (E and F) were assessed in the medium by direct RIA. Open circles and dashed lines correspond to controls. Results are expressed in percentage of the basal value measured in controls, which approximately amounted to 5 pmol/mg cell protein·h for aldosterone and 49 pmol/mg protein·h for pregnenolone. Curves were fitted to data according to a four-parameter logistical model. *, P < 0.05 and **, P < 0.01, vs. control.

 
In this case, a strong inhibition of pregnenolone production was observed. Indeed, Hint2 silencing resulted in a marked decrease, by approximately 45%, of the response to AngII (Fig. 4CGo), whereas the response to K+ was reduced by 65% (Fig. 4DGo). Later steps of steroidogenesis were also evaluated by measuring aldosterone secretion. As expected, a similar effect of Hint2 silencing was observed on aldosterone secretion, although slightly less pronounced than for pregnenolone (Fig. 4Go, E and F). Although in each case the maximal steroidogenic response was affected by Hint2 silencing, the sensitivity to the agonists (EC50 values) was not modified.

Mechanism of Hint2 action
Because Ca2+ mobilization and Ca2+ entry into the mitochondria were required for the steroidogenic response to AngII or K+, we investigated whether the mitochondrial Ca2+ signal induced by these agonists was affected by the levels of Hint2 expression. For this purpose, Ca2+ fluctuations within the mitochondrial matrix were monitored with the fluorescent probe mitochondrial ratiometric pericam, as previously described (7). Cells were exposed to the same protocol, as illustrated in Fig. 5AGo. After 3 min resting, cells were stimulated with AngII (100 nM) for 4 min, and CCCP, a proton ionophore, was added for depolarizing the mitochondria and releasing Ca2+ within the cytosol. A decrease of the signal upon CCCP addition confirmed the intramitochondrial location of the probe. The probe signal was then internally calibrated by the consecutive addition of ionomycin, a Ca2+ ionophore saturating mitochondria with Ca2+, and of EGTA, to reach the minimal levels.


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 5. Effect of Hint2 on the mitochondrial calcium fluctuations. Cells expressing different amounts of Hint2 were transfected with a plasmid coding for the mt ratiometric pericam and mitochondrial calcium fluctuations in response to AngII were recorded in single cells, as indicated in the Materials and Methods. A, A typical trace of mitochondrial calcium variations induced in control (Ctrl) H295R cells consecutively by AngII (100 nM), CCCP (10 µM), ionomycin (2 µM), and EGTA (4 mM). Letters a–d refer to parameters analyzed in B. B, Quantification of various parameters obtained from traces similar to that shown in A but recorded in H295R cells overexpressing Hint2 (left panel, black bars) or with siRNA-treated cells (right panel, black bars). Control values (open bars) were determined in the same experiments from recordings obtained with cells transfected with either an empty plasmid (left) or with nonsilencing siRNA (right). Basal refers to the mean fluorescence ratio value recorded before the addition of AngII [corresponding to (a) in panel A], reported to the whole range of values defined by the addition of ionomycin (maximum) and EGTA (minimum). b, Amplitude corresponds to the difference between the peak response to AngII, determined after signal smoothing, and the basal value, expressed in fluorescence ratio units. c, Time to peak refers to the delay, in minutes, between the addition of AngII and the peak response, and decay (d/b) refers to the ratio between the signal amplitude measured exactly 3 min after the addition of AngII and the peak response. Data are the mean values ± SEM from 95 (left) and 45 (right) independent cells. **, P < 0.01 and ***, P < 0.005, vs. control. Inset, Representative traces recorded in a Hint2 siRNA expressing cell (dark trace) and in a control cell transfected with a nonsilencing siRNA. Scale bars, 2 min (x-axis) and 0.3 fluorescence ratio units (y-axis). Mit., Mitochondrial; arbitr., arbitrary.

 
Several parameters were then measured on each recording, including the: 1) basal Ca2+ concentration; 2) amplitude of the peak response to AngII; 3) delay of the response (time to peak) corresponding to the period (in minutes) between AngII addition and the peak response; and, finally, 4) decay of the signal, determined 3 min after the stimulation with AngII.

