help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.
Right arrow Articles by Stocco, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.
Right arrow Articles by Stocco, D. M.
Endocrinology Vol. 138, No. 7 2722-2728
Copyright © 1997 by The Endocrine Society

ARTICLES

Heat Shock-Induced Inhibition of Acute Steroidogenesis in MA-10 Cells Is Associated with Inhibition of the Synthesis of the Steroidogenic Acute Regulatory Protein1

Zhiming Liu and Douglas M. Stocco

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Dr. Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430. E-mail: cbbdms{at}wpoffice.ttuhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The synthesis of heat shock proteins (HSPs) rapidly increases in cells under a broad range of stress conditions in addition to heat shock. Previous studies have shown that the induction of HSPs severely impairs the ability of steroidogenic cells to synthesize steroids in response to acute stimulation. De novo synthesis of the steroidogenic acute regulatory (StAR) protein has been shown to be indispensable for acute steroid hormone biosynthesis; however, the effect of HSP induction on the synthesis of the StAR protein has not yet been studied. In the present study we investigated whether HSP induction might influence the steroidogenic activity of MA-10 mouse Leydig tumor cells, and whether this effect may involve the synthesis of StAR protein. MA-10 cells exposed to 45 C for 10 min and allowed to recover for 2 h at 37 C displayed a 6-fold increase in HSP-70 at 3 h postrecovery and a 20-fold increase in this protein at 6 h postrecovery. This heat shock regimen also acutely inhibited both progesterone production and StAR protein synthesis in MA-10 cells in response to LH and cAMP analog stimulation. The activity and quantity of cytochrome P450 side-chain cleavage and 3ß-hydroxysteroid dehydrogenase were not affected by this heat shock treatment, indicating that the loss of steroidogenic capacity was not a result of inhibition of the enzymes involved in the conversion of cholesterol to progesterone. The results suggest that the previously observed antisteroidogenic effects of heat shock treatment may be due mainly to the acute inhibition of StAR protein synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ADDITION to temperature increases, a wide variety of stress agents increases the synthesis of heat shock proteins (HSPs) in cells. Environmental stresses, including heavy metals, amino acid analogs, toxins, anoxia, and mediators of inflammation, such as cytokines and PGs, stimulate HSP synthesis (1, 2, 3, 4, 5, 6). HSPs are highly conserved molecules, and their induction requires activation of the transcription of heat shock genes by heat shock factors (7). Although the precise function of the HSPs remains obscure, it has been proposed that these proteins play an important role in providing cells with a protective mechanism against environmental insults and in aiding cellular recovery after such trauma, possibly through the refolding of proteins denatured by heat shock (8).

Several lines of evidence demonstrated that agents known to induce the heat shock response block steroid biosynthesis in steroidogenic cells. Khanna et al. demonstrated that in rat luteal cells, temperature-induced heat shock resulted in the induction of HSP-70 as well as the cessation of progesterone biosynthesis (9, 10, 11). Also, PGF2{alpha} causes a rapid and sustained accumulation of HSP-70 and abruptly inhibits hormone-sensitive progesterone synthesis, leading to the suggestion that HSP-70 mediates the intracellular protein processing underlying luteal regression (12, 13). Additional heat shock protein-producing agents also known to inhibit steroid hormone biosynthesis include phorbol esters (14, 15) and cytokines such as tumor necrosis factor-{alpha} (16, 17). Further, it has been suggested that the mechanism of the heat shock-induced inhibition of steroidogenesis appears to be interference with the translocation of the substrate for all steroid hormone biosynthesis, cholesterol, to the inner membrane of the mitochondria (9, 10, 11).

The heat shock response in steroidogenic MA-10 mouse Leydig tumor cells has not been investigated. Under normal conditions, mediated by the intracellular second messenger cAMP, MA-10 cells rapidly synthesize progesterone in response to stimulation by trophic hormone. This stimulation also results in the rapid synthesis of the steroidogenic acute regulatory (StAR) protein, which is believed to regulate cholesterol transfer from the outer to the inner mitochondrial membrane where it is converted into pregnenolone by cytochrome P450 side-chain cleavage (P450scc) (18, 19, 20, 21). It is this transfer of cholesterol that is the rate-limiting step in hormone-stimulated acute steroidogenesis (22, 23, 24, 25).

