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Pediatric Surgical Research Laboratories, Massachusetts General Hospital and Harvard Medical School (J.T., E.F.T., P.K.D.), Boston, Massachusetts 02114; and the Division of Reproductive Biology, Department of Gynecology/Obstetrics, Stanford University (A.H.P.), Palo Alto, California 94305
Address all correspondence and requests for reprints to: Jose Teixeira, Ph.D., Pediatric Surgical Research Laboratories/WRN1024, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114. E-mail: teixeira{at}helix.mgh.harvard.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leydig cells or interstitial cells are found in the testes surrounding
the seminiferous tubules. Their major function is to produce
testosterone, which is essential for the normal male phenotype.
Testosterone is synthesized from cholesterol in five steps by the
activity of four enzymes (Fig. 1
), three
of which we have studied: P450scc, P450c17, and 3ß-hydroxysteroid
dehydrogenase/
5-4-isomerase (3ßHSD)
(15). P450scc (cytochrome P450-side chain cleavage, also known as
CYP11A) is a member of the superfamily of cytochrome P450 hemeproteins
(16), is located on the inner mitochondrial membrane, and catalyzes the
committed step of cholesterol conversion to steroid hormones by
converting the 27-carbon cholesterol molecule to the 21-carbon
pregnenolone. Pregnenolone moves out of the mitochondria and is
converted to progesterone by the activity of 3ßHSD, a nonP450 enzyme.
Cytochrome P450c17
hydroxylase/C17–20 lyase (P450c17,
CYP17) has dual activities; it hydroxylates progesterone at the 17
position and converts the 21-carbon 17-hydroxyprogesterone to the
19-carbon androstenedione. Androstenedione is then converted to
testosterone by the activity of 17-ketosteroid reductase, a non-P450
enzyme that reduces the ketone at the carbon 17 position.
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Signal transduction by members of the TGFß family of glycoprotein homodimers occurs when the ligand binds to a heteromeric complex of single transmembrane, serine/threonine kinases. Ligand specificity within the family is determined by the type II receptor, which, in turn, recruits and phosphorylates the appropriate type I receptor for subsequent downstream signaling via subsets of ligand-specific Smads (19). Efforts to determine the molecular mechanisms of MIS signal transduction have led us and others to the cloning of the MIS ligand and its MIS type II receptor and their characterization (4, 20, 21, 22, 23). To understand the downstream pathways that are activated by the MIS ligand binding to its receptor, we are dissecting the role that MIS plays in Leydig cell function and steroidogenesis. Using the rodent Leydig tumor cell lines R2C and MA-10, we have established a system for studying MIS signal transduction and have been able to show that MIS regulates steroidogenesis at the transcriptional level.
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Materials and Methods |
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Cell culture
R2C, a rat Leydig tumor cell line (American Type Culture Collection, Manassas, VA), was maintained in F-10 medium
containing 15% horse serum and 2.5% female FCS (Aires
Scientific/Biologos). The mouse MA-10 Leydig tumor cell line, a gift
from Dr. Mario Ascoli (University of Iowa, Ames, IA), was maintained in
Waymouth’s MB 752/1 medium (Life Technologies, Inc.)
modified to contain 20 mM HEPES (pH 7.4), 50 µg/ml
gentamicin, and 15% horse serum. For these experiments, R2C and MA-10
cells were incubated in a humidified atmosphere with 5%
CO2 at 37 C and fed every other day until reaching 80–90%
confluence.