None of these parameters was significantly affected upon overexpression of Hint2, as shown by comparing 49 cells overexpressing the protein with 46 control cells transfected with the empty plasmid (Fig. 5BGo, left panel). In contrast, Hint2 silencing appeared to modify some of these parameters. Indeed, whereas the basal concentration and the peak amplitude were only minimally affected, both the time to peak and the decay parameters were significantly reduced, suggesting the occurrence of a slightly shorter calcium response within the mitochondria of cells defective in Hint2 protein, as illustrated in the inset of the right panel in Fig. 5BGo.

Because mitochondrial calcium signaling is known to be strongly dependent on the organelle inner membrane potential, we determined whether Hint2 levels could affect this parameter. For this purpose we used rhod123 as a fluorescent marker (19). We found, by fluorescence-activated cell sorter analysis, that Hint2 silencing resulted in an increase of the median rhod123 cell fluorescence by 13.5 ± 5.2% (n = 4), indicating partial mitochondrial depolarization under these conditions. By comparison, treatment of control cells with CCCP (10 µM) in the same experiments induced a 19.9 ± 9.1% fluorescence increase. In contrast, Hint2 overexpression had no effect on mitochondrial potential.

To determine whether Ca2+-independent pathways of steroidogenesis were also affected by a lack of Hint2, siRNA-treated cells were stimulated with forskolin, an activator of the adenylyl cyclase enzyme and, therefore, of the cAMP pathway, and with 25-OHChol, a precursor of steroids, like cholesterol, but able to bypass the rate-limiting and Ca2+-dependent step of mitochondrial import (1).

As shown in Fig. 6AGo, 10 µM forskolin increased approximately 15-fold pregnenolone production in control cells treated with nonsilencing siRNA. However, the stimulation by forskolin was significantly lower in cells defective in Hint2. Similar results were obtained when nicardipine (1 µM), a blocker of voltage-gated calcium channels, was present in the medium, excluding the possibility that forskolin in these experiments indirectly mobilized calcium through regulation of calcium channels and exerted its steroidogenic action partly through a Ca2+-dependent mechanism.


Figure 6
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 6. Effect of Hint2 on the steroidogenic response to forskolin and 25-OHChol. H295R cells were transfected for 2 d with siRNA and then incubated for 2 h with 10 µM forskolin (Fsk), in the presence or absence of 1 µM nicardipine (Nic) (A), or with increasing concentrations of 25-OHChol (B). Pregnenolone was determined in the medium after the incubation by direct RIA. Open bars and circles correspond to control conditions with nonsilencing siRNA. Data are the mean value ± SEM from six independent experiments. *, P < 0.05, vs. control.

 
Finally, the transformation of 25-OHChol into pregnenolone by intact H295R cells, a reaction generally considered as reflecting the activity of the P450 side chain cleavage enzyme and to be completely Ca2+ independent, was markedly reduced in cells with down-regulated Hint2 (Fig. 6BGo). This observation confirmed that Hint2 was required also in steroidogenic steps independent of Ca2+ signaling and possibly regulated the activity of the enzyme responsible for the first step common to all steroid biosynthetic processes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that Hint2 is expressed within the mitochondria of H295R cells and that it is required for an optimal steroidogenic activity of these cells in response to various agonists. The protein appears to regulate both Ca2+-dependent and Ca2+-independent processes involved in steroidogenesis.

The relative resistance of Hint2 to degradation by proteinase K in intact mitochondria (Fig. 1BGo), as well as the protein distribution upon mitochondrial membrane fractionation on density gradient (Fig. 2Go), suggests that Hint2 could be associated with contact sites, and could face the matrix of the organelle. Because of the hydrophobic nature of cholesterol and, therefore, its inability to cross the aqueous mitochondrial intermembrane space, contact sites between external and internal membranes are considered as privileged sites for cholesterol transport and, therefore, contain an important pool of steroidogenic cholesterol that is available for the P450 scc enzyme activity (2, 15, 20). Mitochondrial calcium has induced matrix swelling and promoted the fusion between membranes, thus increasing the number of contact sites and the rate of cholesterol transfer (21, 22). Given the putative location of Hint2 within the mitochondria, a role in membrane fusion or cholesterol transport could be envisaged and experimentally tested.