Although inhibition of steroid synthesis by heat shock has been clearly demonstrated in steroidogenic cells (9, 10, 11), the mechanism of action of this inhibition remains unclear, although as mentioned above, cholesterol transfer has been implicated (9, 10, 11). As the StAR protein has been shown to be a critical and indispensable component in the acute regulation of steroidogenesis (26), it was reasoned that examination of the effects of heat shock on StAR synthesis may provide an answer for the observed inhibition of steroid production. Therefore, in the present study, we investigated whether heat shock had any effect on progesterone production in MA-10 cells and, if so, whether this effect involved synthesis of the StAR protein. The results of our study demonstrate that in MA-10 cells, heat shock induces a large increase in HSP-70 synthesis and concomitantly inhibits both progesterone and StAR protein biosynthesis. Thus, these findings suggest that in MA-10 cells, and perhaps in other steroidogenic cells, the heat shock-induced inhibition of steroid production is due to the inhibition of StAR protein synthesis, which results in a loss of cholesterol transfer to the inner mitochondrial membrane.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Waymouth’s MB/752 medium, DMEM, horse serum, antibiotics, lyophilized trypsin-EDTA, PBS with Ca2+ and Mg2+ (PBS+), and sodium bicarbonate were purchased from Life Technologies (Gaithersburg, MD); [1,2,6,7-N-3H]progesterone (SA, 104 Ci/mmol) and [35S]methionine-cysteine (Trans35S-Label; SA, 1000 Ci/mmol) were obtained from DuPont-New England Nuclear (Boston, MA); Dextran T-70 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden); acrylamide, bis-acrylamide, and SDS were purchased from Bio-Rad (Hercules, CA); charcoal (Norit), trichloroacetic acid, and scintiverse BD were obtained from Fisher Scientific (Fairlawn, NJ); (Bu)2cAMP, 22(R)-hydroxycholesterol (22R-HC), and progesterone were purchased from Sigma Chemical Co. (St. Louis, MO). LH (preparation hLH-1–3; 5900 IU/mg) was obtained from the National Hormone and Pituitary Program, NIDDK (Bethesda, MD). Anti-StAR antisera to amino acids 88–98 of mouse StAR protein were produced in rabbits by Research Genetics (Huntsville, AL). Antibodies to progesterone were obtained from Holly Hills Biological (Hillsboro, OR). Mouse monoclonal antibody generated against the inducible form of human HSP-70 protein was obtained from StressGen Biotechnologies (Victoria, Canada). Goat antimouse IgG conjugated with horseradish peroxidase was used as the secondary antibody and purchased from Southern Biotechnology Associates (Birmingham, AL). P450scc and 3ß-hydroxysteroid dehydrogenase (3ßHSD) antisera were gifts from Dr. Alessandro Capponi, University of Geneva (Geneva, Switzerland). Cytochrome oxidase mouse monoclonal antibody was obtained from Molecular Probes (Eugene, OR).

MA-10 cell culture
The MA-10 mouse Leydig tumor cell line was a gift from Dr. M. Ascoli, University of Iowa College of Medicine (Iowa City, IA). The cells were grown in Waymouth’s MB/752 medium containing 15% horse serum at 37 C in a humid atmosphere under 5% CO2 and maintained in culture using standard techniques as previously described (27). In all experiments, 2 x 106 cells were plated into each 100-mm culture dish (Corning, Corning, NY) and grown for 12 h in 10 ml Waymouth’s medium. After this, medium was removed, cells were washed once with PBS+, and fresh Waymouth’s medium was placed back on the cells. Heat shock, stimulation, and radiolabeling of the cells were performed as described below.

Heat shock treatment
Cells were heat shocked by incubation in a water bath at 45 C for 10 min. The cells were then routinely allowed to recover for 2 h at 37 C in a humidified atmosphere containing 5% CO2. After recovery, cells were rinsed with PBS+, covered in Waymouth’s medium without serum, and used immediately for subsequent experiments. In some experiments 1 mM (Bu)2cAMP was placed on the cells, and incubation was continued at 37 C. At 0, 3, and 6 h after the start of incubation, medium was removed from the cells to be assayed for progesterone content, and the cells were collected for preparation of protein samples for Western analysis as described below. In other experiments, 50 ng/ml LH or 1 mM (Bu)2cAMP were placed on the cells after recovery, and they were incubated for an additional 2 h at 37 C. The medium was removed from these cells and assayed for progesterone content. In experiments designed to determine P450scc and 3ßHSD activity, 25 µM 22R-HC was placed on both control and heat-shocked cells at 2 h after recovery and incubated for an additional 2 h at 37 C. At the end of the treatment period, the medium was removed and assayed for progesterone by RIA.

Preparation of protein samples for Western analysis
After the medium was removed, cells were rinsed with PBS+ and collected in buffer containing 10 mM Tris-base, 250 mM sucrose, and 1 mM EDTA, pH 7.4, by scraping with a rubber policeman. Cells were collected by centrifugation at 600 x g for 10 min at 4 C, resuspended in homogenization buffer containing 10 mM Tris-base and 1 mM EDTA, pH 7.4, and homogenized using a Potter Elvehjem homogenizer fitted with a Teflon pestle. Aliquots of the homogenate were used for protein determination using BSA as a standard (28). The homogenate was then centrifuged at 600 x g for 10 min, and the pellet was discarded. The supernatant containing cytosol and mitochondria was used for Western blot analysis of several proteins after its protein content was determined.

Western blot analysis
The presence and quantity of StAR, HSP-70, P450scc, 3ßHSD, and cytochrome oxidase in the 600 x g supernatant were assessed by Western blot analysis essentially using methods previously described (19).

