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Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center (C.P.L., S.Y.H., A.J.W.H.), Stanford, California 94305-5317; and the Hormone Research Center, Chonnam National University (S.Y.C., H.W.B.), Kwangju 500–757, Republic of Korea
Address all correspondence and requests for reprints to: Dr. Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu
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Introduction |
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One key group of intracellular factors regulating apoptosis is the Bcl-2 family of proteins (12). The members of this family can be subdivided into antiapoptotic proteins (such as Bcl-2 and Bcl-xL) and proapoptotic proteins (such as Bax and BAD). It has been proposed that anti- and proapoptotic proteins regulate cell death by binding to each other and forming heterodimers (13, 14). According to this model, a delicate balance between anti- and proapoptotic Bcl-2 family members exists in each cell, and the relative concentrations of these two groups of proteins determine whether the cell survives or undergoes apoptosis.
Of the more than 15 known Bcl-2 family members, each tissue or cell type expresses a specific subset. This laboratory has previously shown that BAD is a proapoptotic Bcl-2 family member expressed in the rat ovary (15). The function of BAD is controlled through its phosphorylation by survival factor-dependent kinases (16, 17). Only the hypophosphorylated form of BAD can heterodimerize with the antiapoptotic proteins Bcl-xL and Bcl-2, thereby leading to cell death. By contrast, the hyperphosphorylated BAD preferentially binds to 14–3-3 proteins, resulting in diminished cell killing (16, 18).
To identify the ovarian binding partners for hypophosphorylated BAD, we screened an ovarian fusion complementary DNA (cDNA) library using the yeast two-hybrid system. As bait, we used a modified rat BAD molecule in which the critical phosphorylation site for 14–3-3 interaction was mutated (serine 137 to alanine). This mutation abolishes the interaction of BAD with 14–3-3 while allowing its interaction with Bcl-xL and thus mimics the hypophosphorylated state of BAD (16, 18). The library screening yielded several positive clones encoding the full-length rat ortholog of human Mcl-1 (myeloid cell leukemia-1).3 Mcl-1 was first discovered as an early induction gene during the differentiation of a human myeloblastic leukemia cell line (19). Subsequent studies established Mcl-1 as an antiapoptotic Bcl-2 family protein with an expression pattern differing from that of Bcl-2 and capable of suppressing cell death induced by various stimuli (20, 21, 22, 23). The expression of Mcl-1 in hemopoietic cells can be induced by various survival factors, leading to enhanced cell viability (24, 25, 26). Here, we describe the cloning of the rat Mcl-1 cDNA, dimerization properties of Mcl-1 with diverse pro- and antiapoptotic Bcl-2 family members, as well as the cellular localization of Mcl-1 messenger RNAs (mRNAs) in the rat ovary and their induction by gonadotropins, which are known follicle survival factors.
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Materials and Methods |
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Protein interaction assays in the yeast two-hybrid system
Interactions between rat Mcl-1 and diverse other Bcl-2 members
were assessed in the yeast two-hybrid system using a pGADGH/Mcl-1
construct and pGBT9 vectors containing the cDNAs of various pro- and
antiapoptotic Bcl-2 family proteins (27). Specific binding of different
protein pairs was evaluated based on the activation of the GAL1-HIS3
reporter gene in the presence of 30 mM 3-aminotriazole
(18).
Animal treatment
For time-course analyses of mRNA expression, female Sprague
Dawley rats (Simonsen Laboratories, Inc., Gilroy, CA) were injected sc
with 10 IU PMSG (Calbiochem, La Jolla, CA) at 26 days of
age and received an ip injection of 10 IU hCG (Schein Pharmaceuticals,
Florham Park, NJ) 48 h later. Rats were killed at different time
points, and the ovaries were collected for total RNA extraction or were
fixed for in situ hybridization analysis. For analysis of
Mcl-1 message in enriched granulosa cells, total RNA was extracted from
granulosa cells isolated by needle puncture (28). All animal protocols
were approved by the administrative panel on laboratory animal care at
Stanford University.
For quantification of Mcl-1 message in preovulatory follicles after hCG or FSH administration, 25-day-old female rats received a sc injection of 10 IU PMSG. In addition, sc injections of the GnRH antagonist Org30850 (40 µg/kg BW; Organon, Oss, The Netherlands) were administered on days 25 and 26 to suppress the endogenous secretion of pituitary gonadotropins (29). On day 27, a single ip injection of 10 IU hCG or 30 IU recombinant FSH (Org32489E, Organon) was administered. These doses were chosen because they were found to reliably induce ovulation in this model system (30). Rats were killed before or 6 h after gonadotropin administration, and ovaries were collected for RNA extraction.
RNA extraction and Northern blot analysis
Ovaries were dissected free of adherent tissue, snap-frozen in a
dry ice/ethanol bath, and stored at -70 C. Total RNA was extracted
using the TRIzol reagent (Life Technologies, Inc.,
Gaithersburg, MD) according to the manufacturer’s instructions. Twenty
micrograms of total RNA per lane were run on agarose-formaldehyde gels
before transfer to nitrocellulose membranes and UV cross-linking. Blots
containing 2 µg/lane polyadenylated [poly(A)+] RNA
extracted from various rat tissues were obtained from CLONTECH Laboratories, Inc. cDNA probes for rat Mcl-1, tissue plasminogen
activator (tPA), ß-actin, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were 32P radiolabeled by random
priming using a commercial kit (Life Technologies, Inc.).
Northern blots were prehybridized and hybridized using ExpressHyb
solution (CLONTECH Laboratories, Inc.) following the
manufacturer’s instructions. After several washes in 0.1 x
SSC-0.5% SDS at 60 C, membranes were exposed to film (at -70 C) or to
phosphorimager screens.
Quantitation of Mcl-1 mRNA levels
After exposure of membranes to phosphorimager screens, the
signal intensities for the Mcl-1 transcripts (as well as for the GAPDH
transcript from a subsequent hybridization) were quantified for each
RNA sample using the Storm 860 PhosphorImager and ImageQuant image
analysis software (Molecular Dynamics, Inc., Sunnyvale,
CA). Alternatively, films were scanned on a GS-710 Imaging Densitometer
and analyzed using the Quantity One Software package (Bio-Rad Laboratories, Inc., Hercules, CA). The values for the 2.3- and
3.7-kb Mcl-1 transcripts in RNA samples extracted from rat ovaries
before and 6 h after injection of hCG or FSH were then normalized
to the respective GAPDH signal. The results are expressed as fold
induction compared with the 0 h control, which was arbitrarily set
at 1.
In situ hybridization studies
Ovaries were fixed at 4 C for 6 h in 4%
paraformaldehyde in PBS, followed by immersion in 0.5 M
sucrose in PBS overnight. Cryostat sections (14 µm thick) were
mounted on microscope slides coated with poly-L-lysine
(Sigma Chemical Co., St. Louis, MO), fixed in 4%
paraformaldehyde in PBS, and stored at -80 C until analyzed. To allow
for direct comparison between ovarian sections from different
experimental groups, all slides were processed simultaneously under
identical conditions. The hybridization procedure was essentially the
same as that previously described (31). In brief, sections were
pretreated serially with 0.2 M HCl, 2 x SSC, pronase
(0.125 mg/ml), 4% paraformaldehyde, and acetic anhydride in
triethanolamine. Hybridization was carried out at 52–55 C overnight in
a mixture containing 35S-labeled rat Mcl-1 complementary
RNA probe (108 cpm/ml), 50% formamide, 0.3 M
NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1 x
Denhardt’s solution, 10% dextran sulfate, 1 µg/ml carrier transfer
RNA, and 10 mM dithiothreitol. Posthybridization washing
was performed under stringent conditions that included ribonuclease A
(25 µg/ml) treatment at 37 C for 30 min and a final stringency of
0.1 x SSC. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed at 4 C for 3–4 weeks
before development. The slides were stained with hematoxylin and eosin
and examined under a light microscope with bright- and darkfield
illumination.
Statistical analysis
Results are presented as the mean ± SEM. For
the ovulation induction experiments using hCG or FSH, differences in
normalized levels of Mcl-1 transcript expression among groups were
assessed by ANOVA followed by Fisher’s protected least significant
differences post-hoc test. Differences in Mcl-1 transcript
expression before and after hCG treatment in granulosa cells were
analyzed using the unpaired t test. Statistical significance
was inferred at P < 0.05.
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).
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). In addition, rat Mcl-1 retains the
long N-terminal sequence containing a proline-glutamic
acid-serine-threonine-rich (PEST) domain that has been
implicated in targeting the protein for rapid intracellular turnover.
Furthermore, use of the rat Mcl-1 amino acid sequence as a query to
search the GenBank expressed sequence tag (EST) division identified a
zebrafish EST (AI544581) that encodes a protein fragment with closest
homology to the mammalian Mcl-1 proteins. Figure 1 shows an alignment
of the deduced amino acid sequences for rat and human Mcl-1 as well as
for the recently published partial sequence of chicken Mcl-1 (32) and
the zebrafish EST.
Interaction of Mcl-1 with diverse pro- and antiapoptotic Bcl-2
family proteins in the yeast two-hybrid system
The dimerization properties of rat Mcl-1 with different pro- and
antiapoptotic Bcl-2 members were assessed in the yeast two-hybrid
system (Fig. 2). In agreement with its
identification in the yeast two-hybrid screening, Mcl-1 interacted
strongly with the phosphorylation site mutant of BAD. However, under
the conditions employed, Mcl-1 did not bind to wild-type BAD, which
appears to be constitutively hyperphosphorylated in yeast cells as
suggested by its strong interaction with the 
-isoform of 14–3-3. In
addition, Mcl-1 also interacted with the proapoptotic Bcl-2 family
members Bax, Bak, Bok/Mtd, Bik, and BOD/Bim. By contrast, Mcl-1
dimerized only weakly (Bcl-w, Bfl-1, CED-9, and BHRF-1) or not at all
(Mcl-1, Bcl-2, and Bcl-xL) with the antiapoptotic Bcl-2 family members
tested in this assay.
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, two Mcl-1 transcripts with sizes of
2.3 and 3.7 kb, respectively, were expressed at varying levels in all
rat tissues examined. The highest levels of expression were found in
spleen, lung, heart, and kidney [Fig. 3A, poly(A)+ RNA
analyzed], whereas lower expression levels were observed in
reproductive tissues, including ovary, oviduct, uterus, and testis
(Fig. 3B, total RNA analyzed). To further investigate developmental
expression of Mcl-1 in the female gonad, total RNA extracted from
ovaries at different points of postnatal development was hybridized
with the same probe. As shown in Fig. 4A, the Mcl-1 message is expressed from day 3 after birth and increased
after day 12 of age.
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, B and C, the
two Mcl-1 transcripts were found to be expressed throughout the
treatment time course. After PMSG injection, expression of the 2.3-kb
transcript increased, peaking at 3–6 h and then returning to baseline
levels by 24 h. Two days after PMSG priming, ovulation induction
with hCG resulted in an 8.5-fold induction (P < 0.05)
of the 2.3-kb transcript (normalized to GAPDH expression; n = 3)
at the 6 h point, whereas expression of this transcript at 12
h was not significantly different from the 0 h expression level
(P > 0.10). There were no statistically significant
differences among the expression levels of the larger 3.7-kb transcript
at 0, 6, and 12 h after hCG administration (P >
0.05).
Both hCG and FSH cause a preovulatory induction of Mcl-1 message in
the rat ovary
As both hCG and FSH can act as follicle survival factors in
preovulatory follicles (7) as well as induce follicle rupture in
PMSG-primed immature rats (30), we further compared the effects of
these two gonadotropins on the induction of Mcl-1 mRNA in the ovary.
Immature rats were primed with PMSG, but additionally received two
injections of the GnRH antagonist Org30850 to suppress endogenous
pituitary gonadotropin secretion. At 48 h after PMSG
administration, the rats were injected with an ovulatory dose of either
hCG or FSH. As the earlier time-course studies had demonstrated a
maximal induction of Mcl-1 message after 6 h, expression at this
time point was compared with levels before the injection of hCG or
FSH.
Northern blots of total RNA isolated from rat ovaries before treatment
or at 6 h after hCG or FSH administration were hybridized with a
radiolabeled Mcl-1 cDNA probe. Subsequently, the same blots were also
hybridized with control probes for tPA and GAPDH (Fig. 5A). The message for tPA had previously
been shown to be induced by both hCG and FSH (30) and therefore served
as a positive control, whereas the housekeeping gene GAPDH was used as
a control for RNA loading. The expression of each of the two Mcl-1
transcripts was quantified separately and normalized to the GAPDH
signal. As shown in Fig. 5B, expression of the 2.3-kb Mcl-1 transcript
in the ovaries of GnRH antagonist-pretreated rats was induced 3.3- and
2.4-fold (P < 0.01) by hCG and FSH, respectively,
whereas expression of the 3.7-kb transcript remained essentially
unchanged (P > 0.05).
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, ovaries
from immature rats contained multiple early antral follicles, preantral
follicles, and some atretic follicles. Mcl-1 mRNA was mainly detected
in the thecal cells of early and preantral follicles (Fig. 6B).
Although treatment with PMSG for 6 h did not cause obvious changes
in ovarian morphology (Fig. 6C), a major increase in Mcl-1 message
could be detected in both granulosa and thecal cells (Fig. 6, D and E).
The specificity of the signal obtained with the antisense Mcl-1 probe
was demonstrated by the lack of hybridization in sections treated with
the sense Mcl-1 probe (Fig. 6F). After PMSG priming for 48 h, the
rat ovaries contained multiple large antral follicles, and the Mcl-1
message was again mainly expressed in the thecal cells of these
follicles (Fig. 6, G and H). Six hours after the hCG injection,
hybridization with the Mcl-1 antisense probe demonstrated an increase
in Mcl-1 mRNA in both thecal and granulosa cells of these follicles
(Fig. 6, I and J).
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Discussion |
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Among the intracellular regulators of apoptosis, the Bcl-2 family of
proteins occupies a central position by integrating diverse survival
and death signals and linking them to downstream apoptotic events such
as mitochondrial cytochrome c release and caspase activation
(12). The importance of Bcl-2-mediated pathways to the regulation of
follicle survival and atresia is illustrated by the phenotype of mice
overexpressing Bcl-2 under the control of the inhibin-
promoter/enhancer. The targeted overexpression of this antiapoptotic
gene in somatic ovarian cells leads to diminished follicular cell
apoptosis and increased ovulation rates and litter sizes in transgenic
animals (37). This phenotype is somewhat reminiscent of the effects
seen in wild-type animals hyperstimulated with gonadotropins, raising
the possibility that gonadotropins might induce an antiapoptotic Bcl-2
family protein in follicular cells.
Although the mRNAs for the antiapoptotic molecules Bcl-2 and Bcl-xL have been found in the ovary, their expression appears to be unaffected by PMSG treatment in the immature rat model (38). To identify additional antiapoptotic Bcl-2 family members in the ovary, we performed a yeast two-hybrid screening of a rat ovary cDNA library using a mutant BAD molecule as bait. The proapoptotic BAD is constitutively expressed in the rat ovary (15). Although presumably hyperphosphorylated BAD preferentially binds different isoforms of 14–3-3, a BAD mutant mimicking the hypophosphorylated form of BAD was found to interact with P11, a survival gene induced by nerve growth factor in the PC12 pheochromocytoma cell line (18). Here, we describe the identification of Mcl-1 as an additional interaction partner for this phosphorylation site mutant of BAD.
In living eukaryotic cells, Mcl-1 is capable of interacting with the wild-type forms of several other proapoptotic Bcl-2 family proteins (Bax, Bak, Bok/Mtd, Bik, and BOD/Bim), as demonstrated by yeast two-hybrid assays. Both Bok/Mtd (27, 39) and BOD/Bim (40, 41) were previously shown to be expressed in the rat ovary. In a recently performed yeast two-hybrid screening using BOD/Bim as bait, several of the clones with the strongest interaction were also found to encode Mcl-1 (data not shown). The dimer formation between pro- and antiapoptotic Bcl-2 family proteins, mediated by their highly conserved Bcl-2 homology domains, is thought to be a critical determinant of their mutual functional antagonism (12, 13, 14). In this respect, the observation that Mcl-1 can interact promiscuously with different proapoptotic Bcl-2-related proteins in yeast cells complements previous findings showing that Mcl-1 coimmunoprecipitates with and suppresses apoptosis induced by diverse proapoptotic Bcl-2 members (22, 27).
In addition to its heterodimerization with proapoptotic Bcl-2 family members, Mcl-1 could inhibit apoptosis by at least two other mechanisms. Similar to Bcl-xL and Bcl-2, Mcl-1 could form pores in the outer mitochondrial membrane to regulate cytochrome c release (42, 43) and/or inhibit the activation of caspases by retaining the adaptor molecule Apaf-1 in an inactive conformation (12, 44). Although its exact mechanism of action remains to be examined, Mcl-1 has been shown to increase cell viability under different apoptosis-inducing conditions, including growth factor deprivation and exposure to chemotherapeutic agents or UV irradiation (21, 22, 23).
The complete conservation of the Bcl-2 homology domains BH1, BH2, and BH3 (located in the C-terminal portion of the protein) between rat and human is consistent with their suggested importance in providing the structural basis for dimerizations between Bcl-2 family proteins (12, 45). The long N-terminal portion of Mcl-1, which bears no homology to other Bcl-2-related proteins, is also conserved between rat and human, although to a lesser degree. The functional significance of this domain is unclear, except for its PEST sequence, which is thought to account for the short half-life of the protein (see below). The partial amino acid sequences, deduced from a published chicken nucleotide sequence (32) and a recently released zebrafish EST, offer further insights into the conservation of Mcl-1-related sequences in nonmammalian species. The chicken sequence and zebrafish EST encode protein fragments with 61% and 53% identity to rat Mcl-1, respectively. The zebrafish sequence described here, therefore, most likely represents the first Bcl-2 family gene to be found in teleosts.
Several antiapoptotic Bcl-2 family genes (Bcl-2, Bcl-xL, A1/Bfl-1, and Mcl-1) are transcriptionally induced by specific cytokines in hemopoietic cell types (12). Prosurvival factors inducing Mcl-1 in hemopoietic cells include granulocyte-macrophage colony-stimulating factor, interleukin-1ß, and vascular endothelial growth factor (24, 25, 46). The stimulation of Mcl-1 expression by gonadotropins described here represents the first evidence of a Bcl-2 family gene being induced by follicle survival factors in the ovary. The rapid and transient increase in the ovarian Mcl-1 message after gonadotropin treatment mirrors the pattern of induction observed in hemopoietic cells (24, 47). This pattern was shown to reflect a transient transcriptional activation of the Mcl-1 gene, generating a message with a short half-life of less than 2 h (47). In hemopoietic cells, these changes in Mcl-1 mRNA levels closely correlate to similar alterations in the levels of the Mcl-1 protein, which is also rapidly turned over, probably due to its N-terminal PEST sequence (24, 47, 48). It has been suggested that the transient induction of Mcl-1 message and protein by survival factors serves to protect cells during specific stages of differentiation when they are particularly vulnerable to apoptosis (23).
Because of its induction by PMSG, FSH, or hCG, the gonadotropin-dependent expression of Mcl-1 in ovarian cells is probably mediated by a mechanism in the common downstream signaling pathways of the LH/hCG and FSH receptors. The murine Mcl-1 promoter contains a DNA sequence similar to the consensus site recognized by the CRE-2-binding protein, which is involved in transcriptional induction by cAMP (24, 49). However, a detailed molecular analysis of the Mcl-1 gene promoter will be necessary to delineate potential cis-responsive elements mediating the gonadotropin induction of Mcl-1. Interestingly, the induction of the 2.3-kb Mcl-1 transcript at 6 h after hCG injection was lower in Org30850-pretreated rats (3.3 ± 0.30-fold) than in rats not pretreated with the GnRH antagonist (8.5 ± 0.17-fold). This observation could be due to the different age of animals in these two series of experiments (27 vs. 28 days) and/or differences in their endogenous gonadotropin levels.
The two Mcl-1 transcripts observed in rat tissues (2.3 and 3.7 kb) correspond in size to the two known human Mcl-1 messages (2.5 and 3.8 kb) (19). Both Mcl-1 transcripts in the rat are long enough to contain the full-length coding sequence. Although one cannot exclude the possibility that the rat mRNAs may encode different ORFs, the two human Mcl-1 transcripts probably result from the use of alternative polyadenylation sites in the 3'-untranslated region of the gene (19). Although the significance of selective induction of the 2.3-kb Mcl-1 transcript by gonadotropins remains unknown, it is interesting to note that a similar differential induction of two splicing variants by gonadotropins in granulosa cells has recently been observed for the kit ligand gene (50).
During follicle atresia in the rodent ovary, apoptotic cell death is confined to granulosa cells (9, 11). The basal expression and gonadotropin induction of Mcl-1 in granulosa cells are therefore consistent with its potential role in regulating follicle atresia. Whether the levels of Mcl-1 expression in individual follicles correlate with granulosa cell apoptosis remains to be determined. Interestingly, levels of Mcl-1 mRNA expression in thecal cells, a cell type resistant to apoptosis in this model, were higher than those in granulosa cells. A similar differential ovarian expression pattern has been observed for the inhibitor of apoptosis proteins, Hiap-2 and Xiap, which are also induced by gonadotropins (51). It is likely that the high expression of antiapoptotic genes in thecal cells confers a protection from apoptosis.
From the data presented, we propose the following model. Mcl-1 is an antiapoptotic Bcl-2 member expressed in ovarian follicles that can dimerize with BAD and other proapoptotic ovarian Bcl-2 family members (e.g. Bax, BOD, and Bok). Gonadotropins induce a transient increase in the expression of Mcl-1 and may thereby shift the balance of ovarian anti- and proapoptotic Bcl-2 family proteins to favor survival and rescue follicles from atresia. The induction of Mcl-1 along with other antiapoptotic genes such as inhibitor of apoptosis proteins may therefore represent a molecular basis for the suppression of follicular apoptosis and atresia by gonadotropins.
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Acknowledgments |
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Footnotes |
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2 Supported by a postdoctoral fellowship from the German Academic
Exchange Service. Present address: Department of Obstetrics and
Gynecology, University of Leipzig, Leipzig, Germany 04207.
3 The GenBank accession number for rat Mcl-1 is
AF115380.
Received May 6, 1999.
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