Hint2 overexpression had little effect on steroidogenesis, whereas protein silencing markedly reduced pregnenolone and aldosterone production (Fig. 4Go). A similar observation was made when measuring the mitochondrial Ca2+ signaling elicited by AngII (Fig. 5BGo). Hint2 overexpression did not significantly affect the various evaluated parameters of this response, whereas Hint2 down-regulation resulted in a shorter time to peak response and a more rapid decay of mitochondrial calcium. These data suggest that, under basal conditions, Hint2 is optimally expressed for its function in steroidogenesis and is not rate limiting. In fact, H295R cells are among the cells expressing Hint2 the most abundantly, at levels at least comparable to those found in hepatic or pancreatic cells, according to our TaqMan assays. By comparison, in control H295R cells, mRNA levels coding for the steroidogenic acute regulatory protein, responsible for the import of mitochondrial cholesterol, the rate-limiting step in steroidogenesis, were approximately 8-fold lower than those of Hint2 (data not shown).

Moreover, we did not observe any induction of Hint2 expression upon stimulation with AngII, even after 48 h incubation (data not shown), demonstrating that the protein is probably permissive for steroidogenesis but not regulated.

By decreasing the rate of calcium decay upon stimulation with AngII, Hint2 significantly prolongs the signal required for steroidogenesis and could, through this simple mechanism, potentiate steroid production. This is particularly relevant for an agonist like AngII, which mobilizes calcium in an oscillatory manner (23). Therefore, a more sustained elevation of mitochondrial calcium is expected to be directly translated in higher pregnenolone production (1, 5). Moreover, this mode of action proposed for Hint2 fits well with the high sensitivity of the response elicited by K+ to a reduction of the protein because this agonist is known to be more dependent on the Ca2+ signal than AngII, which also activates additional pathways (24).

Interestingly, a part of Hint2 action on steroid production cannot be explained by its effect on the mitochondrial calcium signal. Indeed, pregnenolone formation in response to forskolin (Fig. 6AGo) or 25-OHChol (Fig. 6BGo) is also significantly affected by a reduction of Hint2 expression. Forskolin acts through the activation of adenylyl cyclase and the cAMP pathway, normally in a Ca2+-independent manner; however, because L-type Ca2+ channels have been positively modulated by protein kinase A (25, 26), we also tested the same conditions but in the presence of nicardipine for preventing any Ca2+ mobilization that could be transmitted to the mitochondria. Results being very similar in the presence or absence of nicardipine, and because Hint2 silencing was not affecting the basal mitochondrial Ca2+ concentration (Fig. 5BGo), we can conclude that Hint2 is also required for an optimal steroidogenic response to stimuli completely independent of Ca2+ mobilization. This hypothesis was confirmed by the observation that the conversion into pregnenolone of 25-OHChol, a more hydrophilic form of cholesterol able to cross freely the intermembrane barrier and enter the mitochondria, was affected by Hint2 siRNA treatment, despite the fact that with 25-OHChol as substrate, pregnenolone formation is independent of second messenger production. Therefore, the activity of the P450 scc enzyme itself seems affected by Hint2 reduction. Although we do not know yet the precise molecular mechanisms of Hint2 action, our finding that Hint2 levels are important for maintaining mitochondrial potential is probably an important clue for the discovery of these mechanisms.

The action of Hint2 on the mitochondrial calcium homeostasis could also account for its proapoptotic role in nonsteroidogenic cells. Indeed, a sustained Ca2+ uptake into the mitochondria, leading to Ca2+ overload in the organelle, is associated with apoptosis in many cell types (27, 28). This phenomenon is believed to be due to the opening, in the presence of excessive calcium, of the mitochondrial permeability transition pore followed by depolarization of the organelle, swelling and release of cytochrome c and of proapoptotic proteins in the cytosol (29). It is not clear whether steroid-producing cells, which experience frequent calcium increases within their mitochondria as steroidogenic signals, are more resistant, by nature, to apoptosis than other cell types. Nevertheless, preliminary investigations in our laboratory, consisting of measuring the expression of caspase 3 as a proapoptotic marker, suggest that Hint2 levels could also affect apoptosis in H295R cells (data not shown). Thus, it is not really surprising in this context that the same protein, Hint2, is described as being both prosteroidogenic in adrenal cells and proapoptotic in hepatocytes (13).