The membrane used for detection of StAR was repeatedly stripped and reblotted with specific antibodies for subsequent detection of HSP-70, P450scc, 3ßHSD, and cytochrome oxidase. Briefly, for stripping, the membrane was soaked in buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol at 70 C for 20 min. The membrane was then washed with buffer containing 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl twice for 10 min each time. The remainder of the procedures were the same as those for StAR detection, except different antibodies were used.

The bands of interest were quantitated using a BioImage Visage 2000 (BioImage Corp., Ann Arbor, MI) imaging system after correction of the samples for protein loading differences using cytochrome oxidase (Western blot not shown) as the reference. Values are expressed as integrated optical density units, as previously described (19).

It is important to note that the data presented in the figures describing the quantitation of StAR, P450scc, 3ßHSD, and HSP-70 by Western analysis represent the results of a typical experiment that was performed at least twice and in which essentially identical results were obtained. As experiments were performed at different times, and the integrated optical densities in the bands are a direct result of the exposure time of the blots and not of the protein content, it is not legitimate to pool data from separate experiments and perform statistical analyses on them.

Protein synthesis determination
In experiments designed to determine the effect of heat shock on the synthesis of total cellular proteins, Waymouth’s medium without serum and containing 25 µCi/ml [35S]methionine-cysteine was placed on 1 x 105 cells in separate wells of 12-well plates in the presence of 1 mM (Bu)2cAMP and incubated for 2 h. At the end of the incubation, cells were washed twice with PBS+, followed by the addition of 100 µl 0.25 M NaOH to solubilize the cells and, later, by the addition of 100 µl 20% cold trichloroacetic acid to the wells. After 2 h at 4 C, the acid-insoluble material was removed from the wells and washed onto glass fiber filters (Whatman, Clifton, NJ) with several volumes of 5% trichloroacetic acid. The filters were dried and assayed for radioactivity in a liquid scintillation counter.

RIA
Quantitation of progesterone in the medium was performed by RIA as previously described (29). Analysis of the RIA data was performed using a computer program specifically designed for this purpose. The data were expressed as nanograms of progesterone per mg protein or as nanograms of progesterone per mg protein/unit time.

Statistical analysis
To assess the consistency of results, each experiment was repeated at least three times. Student’s t test, one- and two-way ANOVAs, and Duncan’s multiple range test were used as appropriate for analysis of the data using the Statistical Analysis System (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of heat shock on hormone-stimulated progesterone synthesis
Treatment of the cells at 45 C for 10 min inhibited the ability of (Bu)2cAMP to stimulate progesterone production in MA-10 cells. In Fig. 1Go, it is shown that 45 C heat shock treatment resulted in a 70% decrease in progesterone production during a 2-h period immediately following recovery, whereas the effect of heat shock on total cellular protein synthesis indicated only a 7% decrease during this same period. As shown in Fig. 2Go, although progesterone production was drastically inhibited during the 3-h period immediately following recovery, this inhibition was reversed, and at 6 h significant levels of progesterone synthesis were observed. This inhibition was seen with LH-stimulated as well as (Bu)2cAMP-stimulated progesterone production, as shown in Fig. 3Go. Further, that inhibition of progesterone synthesis was not a result of impaired activities of P540scc and/or 3ßHSD due to heat shock treatment is also shown in Fig. 3Go. When 22R-HC, which is freely permeable to the cell and mitochondrial membranes, was added to cells, there was no difference in the amount of progesterone production in heat-shocked and control cells, indicating that the activities of these enzymes were unaffected by heat shock.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. The effects of temperature on progesterone production and protein synthesis in MA-10 cells. In this experiment, cells were plated in multiwell dishes and grown as described in Materials and Methods. After 24 h, some of the cells were subjected to a heat shock of 43, 44, or 45 C for 10 min; returned to 37 C; and allowed to recover for 2 h. In some cases the medium was removed and replaced with medium without serum containing 1 mM (Bu)2cAMP. At the end of 2 h, the medium from the dishes was removed and assayed for progesterone. In other cases, 25 µCi/ml [35S]methionine-cysteine was placed on the cells. At the end of 2 h, the medium was removed, the cells were washed and solubilized in 0.25 M NaOH, and the acid-insoluble material was precipitated by adding cold 20% trichloroacetic acid to the wells. Acid-insoluble material was collected on glass fiber filters, washed thoroughly, and assayed for radioactivity.

 


View larger version (45K):
[in this window]
[in a new window]
  		Figure 2. The effects of 45 C heat shock on progesterone production in MA-10 cells. In this experiment, cells were plated in 100-mm dishes and grown as described in Materials and Methods. After 24 h, some of the cells were subjected to a heat shock of 45 C for 10 min, returned to 37 C, and allowed to recover for 2 h. At this time the medium was removed, and medium minus serum containing 1 mM (Bu)2cAMP was placed back on the cells. Incubation was continued for an additional 3 or 6 h. At each time point the medium was removed from the cells and assayed for progesterone content. The data shown represent the results of triplicate samples from one experiment that was repeated twice with similar results.