Northern analyses
Total RNA was extracted using the guanidine
isothiocyanate-cesium chloride method (24). All RNA was extracted with
successive rounds of phenol, chloroform, and ether; ethanol
precipitated; and quantitated by absorbance at A260. Ten
micrograms of each RNA sample were denatured with dimethylsulfoxide and
glyoxal at 65 C, separated in a 1.5% agarose gel, blotted overnight
onto nylon membranes, and either baked at 80 C in a vacuum oven or UV
cross-linked. Testes were surgically extracted from 30-day postnatal
rats and homogenized, and RNA was extracted as before for use as a
positive control. RNA from isolated primary Leydig cells of 21-day-old
rats purified by Percoll density gradient centrifugation to 95%
homogeneity was provided by Dr. Mary Lee (25). Blots were prehybridized
with 100 µg/ml sonicated salmon sperm DNA in 50% formamide
hybridization solution for 3 h at 65 C for riboprobes and at 42 C
for random primed probes. The MIS type II receptor plasmid was
linearized with EcoRV and riboprobes made with SP6
polymerase using standard techniques. 32P-Labeled random
primed cDNA probes were made for P450scc and 3ßHSD. The P450c17
plasmid was linearized with EcoRI and the
32P-labeled riboprobe made with T7 RNA polymerase using
standard techniques. Blots were hybridized overnight with 2 x
106 cpm/ml probe with riboprobes at 65 C and washed at 72 C
or with random primed human probes at 42 C and washed at 60 C with
0.1 x SSC (1 x SSC = 150 mM sodium
chloride and 15 mM sodium citrate)-0.1% SDS and exposed to
radiographic film with intensifying screens at -70 C for 3 days. All
animal studies were performed in accordance with the NIH Guide for the
Care and Use of Laboratory Animals, with institutional approval on
8–19-98 under accession no. 97–4216.
RIAs
MA-10 cells were plated on day 0 in sextuplicate at 5 x
104/well in 12-well plates for each time point indicated.
Recombinant human MIS (rhMIS) was prepared from Chinese hamster ovary
(CHO) cells stably transfected with a linear construct of the human
MIS gene (4). Active, secreted protein in the growth medium
was then passed over an immunoaffinity column prepared with the
monoclonal MIS antibody, 6E11, conjugated to Affi-Gel-10 (26). Bound
MIS was then eluted off the column with 3 M ammonium
thiocyanate solution, pH 7.4, or 1 M acetic acid, pH 3.0;
protein concentrations were determined by Bradford assays after pH
neutralization and desalting by centrifugal filtration (27). The
bioactivity of the MIS was then verified using an established organ
culture assay, which grades the regression of the 14.5-day gestation
rat urogenital ridge (28). Vehicle control or 105 nM MIS in
the presence or absence of (Bu)2cAMP (50 µM)
was added to MA-10 cells 2 days after plating and incubated for the
indicated number of days. Culture medium was collected and assayed by
RIA for total accumulated progesterone and testosterone by the NICHHD
P30 Reproductive and Endocrine Sciences RIA Core Laboratory at the
Massachusetts General Hospital.
Luciferase reporter assays
The P450c17 promoter fragment of 1018 bp was amplified by PCR
from mouse genomic DNA using 5'-GAGCTCGAGTATTGGCATTGCGTCCC and
5'-CTCGAGGGCAGATGGCCAGCTGTGGA as primers with complimentary
SacI and XhoI sites (29). Advantage polymerase
enzyme was used in the PCR amplification (CLONTECH Laboratories, Inc., Palo Alto, CA). The PCR fragment was cloned into pCRII
(Invitrogen, San Diego, CA) for sequence analysis and cut
out with SacI/XhoI. pGL3B vector was digested
with SacI and XhoI and ligated to the P450c17
promoter fragment, generating P450c17-Luc. The constructs
were purified by cesium chloride ultracentrifugation. MA-10 cells were
plated at 2 x 105 in triplicate and were transfected
with P450c17-Luc using the FuGene 6 lipid protocol
(Roche Molecular Biochemicals, Indianapolis, IN).
pGL3B, the promoterless parent plasmid was used as a negative control.
pRL-TK encoding Renilla luciferase (Promega Corp.) under the control of a HSV thymidine kinase
promoter was cotransfected as an internal control for transfection
efficiency and steroid pathway specificity. Cell lysates were made and
assayed for luciferase activity using a Berthold Automatic luminometer
(Wallac, Inc., Turku, Finland).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) was performed to determine whether
there was requisite expression of the MIS type II receptor in total RNA
isolated from rat testes, R2C cells, MA-10 cells, and purified primary
Leydig cells. These findings indicate that Leydig cells could respond
directly via the MIS type II receptor from paracrine production of MIS
in the nearby Sertoli cells. The bands observed under stringent
hybridization conditions in the cell lines and purified Leydig cells
migrated a distance equal to that seen with testis RNA, suggesting that
the MIS type II receptor probe hybridized to the same mRNA species.
MA-10 cells also show a faster migrating band that has been observed
with R2C cells in other Northern experiments. Attempts to clone
the faster migrating band by rapid amplification of cDNA 5'-ends have
resulted in the isolation of type II receptor cDNAs lacking the first
two exons whose functional physiological relevance has not yet been
tested (Teixeira, J., and P. K. Donahoe, unpublished).
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). MA-10 cells are a
mouse Leydig cell tumor line that, in addition, have functional
gonadotropin receptors resulting in enhanced steroid production in
response to cAMP or LH/hCG, thus mimicking the physiological state of
Leydig cells in vivo (15, 30). Figure 3A shows that
incubation of MA-10 cells with MIS for 2 days resulted in a modest, but
significant, 40% reduction in progesterone secretion in both the
cAMP-stimulated and unstimulated states. Figure 3B shows the
concentration of testosterone secreted by MA-10 cells incubated with
MIS over a 2-day time course was 10-fold lower than that of cells not
treated with MIS. The level and time course of steroid hormone
reduction were similar to those seen when human follicular cells,
harvested at the time of in vitro fertilization, were
incubated with recombinant MIS (9) and when primary Leydig cells were
incubated with MIS (18).
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). Northern analysis revealed that
the levels of mRNA, although marginally lower for P450scc and 3ßHSD
for both R2C after 3-h treatment and MA-10 cells after overnight
treatment with MIS (Fig. 4, A and B, respectively), were dramatically
reduced for P450c17 to undetectable levels in cAMP-stimulated MA-10
cells.
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). RNA was prepared from MA-10 cells,
incubated with MIS in the presence or absence of cycloheximide and
analyzed by Northern blot for c17 mRNA. Surprisingly, addition of
cycloheximide alone was sufficient to induce expression of c17 mRNA
above levels seen with cAMP induction.
MIS regulates c17 expression at the transcriptional level
Changes in mRNA for the steroidogenic enzymes could be
reflected in, among others, differences in message stability, nuclear
export, or transcription, all of which could be potential points for
regulation by MIS. To study transcriptional regulation of the
Cyp17gene by MIS, we made DNA constructs containing
approximately 1 kb of the Cyp17 promoter fused with the
firefly luciferase gene, transfected them into MA-10 cells,
and assayed for luciferase activity (Fig. 5). In the first set of experiments, on
the day after transfection with the reporter construct, MA-10 cells
were incubated with varying concentrations of MIS and harvested either
18 or 29 h later to permit induction of luciferase activity, which
was measured and compared (Fig. 5A). MIS appears to exert its effect on
the exogenous Cyp17 promoter/luciferase reporter after only
18 h with 70 nM MIS. By 29 h, 35 nM
MIS was able to markedly lower luciferase activity and, with 105
nM MIS, luciferase activity was reduced to background
levels. These concentrations, which are high compared with those
required for transcriptional activation by other members of the TGFß
family, are similar to those required for an MIS effect in other assays
and with the 35-nM MIS concentration required for complete
regression of the Müllerian duct in the organ culture bioassay
used to test the potency of MIS preparations (17, 31). Also, firefly
luciferase activation is normalized to a cotransfected
Renilla reporter to rule out nonspecific effects on the cell
by MIS. It is also important to note that the MIS preparations were
tested and were found to be free of activin and TGFß by enzyme-linked
immunosorbent assay, and that when TGFß was added to MA-10, the
Cyp17-driven luciferase activity was not significantly
different from that of untreated cells (not shown).
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shows that MIS incubation on the day after transfection has a
slight, but significant, effect on luciferase after 3 h and
becomes pronounced after 18 h. In another experiment, MA-10 cells
when incubated with L9, an inactive form of the MIS ligand that has
been mutated so that pro-MIS could not be cleaved to generate the
bioactive C-terminal portion of the hormone (32), did not affect
luciferase activity as did bioactive, cleavable MIS at the same
concentration and time (shown with an asterisk in Fig. 5).
As L9 is produced in CHO cells and purified in the same manner as
wild-type MIS, we can conclude that the effect on P450c17 transcription
is due to MIS and not to a possible copurified contaminant.
Luciferase expression appears maximal after overnight transfection, as
luciferase activity was not significantly different in the control
MA-10 cells with 3- to 18-h additional incubation (Fig. 5B). To take
advantage of an earlier MIS effect, we transfected the Cyp17 reporter
construct and added MIS at the same time. As shown in Fig. 5C, 18
h after transfection, we observed a 4-fold decrease in luciferase
activity with added MIS, which is considerably greater than that
observed when MIS was added 1 day later. This observation suggests that
MIS can more effectively repress expression of the Cyp17
promoter-driven reporter before it is fully activated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have also observed an effect of MIS on progesterone secretion by MA-10 cells that was reflected in a decrease in steady state mRNA levels for both P450scc and 3ßHSD. Although the effect was modest compared with that of MIS on testosterone and Cyp17 mRNA, it suggests strongly that MIS might also regulate the expression or activity of other enzymes in the steroid biosynthetic pathway.
The MIS regulation of steroidogenesis in Leydig cells by transcriptional control of Cyp17 exposes a conundrum in MIS-mediated suppression of testosterone synthesis that may indicate it is developmentally regulated. Fetal Leydig cells proliferate and show increased androgen synthesis, which is required for male phenotypic development, independent of gonadotropin stimulation in the rodent (38, 39). We would have expected that these fetal Leydig cells must be refractory to MIS-mediated inhibition of androgen synthesis, because the level of MIS is also high at this time. However, in MIS-overexpressing mice (11) impairment of the testosterone-regulated differentiation of the fetal Wolffian duct, which is the precursor of the vas deferens, epididymides, and seminal vesicles, was observed. Also, incubation of fetal rat Leydig cells with MIS in vitro results in suppression of testosterone synthesis (18). After birth, when MIS levels remain high, fetal-derived Leydig cells begin regressing, and lower levels of androgens ensue. The adult Leydig cells arise and proliferate from mesenchyme precursors at puberty, when MIS levels reach their nadir (35, 40), and begin producing androgens after stimulation with LH (41). The MIS-regulated cell lines used in our studies were originally derived from this adult Leydig cell population. This distinction is important when attempting to understand why fetal Leydig cells, which are in an environment with high levels of MIS, continue to produce androgens. Study of these different Leydig cell populations for type II receptor expression and MIS signal transduction could address the role of MIS-mediated inhibition of steroidogenesis in fetal Leydig cells (25).
Although the amount of testosterone secreted by MA-10 cells is much lower than that secreted by purified Leydig cells (18) and significantly lower than the level of progesterone, prevailing opinion is that MA-10 cells do not produce testosterone. Our results clearly indicate not only that MA-10 cells secrete measurable amounts of testosterone into the medium, but that it can be enhanced 3-fold by cAMP addition. The original report detailing the cloning and characterization of the MA-10 cell line also shows low levels of testosterone secretion, which was induced with hCG (30). MA-10 cells have proven enormously valuable to understand MIS-mediated inhibition of Cyp17 expression and will be very useful in our future efforts to understand the molecular mechanisms of that inhibition.
In our studies to determine whether the MIS signal transduction directly mediated suppression of Cyp17 mRNA expression in the absence of protein synthesis, we observed that cycloheximide alone was able to induce expression of Cyp17 mRNA. The increase in Cyp17 mRNA with cycloheximide treatment is probably not artifactual and is commonly observed with labile mRNAs (42); it could indicate the presence of a protein that inactivates or turns over a crucial component of Cyp17 expression. In the case of c-myc mRNA, mRNA stability has been linked to translation of the c-myc mRNA itself, i.e. c-myc mRNA decay is accelerated by its translation. Cycloheximide blocks c-myc translation and therefore prolongs the c-myc mRNA half-life (43).
Many advances in understanding TGFß signal transduction mechanisms have been made because of the availability of both a cell line that responds to TGFß and a reporter gene with which to measure that response. Until now such a model system for studying MIS signal transduction has been lacking. Evidence of transcriptional regulation of steroidogenic enzymes in rodent Leydig cells by MIS is a significant advance in our understanding of MIS signal transduction. This first example of transcriptional regulation of a gene by MIS can be used to advantage to dissect and define the relevant MIS-specific cis-elements and trans-acting factors and the upstream pathways that are responsible for MIS receptor-mediated molecular events.
There are a number of potential clinical implications that could
emanate from the study of this MIS signal transduction pathway. For
example, MIS-specific checkpoints in the MIS-mediated down-regulation
of testosterone synthesis could be activated and used to lower
endogenous testosterone in such clinical settings as prostatic cancer
and benign prostatic hypertrophy or to lower elevated steroids in
precocious puberty syndromes. Current treatments with GnRH long acting
analogs to down-regulate the GnRH receptor have been successful in the
treatment of central precocious puberty (44), but it is the downstream
variants for which treatment is not yet optimal and could be augmented
by MIS-related treatments. Over 60% of McCune-Albright patients, who
for unknown reasons have a 3:1 ratio of females to males, have elevated
sex steroids in the absence of elevated gonadotropins due to activating
mutations in the -subunit of the G protein-regulating adenylyl
cyclase (45). In these patients with gonadotropin-independent
precocious puberty, suppression of multiple enzymes in the steroid
production pathway by MIS might, for example, be used to augment the
currently used aromatase inhibitors, which has been helpful over the
short term, but less effective over the long term (1–3 yr) (46, 47).
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Acknowledgments |
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Footnotes |
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Received April 26, 1999.
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P. T. Masiakos, D. T. MacLaughlin, S. Maheswaran, J. Teixeira, A. F. Fuller Jr., P. C. Shah, D. J. Kehas, M. K. Kenneally, D. M. Dombkowski, T. U. Ha, et al. Human Ovarian Cancer, Cell Lines, and Primary Ascites Cells Express the Human Mullerian Inhibiting Substance (MIS) Type II Receptor, Bind, and Are Responsive to MIS Clin. Cancer Res., November 1, 1999; 5(11): 3488 - 3499. [Abstract] [Full Text] [PDF]
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 L. Gouedard, Y.-G. Chen, L. Thevenet, C. Racine, S. Borie, I. Lamarre, N. Josso, J. Massague, and N. di Clemente Engagement of Bone Morphogenetic Protein Type IB Receptor and Smad1 Signaling by Anti-Mullerian Hormone and Its Type II Receptor J. Biol. Chem., September 1, 2000; 275(36): 27973 - 27978. [Abstract] [Full Text] [PDF]
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D. L. Segev, T. U. Ha, T. T. Tran, M. Kenneally, P. Harkin, M. Jung, D. T. MacLaughlin, P. K. Donahoe, and S. Maheswaran Mullerian Inhibiting Substance Inhibits Breast Cancer Cell Growth through an NFkappa B-mediated Pathway J. Biol. Chem., September 8, 2000; 275(37): 28371 - 28379. [Abstract] [Full Text] [PDF]
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A. M. Trbovich, P. M. Sluss, V. M. Laurich, F. H. O'Neill, D. T. MacLaughlin, P. K. Donahoe, and J. Teixeira Mullerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents PNAS, March 13, 2001; 98(6): 3393 - 3397. [Abstract] [Full Text] [PDF]
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D. L. Segev, Y. Hoshiya, M. Hoshiya, T. T. Tran, J. L. Carey, A. E. Stephen, D. T. MacLaughlin, P. K. Donahoe, and S. Maheswaran Mullerian-inhibiting substance regulates NF-kappa B signaling in the prostate in vitro and in vivo PNAS, January 8, 2002; 99(1): 239 - 244. [Abstract] [Full Text] [PDF]
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