In conclusion, we have shown that Hint2, a proapoptotic mitochondrial protein previously characterized in hepatic and pancreatic cells, is expressed in the mitochondria of adrenocortical cells and participates in the steroidogenic response of calcium-dependent and calcium-independent agonists. In addition to its action on the kinetics of the mitochondrial calcium signaling, Hint2 also seems to exert its action through Ca2+-independent mechanisms, e.g. by maintaining some degree of P450 scc activation. Although not regulated, this protein nevertheless appears involved in ensuring an optimal steroidogenic response.


   Acknowledgments
 
We thank Dr. H. Lefebvre for providing us with human adrenal cortex, Dr. Pozzan for the plasmid coding the mitochondrial ratio pericam, and Dr. A. M. Capponi for PCR primers for steroidogenic acute regulatory protein. We also thank Ms. M. Python for her excellent technical assistance.


   Footnotes
 
This work was supported by the Swiss National Science Foundation Grants 310000-111808 (to M.F.R.) and 3100A0-112173 (to J.-F.D.), and by the Fondation pour la Lutte contre le Cancer et pour les Recherches Médico-Biologiques.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: AngII, Angiotensin II; CCCP, carbonyl cyanide m-chlorophenylhydrazone; COX, cytochrome C oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 25-OHChol, 25-hydroxycholesterol; Mito, mitochondrial fraction; RT, reverse transcription; siRNA, small interfering RNA.

Received March 21, 2008.