 


View larger version (47K):
[in this window]
[in a new window]
  		Figure 3. The effects of 45 C heat shock on progesterone production in MA-10 cells. In this experiment, cells were plated in multiwell dishes and grown as described in Materials and Methods. After 24 h, some of the cells were subjected to a heat shock of 45 C for 10 min, returned to 37 C, and allowed to recover for 2 h. At this time the medium was removed from the cells and replaced with medium without serum containing 50 ng/ml LH, 1 mM (Bu)2cAMP, or 25 µM 22R-HC. After an additional 2-h incubation, the medium was collected and assayed for progesterone. The data shown represent the results of triplicate samples from one experiment that was repeated three times with similar results.

 
Effect of heat shock on the contents of HSP-70, P450scc, 3ßHSD, and StAR proteins
To determine the effects of heat shock on the contents of several key proteins involved in steroidogenesis, Western analysis of these proteins was performed. As shown in Fig. 4Go, incubation of MA-10 cells at 37 C resulted in no detectable change in HSP-70 protein for a period of up to 6 h. However, incubation at 45 C resulted in a 6-fold increase in HSP-70 at 3 h and a 20-fold increase at 6 h after recovery. When the P450scc enzyme was similarly analyzed, it was seen that although some variation in the amount of this protein occurred, there were large quantities of this enzyme present at all time points in both control and heat-shocked cells (Fig. 5Go). Western analysis of 3ßHSD revealed the presence of two isoforms of this enzyme in MA-10 cells. As shown in Fig. 6Go, although it appeared that heat shock treatment resulted in a slight decrease in the content of the higher mol wt isoform at the point immediately following recovery, this decrease was essentially reversed in the 3 and 6 h samples. Also, there appeared to be no difference in the content of the lower mol wt isoform of 3ßHSD in either control or heat-shocked cells at any of the time points examined.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 4. The effects of 45 C heat shock on HSP-70 protein levels in MA-10 cells. In this experiment, cells were plated in 100-mm dishes and grown as described in Materials and Methods. After 24 h, some of the cells were subjected to a heat shock of 45 C for 10 min, returned to 37 C, and allowed to recover for 2 h. At this time the medium was removed, and medium without serum containing 1 mM (Bu)2cAMP was placed back on the cells. Incubation was continued for an additional 3 or 6 h. At each point the medium was removed, and the cells were washed twice with PBS+. The cells were collected from the dish by scraping as described in Materials and Methods, and protein samples were prepared for Western analysis, also as described in Materials and Methods. After Western blotting, the samples were stained specifically for HSP-70, and the integrated optical density was determined using a BioImage Visage 2000 image analysis system. The data shown represent essentially identical results obtained in two separate experiments.

 


View larger version (67K):
[in this window]
[in a new window]
  		Figure 5. The effects of 45 C heat shock on P450scc protein levels in MA-10 cells. The conditions and methods used are identical to those described in Fig. 4Go. In this case, the Western blot was stripped as described in Materials and Methods, the membrane was stained for P450scc protein using a specific antiserum for this protein, and the integrated optical density was determined using a BioImage Visage 2000 image analysis system. The data shown represent essentially identical results obtained in two separate experiments.

 


View larger version (58K):
[in this window]
[in a new window]
  		Figure 6. The effects of 45 C heat shock on 3ßHSD protein levels in MA-10 cells. The conditions and methods used are identical to those described in Fig. 4Go. In this case, the Western blot was stripped as described in Materials and Methods, the membrane was stained for 3ßHSD protein using a specific antiserum for this protein, and the integrated optical density was determined using a BioImage Visage 2000 image analysis system. The data shown represent essentially identical results obtained in two separate experiments.

 
The situation with the StAR protein, however, was quite different. Although control cells displayed a large increase in the synthesis of StAR at 3 h, heat shock resulted in a complete inhibition of the appearance of the StAR protein at this time (Fig. 7Go). As expected, the StAR protein content was further increased in control cells at 6 h after stimulation. Heat-shocked cells mimicked the results obtained for progesterone production, demonstrating that although both progesterone and StAR protein were virtually undetectable at 3 h post heat shock, the level of StAR protein was increased at 6 h after recovery from heat shock. Thus, a tight correlation between the synthesis of progesterone and StAR protein content was observed in these studies.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 7. The effects of 45 C heat shock on StAR protein levels in MA-10 cells. The conditions and methods used are identical to those described in Fig. 4Go. In this case, the Western blot was stripped as described in Materials and Methods, the membrane was stained for StAR protein using a specific antiserum for this protein, and the integrated optical density was determined using a BioImage Visage 2000 image analysis system. The data shown represent essentially identical results obtained in two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Heat shock treatment of steroidogenic cells has been shown to result in the inhibition of steroid hormone biosynthesis (9, 10, 11). This inhibition was directly attributable to the production of the heat shock protein HSP-70, as blocking its expression, either through treatment of the cells with the transcription inhibitor actinomycin D (9) or with antisense oligonucleotides (11), significantly, if not fully, reversed the observed inhibition. In addition, it was found that this inhibition was not the result of an inability of heat-shocked cells to transduce the trophic hormone signal, as intracellular cAMP levels were found to be unaffected by heat shock, and the inhibition could not be alleviated by treatment of the cells with cAMP analog (9). Lastly, it was further demonstrated that the observed inhibition was not due to a direct effect on the steroidogenic enzymes. Use of the cholesterol analog, 22-hydroxycholesterol, which freely diffuses to the inner mitochondrial membrane, the site of the P450scc enzyme, demonstrated that full steroid synthesis was restored, indicating that the enzymes involved in the conversion of cholesterol to progesterone were not affected by heat shock treatment (9). As a result of such observations, Khanna et al. (9, 10, 11) suggested that the locus of the inhibition of steroid synthesis in heat-shocked cells lay at the intramitochondrial transfer of cholesterol from the outer to the inner membrane. This observation is significant in light of the long-held belief that it is this transfer of cholesterol to the inner mitochondrial membrane that is the true rate-limiting step in steroidogenesis (22, 23, 24, 25). Further, it is well accepted that this step requires de novo protein synthesis, and the role of the putative protein is to effect this transfer.

In an effort to further characterize the mechanism by which heat shock inhibits steroid hormone production, we employed the MA-10 mouse Leydig tumor cell line, which synthesizes progesterone as its major steroid product (27). As the site of heat shock induced inhibition of steroid production had essentially been localized to the transfer of cholesterol to the inner mitochondrial membrane (9, 10, 11), we endeavored to determine the effects of heat shock on the expression of the StAR protein. Earlier work demonstrated that StAR is a mitochondrial protein whose expression in MA-10 and COS-1 cells results in increased steroid production (19, 20, 26, 30, 31). Additional studies of the StAR protein have indicated that it has an indispensable role in steroid hormone biosynthesis (26), and it has been further postulated that this role is in regulating cholesterol transfer to the inner mitochondrial membrane (18, 19, 20, 21).

Results from the present studies demonstrate that heat shock treatment acutely inhibits hormone-stimulated progesterone synthesis in MA-10 cells and that this inhibition is coincident with the induction of HSP-70 protein and the inhibition of StAR protein synthesis. Similar to earlier observations, it was demonstrated that the cells maintained the capacity to synthesize steroids if incubated in the presence of the cholesterol analog, 22R-hydroxycholesterol. This observation importantly indicated that, as in rat luteal cells, heat shock treatment did not inhibit steroidogenesis in MA-10 cells by affecting the activity of the enzymes involved in the conversion of cholesterol to progesterone. In the present studies, both the activity, as determined by the conversion of 22R-hydroxycholesterol to progesterone, and the amounts of P450scc and 3ßHSD, as determined by Western analysis, were similar in control and heat-shocked cells. The observation that the higher mol wt isoform of 3ßHSD was slightly decreased after heat shock is of interest, but apparently of no consequence, as full steroid-synthesizing capacity was seen with 22R-HC. The presence of two isoforms of 3ßHSD in MA-10 cells has been previously observed (32), and is similar to observations made earlier in mouse testis (33). Therefore, based on these observations, it would appear that the heat shock-induced depression in progesterone production in MA-10 cells is to be due to the inhibition of StAR protein synthesis. This observation is highly consistent with previous studies in which stimulation and inhibition of steroid hormone biosynthesis have been tightly correlated with StAR synthesis. For example, stimulation of a number of steroidogenic cells with trophic hormone and other agents known to increase steroid production have all resulted in an increased expression of StAR protein (19, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43). Conversely, agents and conditions that have been shown to result in a decrease in steroid hormone biosynthesis, such as cycloheximide (18, 44), lipopolysaccharide (45), diethyumbelliferyl phosphate (46), PGF2{alpha} (33, 47), and estrogen withdrawal (39), have all been demonstrated to decrease StAR protein content.

The mechanism by which heat shock results in decreased steroid synthesis remains unknown. However, in light of the present findings, additional possibilities have arisen. As heat shock in MA-10 cells has been shown to result in an increase in the level of HSP-70 and a decrease in StAR protein in the present study, several scenarios may be important to consider. First, it is known that HSP-70 proteins can act as chaperones and are required for the successful transport of mitochondrial proteins from the cytosol into the matrix of this organelle (48, 49, 50, 51). As it has been proposed that cholesterol transfer to the inner mitochondrial membrane occurred as a result of the synthesis and import of the StAR protein and the concomitant formation of contact sites (20), perhaps the abundant increase in HSP-70 noted sequesters StAR in a manner to render it inoperable. This seems unlikely in view of the observation that StAR protein does not appear to be present in the cell based on Western analysis of the total cellular protein. It is also possible that in reaction to heat stress the cells mobilize HSP-70s to counteract the deleterious effects of heat shock on protein denaturation. Thus, all of the cellular HSP-70 proteins would be occupied and unavailable to act as chaperones for the StAR protein. In this case, StAR protein may be quickly degraded, as previously shown for mitochondrial proteins that are not imported (52). Lastly, it is possible that steroid biosynthesis in response to heat shock is inhibited as a result of a decrease in overall protein synthesis as previously observed (53). Again, this does not appear to be likely to us in view of our observation that in MA-10 cells, heat shock did not result in a significant decrease in total cellular protein synthesis. Therefore, unless the synthesis of StAR was specifically inhibited over that of other cellular proteins, this scenario seems unlikely.

At this point, although the exact reason why heat shock causes an inhibition of steroid production and StAR synthesis remains unknown, it is highly likely that there is a connection between the two events. It would appear reasonable to indicate that the observed decrease in steroidogenesis is due directly to the observed inhibition of StAR synthesis. This observation is strengthened by the finding that at 6 h postrecovery from heat shock, StAR reappears, and steroid hormone biosynthesis is reestablished. It should be noted that although progesterone synthesis at 6 h in the heat shock samples was almost as high as that in the 37 C samples, the amount of StAR protein present at 6 h was only 20% of that seen in the 37 C sample. This observation is in keeping with our earlier observations. StAR is synthesized as a 37-kDa precursor and then is quickly imported into and processed by the mitochondria to the 30-kDa mature form seen here (18). The 30-kDa form continues to accumulate inside the mitochondria; however, in this form it is inactive in further cholesterol transport. This situation results in higher levels of StAR protein at 3 and 6 h in the 37 C samples compared to the 45 C samples, which have synthesized StAR for a much shorter period of time. As nothing is known concerning the stoichiometry of StAR synthesis and steroid production, it is not possible at this time to predict the amount of steroid that will be made in response to StAR synthesis, especially in light of additional changes that may occur in the cell as a result of heat shock. One possibility, however, is that even though StAR and steroids are not being synthesized during the first 3 h of recovery from heat shock, other events, such as cholesterol mobilization to the mitochondria, which is known to occur in response to stimulation of steroidogenic cells, are unaffected. Thus, when recovery from heat shock occurs, a larger pool of cholesterol is available in the outer mitochondrial membrane for transfer and conversion to steroid.

In future studies it will be of interest to determine whether the decrease in StAR protein levels occurs as a result of an inhibition of transcription of the StAR gene or if, as indicated above, StAR is synthesized and quickly degraded as a consequence of not being imported into the mitochondria. Regardless, it is now possible to suggest that heat shock-induced inhibition of steroidogenesis may be due to an inhibition of StAR synthesis.


   Acknowledgments
 
The authors acknowledge the technical assistance of Deborah Alberts and Joseph Marney, and the photographic assistance of Harvey Olney.


   Footnotes
 
1 This work was supported by NIH Grants HD-17481 (to D.M.S.) and HD-07271 (to Z.L.). Back

Received February 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Benjamin IJ, Kroger B, Williams RS 1990 Activation of the heat shock transcription factor by hypoxia in mammalian cells. Proc Natl Acad Sci USA 87:6263–6267[Abstract/Free Full Text]
  2. Van Why SK, Hildebrandt F, Ardito T, Mann AS, Siegel NJ, Kashgarian M 1992 Induction and intracellular localization of hsp-72 after renal ischemia. Am J Physiol 263:F769–F775
  3. Morimoto RI, Sarge KD, Abravaya K 1992 Transcriptional regulation of heat shock genes: a paradigm for inducible genomic response. J Biol Chem 267:21987–21990[Abstract/Free Full Text]
  4. Heufelder AE, Wenzel BE, Bahn RS 1992 Methimazole and propylthiouracil inhibit the oxygen free radical-induced expression of a 72 kilodalton heat shock protein in Graves’ retroocular fibroblasts. J Clin Endocrinol Metab 74:737–742[Abstract]
  5. Cruz TF, Kandel RA, Brown IR 1991 Interleukin 1 induces the expression of a heat-shock gene in chondrocytes. Biochem J 277:327–330
  6. Santoro MG, Garaci E, Amici C 1989 Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. Proc Natl Acad Sci USA 86:8407–8411[Abstract/Free Full Text]
  7. Morimoto RI 1993 Cells in stress: transcriptional activation of heat shock genes. Science 259:1409–1410[Free Full Text]
  8. Pelham HRB 1986 Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 46:959–961[CrossRef][Medline]
  9. Khanna A, Aten RF, Behrman HR 1994 Heat shock protein induction blocks hormone-sensitive steroidogenesis in rat luteal cells. Steroids 59:4–9[CrossRef][Medline]
  10. Khanna A, Aten RF, Behrman HR 1995 Physiological and pharmacological inhibitors of luteinizing hormone-dependent steroidogenesis induce heat shock protein-70 in rat luteal cells. Endocrinology 136:1775–1781[Abstract]
  11. Khanna A, Aten RF, Behrman HR 1995 Heat shock protein-70 induction mediates luteal regression in the rat. Mol Endocrinol 9:1432–1440
  12. Thomas JP, Dorflinger LJ, Behrman HR 1978 Mechanism of the rapid antigonadotropic action of prostaglandins in cultured luteal cells. Proc Natl Acad Sci USA 75:1344–1348[Abstract/Free Full Text]
  13. Jordan AW 1981 Effects of prostaglandin F2{alpha} treatment on LH and dibutyryl cyclic AMP-stimulated progesterone secretion by isolated rat luteal cells. Biol Reprod 25:327–331[Abstract]
  14. Sender Baum M, Rosberg S 1987 A phorbol ester, phorbol 12-myristate 13-acetate, and a calcium ionophore, A23187, can mimic the luteolytic effect of prostaglandin F2{alpha} in isolated rat luteal cells. Endocrinology 120:1019–1026[Abstract]
  15. Musicki B, Aten RF, Behrman HR 1990 The antigonadotropic actions of prostaglandin F2{alpha} and phorbol ester are mediated by separate processes in rat luteal cells. Endocrinology 126:1388–1395[Abstract]
  16. Benyo DF, Pate JL 1992 Tumor necrosis factor-{alpha} alters bovine luteal cell synthetic capacity and viability. Endocrinology 130:854–860[Abstract]
  17. Wang HZ, Lu SH, Han XJ, Zhou W, Shen WX, Sun ZD, Gong YT 1992 Inhibitory effect of interferon and tumor necrosis factor on human luteal function in vitro. Fertil Steril 58:941–945[Medline]
  18. Stocco DM, Sodeman TC 1991 The 30-kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from larger precursors. J Biol Chem 266:19731–19738[Abstract/Free Full Text]
  19. Clark BJ, Wells J, King SR, Stocco DM 1994 The purification, cloning, and expression of a novel LH-induced mitochondrial protein in MA-10 mouse Leydig tumor cells: characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269:28314–28322[Abstract/Free Full Text]
  20. Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244[CrossRef][Medline]
  21. Stocco DM 1997 A StAR search: implication in controlling steroidogenesis. Biol Reprod 56:328–336[Abstract]
  22. Privalle CT, Crivello JF, Jefcoate CR 1983 Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P450scc in rat adrenal gland. Proc Natl Acad Sci USA 80:702–706[Abstract/Free Full Text]
  23. Stevens VL, Aw TY, Jones DP, Lambeth JD 1984 Oxygen dependence of adrenal cortex cholesterol side chain cleavage. Implications in the rate-limiting steps in steroidogenesis. J Biol Chem 259:1174–1179[Abstract/Free Full Text]
  24. Privalle CT, McNamara BC, Dhariwal MS, Jefcoate CR 1987 ACTH control of cholesterol side-chain cleavage at adrenal mitochondrial cytochrome P-450scc. Regulation of intramitochondrial cholesterol transfer. Mol Cell Endocrinol 53:87–101[CrossRef][Medline]
  25. Stevens VL, Xu T, Lambeth JD 1993 Cholesterol trafficking in steroidogenic cells: reversible cycloheximide-dependent accumulation of cholesterol in a pre-steroidogenic pool. Eur J Biochem 216:557–563[Medline]
  26. Lin D, Sugawara T, Strauss III JF, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL 1995 Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267:1828–1831[Abstract/Free Full Text]
  27. Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 10:88–95
  28. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  29. Resko JA, Norman RL, Niswender GD, Spies HG 1974 The relationship between progestins and gonadotropins during the late luteal phase of the menstrual cycle in rhesus monkeys. Endocrinology 94:128–135[Medline]
  30. Sugawara T, Holt JA, Driscoll D, Strauss III JF, Lin D, Miller WL, Patterson D, Clancy KP, Hart IM, Clark BJ, Stocco DM 1995 Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the gene to 8p11.2 and a pseudogene to chromosome 13. Proc Natl Acad Sci USA 92:4778–4782[Abstract/Free Full Text]
  31. Arakane F, Sugawara T, Nishino H, Liu Z, Holt JA, Pain D, Stocco DM, Miller WL, Strauss JF 1996 Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: implications for the mechanism of StAR action. Proc Natl Acad Sci USA 93:13731–13736[Abstract/Free Full Text]
  32. Stocco DM, Wells J, Clark BJ, Stalvey JR 1994 The effects of hydrogen peroxide on steroidogenic enzymes in MA-10 mouse Leydig tumor cells. Adv Contra Deliv Syst 10:37–50
  33. Keeny DS, Naville D, Milewich L, Bartke A, Mason JI 1993 Multiple forms of 3ß-hydroxysteroid dehydrogenase/{Delta}5–4-isomerase in mouse tissues: male specific isoforms are expressed in the gonads and liver. Endocrinology 133:39–45[Abstract]
  34. Clark BJ, Pezzi V, Stocco DM, Rainey WE 1995 The steroidogenic acute regulatory protein is induced by angiotensin II and K+ in H295R adrenocortical cells. Mol Cell Endocrinol 115:215–219[CrossRef][Medline]
  35. Juengel JL, Meberg BM, Turzillo AM, Nett TM, Niswender GD 1995 Hormonal regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in ovine corpora lutea. Endocrinology 136:5423–5429[Abstract]
  36. Hartung S, Rust W, Balvers M, Ivell R 1995 Molecular cloning and in vivo expression of the bovine steroidogenic acute regulatory protein. Biochem Biophys Res Commun 215:646–653[CrossRef][Medline]
  37. Gradi A, Tang-Wai R, McBride HM, Chu LL, Shore GC, Pelletier J 1995 The human steroidogenic acute regulatory (StAR) gene is expressed in the urogenital system and encodes a mitochondrial polypeptide. Biochim Biophys Acta 1258:228–233[Medline]
  38. Pescador N, Korian Soumano S, Stocco DM, Price CA, Murphy BD 1996 Steroidogenic acute regulatory protein in bovine corpora lutea. Biol Reprod 55:485–491[Abstract]
  39. Townson DH, Wang XJ, Keyes PL, Kostyo JL, Stocco DM 1996 Expression of the steroidogenic acute regulatory protein (StAR) in the corpus luteum of the rabbit: dependence upon the luteotrophic hormone, 17ß-estradiol. Biol Reprod 55:868–874[Abstract]
  40. Sandhoff TW, McLean MP 1996 Hormonal regulation of steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary. Endocrine 4:259–267
  41. Selvaraj N, Israeli D, Amsterdam A 1996 Partial sequencing of the rat steroidogenic acute regulatory protein message from immortalized granulosa cells: regulation by gonadotropins and isoproterenol. Mol Cell Endocrinol 123:171–177[CrossRef][Medline]
  42. Kiriakidou M, McAllister JM, Sugawara T, Strauss JF 1996 Expression of steroidogenic acute regulatory protein (StAR) in the human ovary. J Clin Endocrinol Metab 81:4122–4128[Abstract/Free Full Text]
  43. Balasubramanian K, LaVoie HA, Garmey JC, Stocco DM, Velduis JD 1997 Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist. Endocrinology 138:433–439[Abstract/Free Full Text]
  44. Stocco DM, Kilgore MW 1988 Induction of mitochondrial proteins in MA-10 Leydig tumour cells with human choriogonadotropin. Biochem J 249:95–103[Medline]
  45. Bosmann HB, Hales KH, Li X, Liu Z, Stocco DM, Hales DB 1996 Acute in vivo inhibition of testosterone by endotoxin parallels loss of steroidgenic acute regulatory (StAR) protein in Leydig cells. Endocrinology 137:4522–4525[Abstract]
  46. Choi YS, Stocco DM, Freeman DA 1995 Diethylumbelliferyl phosphate inhibits steroidogenesis by interfering with a long-lived factor acting between protein kinase A activation and induction of the steroidogenic acute regulatory (StAR) protein. Eur J Biochem 234:680–685[Medline]
  47. Sandhoff TW, McLean MP 1996 Prostoglandin F2{alpha} reduces steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary. Endocrine 5:183–190
  48. Chirico WJ, Waters MG, Blobel G 1988 70K heat shock related proteins stimulate protein translocation into microsomes. Nature 332:805–810[CrossRef][Medline]
  49. Deshaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R 1988 A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature 332:800–805[CrossRef][Medline]
  50. Hartl FU 1996 Molecular chaperones in cellular protein folding. Nature 381:571–580[CrossRef][Medline]
  51. Kang P-J, Ostermann J, Shilling J, Neupert W, Craig EA, Pfanner N 1990 Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 348:137–143[CrossRef][Medline]
  52. Jaussi R, Sonderegger P, Fluckiger J, Christen P 1982 Biosynthesis and topogenesis of aspartate aminotransferase isoenzymes in chicken embryo fibroblasts. J Biol Chem 257:13334–13340[Abstract/Free Full Text]
  53. Lindquist S 1981 Regulation of protein synthesis during heat shock. Nature 293:311–314[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Ann. N. Y. Acad. Sci.Home page
D. B. HALES, J. A. ALLEN, T. SHANKARA, P. JANUS, S. BUCK, T. DIEMER, and K. H. HALES
Mitochondrial Function in Leydig Cell Steroidogenesis
Ann. N.Y. Acad. Sci., December 1, 2005; 1061(1): 120 - 134.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
B. D. Murphy, E. Lalli, L. P. Walsh, Z. Liu, J. Soh, D. M. Stocco, and P. Sassone-Corsi
Heat Shock Interferes with Steroidogenesis by Reducing Transcription of the Steroidogenic Acute Regulatory Protein Gene
Mol. Endocrinol., August 1, 2001; 15(8): 1255 - 1263.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
N. Pescador, D. M. Stocco, and B. D. Murphy
Growth Factor Modulation of Steroidogenic Acute Regulatory Protein and Luteinization in the Pig Ovary
Biol Reprod, June 1, 1999; 60(6): 1453 - 1461.
[Abstract] [Full Text]

Home page
 Physiol. Rev.Home page
J. A. McCracken, E. E. Custer, and J. C. Lamsa
Luteolysis: A Neuroendocrine-Mediated Event
Physiol Rev, April 1, 1999; 79(2): 263 - 323.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
Y.-J. Chen, Q. Feng, and Y.-X. Liu
Expression of the Steroidogenic Acute Regulatory Protein and Luteinizing Hormone Receptor and Their Regulation by Tumor Necrosis Factor {alpha} in Rat Corpora Lutea
Biol Reprod, February 1, 1999; 60(2): 419 - 427.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.
Right arrow Articles by Stocco, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.
Right arrow Articles by Stocco, D. M.

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