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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
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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- (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.
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Materials and Methods |
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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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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. 2, 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. 3. 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. 3. 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.
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, 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. 5). Western analysis of 3ßHSD revealed the
presence of two isoforms of this enzyme in MA-10 cells. As shown in
Fig. 6, 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.
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). 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (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.
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Acknowledgments |
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Footnotes |
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Received February 18, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment on LH and dibutyryl cyclic AMP-stimulated
progesterone secretion by isolated rat luteal cells. Biol Reprod 25:327–331[Abstract]
in
isolated rat luteal cells. Endocrinology 120:1019–1026 and phorbol
ester are mediated by separate processes in rat luteal cells.
Endocrinology 126:1388–1395
alters bovine luteal cell synthetic capacity and viability.
Endocrinology 130:854–860
5–4-isomerase in mouse tissues: male
specific isoforms are expressed in the gonads and liver. Endocrinology 133:39–45 reduces steroidogenic acute regulatory (StAR) protein
messenger ribonucleic acid expression in the rat ovary. Endocrine 5:183–190[CrossRef]
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