Accepted for publication July 15, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Python CP, Laban OP, Rossier MF, Vallotton MB, Capponi AM 1995 The site of action of Ca2+ in the activation of steroidogenesis: studies in Ca(2+)-clamped bovine adrenal zona-glomerulosa cells. Biochem J 305(Pt 2):569–576
  2. Cherradi N, Rossier MF, Vallotton MB, Capponi AM 1996 Calcium stimulates intramitochondrial cholesterol transfer in bovine adrenal glomerulosa cells. J Biol Chem 271:25971–25975[Abstract/Free Full Text]
  3. Cherradi N, Brandenburger Y, Rossier MF, Capponi AM 1998 Regulation of mineralocorticoid biosynthesis by calcium and the StAR protein. Endocr Res 24:355–362[Medline]
  4. Capponi AM, Rossier MF, Davies E, Vallotton MB 1988 Calcium stimulates steroidogenesis in permeabilized bovine adrenal cortical cells. J Biol Chem 263:16113–16117[Abstract/Free Full Text]
  5. Brandenburger Y, Kennedy ED, Python CP, Rossier MF, Vallotton MB, Wollheim CB, Capponi AM 1996 Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells. Endocrinology 137:5544–5551[Abstract]
  6. Brandenburger Y, Arrighi JF, Rossier MF, Maturana AD, Vallotton MB, Capponi AM 1999 Measurement of perimitochondrial Ca2+ concentration in bovine adrenal glomerulosa cells with aequorin targeted to the outer mitochondrial membrane. Biochem J 341(Pt 3):745–753
  7. Lalevee N, Resin V, Arnaudeau S, Demaurex N, Rossier MF 2003 Intracellular transport of calcium from plasma membrane to mitochondria in adrenal H295R cells: implication for steroidogenesis. Endocrinology 144:4575–4585[Abstract/Free Full Text]
  8. Rossier MF 2006 T channels and steroid biosynthesis: in search of a link with mitochondria. Cell Calcium 40:155–164[CrossRef][Medline]
  9. Gallo-Payet N, Grazzini E, Côté M, Chouinard L, Chorvátová A, Bilodeau L, Payet MD, Guillon G 1996 Role of calcium in the action of adrenocorticotropin in cultured human adrenal glomerulosa cells. J Clin Invest 98:460–466[Medline]
  10. Burnay MM, Vallotton MB, Capponi AM, Rossier MF 1998 Angiotensin II potentiates adrenocorticotrophic hormone-induced cAMP formation in bovine adrenal glomerulosa cells through a capacitative calcium influx. Biochem J 330(Pt 1):21–27
  11. Brenner C, Bieganowski P, Pace HC, Huebner K 1999 The histidine triad superfamily of nucleotide-binding proteins. J Cell Physiol 181:179–187[CrossRef][Medline]
  12. Brenner C 2002 Hint, Fhit, and GalT: function, structure, evolution, and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases. Biochemistry 41:9003–9014[CrossRef][Medline]
  13. Martin J, Magnino F, Schmidt K, Piguet AC, Lee JS, Semela D, St Pierre MV, Ziemiecki A, Cassio D, Brenner C, Thorgeirsson SS, Dufour JF 2006 Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma. Gastroenterology 130:2179–2188[CrossRef][Medline]
  14. Bird IM, Hanley NA, Word RA, Mathis JM, McCarthy JL, Mason JI, Rainey WE 1993 Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin II-responsive aldosterone secretion. Endocrinology 133:1555–1561[Abstract/Free Full Text]
  15. Cherradi N, Rossier MF, Vallotton MB, Timberg R, Friedberg I, Orly J, Wang XJ, Stocco DM, Capponi AM 1997 Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein, P450 side-chain cleavage and 3β-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells. J Biol Chem 272:7899–7907[Abstract/Free Full Text]
  16. Appelmans F, Wattiaux R, De Duve C 1955 Tissue fractionation studies. 5. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem J 59:438–445[Medline]
  17. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-{delta} {delta} C(T)) method. Methods 25:402–408[CrossRef][Medline]
  18. Cherradi N, Brandenburger Y, Rossier MF, Vallotton MB, Stocco DM, Capponi AM 1998 Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells. Mol Endocrinol 12:962–972[Abstract/Free Full Text]
  19. Duchen MR, Biscoe TJ 1992 Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body. J Physiol 450:33–61[Abstract/Free Full Text]
  20. Stevens VL, Xu T, Lambeth JD 1992 Cholesterol pools in rat adrenal mitochondria: use of cholesterol oxidase to infer a complex pool structure. Endocrinology 130:1557–1563[Abstract/Free Full Text]
  21. Brdiczka D 1991 Contact sites between mitochondrial envelope membranes. Structure and function in energy- and protein-transfer. Biochim Biophys Acta 1071:291–312[Medline]
  22. Bakker A, Bernaert I, De Bie M, Ravingerova T, Ziegelhoffer A, Van Belle H, Jacob W 1994 The effect of calcium on mitochondrial contact sites: a study on isolated rat hearts. Biochim Biophys Acta 1224:583–588[Medline]
  23. Rossier MF, Capponi AM 2002 Cytosolic calcium oscillations in signal transduction pathways. Mini Rev Med Chem 2:353–360[CrossRef][Medline]
  24. Capponi AM, Python CP, Rossier MF 1994 Molecular basis of angiotensin II action on mineralocorticoid synthesis. Endocrine 2:579–586
  25. Armstrong D, Eckert R 1987 Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization. Proc Natl Acad Sci USA 84:2518–2522[Abstract/Free Full Text]
  26. Kamp TJ, Hell JW 2000 Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res 87:1095–1102[Abstract/Free Full Text]
  27. Bernardi P, Rasola A 2007 Calcium and cell death: the mitochondrial connection. Subcell Biochem 45:481–506[CrossRef][Medline]
  28. Dong Z, Saikumar P, Weinberg JM, Venkatachalam MA 2006 Calcium in cell injury and death. Annu Rev Pathol 1:405–434[CrossRef][Medline]
  29. Armstrong JS 2006 The role of the mitochondrial permeability transition in cell death. Mitochondrion 6:225–234[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lenglet, S.
Right arrow Articles by Rossier, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lenglet, S.
Right arrow Articles by Rossier, M. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals