Origin and Identity of Adrenocortical Tumors in Inhibin Knockout Mice: Implications for Cellular Plasticity in the Adrenal Cortex
Brendan D. Looyenga and Gary D. Hammer
Cellular and Molecular Biology Graduate Program (B.D.L., G.D.H.), and Department of Internal Medicine (G.D.H.), Division of Endocrinology, University of Michigan, Ann Arbor, Michigan 48109-2200
Inhibin knockout (Inha-/-) mice develop gonadal sex-cord tumors and-when gonadectomized- adrenocortical tumors. Previous reports demon- strated that adrenocortical tumors from Inha-/- mice produce estrogen and depend on gonadotro- pin signaling for initiation. Here we show that, in addition to producing estrogen, the adrenocortical tumors display a global change in cellular identity, composed of two unique cell types expressing dif- fering arrays of genes normally restricted to theca and granulosa cells of the ovary. Many of these genes are also induced in wild-type adrenals after gonadectomy or upon chronic gonadotropin stim- ulation, suggesting that the adrenal cortex nor- mally contains a population of pluripotent cells that can be driven toward an adrenal or gonadal identity given the appropriate pituitary stimuli. A central feature of this altered cellular identity is the switch from predominant expression of Gata6 (endoge-
nous to the adrenal cortex) to Gata4, which defines cellular identity in the ovary. We show that stable transfection of Gata4 in cultured adrenocortical cells is sufficient to activate ovarian-specific genes of both theca and granulose lineages. Spatial anal- ysis of Gata4 expression reveals a distinct pattern of localization to the supcapsular region of the adrenal, which contains undifferentiated progeni- tor cells that continuously populate the adrenocor- tical zones. Although both wild-type and Inha-/- mice display this pattern, only Inha-/- mice pro- duce tumors composed of these Gata4-positive cells. These data suggest that Inha-/- adrenocor- tical tumors cells are derived from pluripotent ad- renocortical progenitor cells that adopt a gonadal fate due to the convergent loss of inhibin and chronic exposure to elevated gonadotropins. (Mo- lecular Endocrinology 20: 2848-2863, 2006)
T HE ADRENAL CORTEX and gonad share a common embryonic origin from tissue of the urogenital ridge. A specific subset of urogenital cells, termed the adreno- gonadal primordium, can first be detected at embryonic d 9.5 in mice by staining for the transcription factor steroidogenic factor-1 (Sf1), which is required for proper specification of both the gonad and adrenal cortex (1, 2). Shortly thereafter, the adrenogonadal primordium is di- vided into two distinct cellular compartments coincident with the ascension of the metanephric kidney and the inward migration of primordial germ cells into the caudal region of the urogenital ridge (3, 4). Positional cues de- rived from spatial separation of the two regions of the adrenogonadal primordium are thought to mediate dif- ferential patterns of gene expression, which ultimately result in the specification of distinct steroidogenic tissues
First Published Online July 27, 2006
Abbreviations: Amh, Anti-Müllerian hormone; Cyp17, P450c17%; Cyp19, P450aromatase; GC, granulosa cells; Inha, inhibin &; Inhbb, inhibin/activin B-B; Lhr, LH receptor; Sf1, steroidogenic factor-1; Star, steroidogenic acute regulatory protein; TC, theca cells.
Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.
of the adrenal cortex and gonad. Further development and differentiation of these tissues occurs upon matura- tion of the pituitary gland, which produces an array of peptide hormones that instructively direct the growth and differential steroidogenic profiles of the adrenal cor- tex and gonad (5-7).
Although the positional signals that direct the diver- gence of the adrenal cortex and gonad are poorly understood, several tissue-specific transcription fac- tors that guide this process have been elucidated by in vivo loss-of-function studies and in vitro genetic screens (3, 8, 9). This group of factors includes Lim1, Dax1, Sry, and members of the Sox and Gata families of transcription factors. Of these factors, the GATA family transcription factors are particularly notable be- cause they play several significant roles in develop- ment of the mammalian pituitary-adrenal-gonadal axis. In addition to helping specify the steroidogenic tissues of the adrenal and gonad, members of the Gata family are also critical for proper differentiation of the pituitary gonadotropes, specification of genitouri- nary ductal system, and sexual dimorphism of the male and female reproductive systems (10-12).
Within the Gata family, Gata4 and Gata6 are specif- ically involved in development and differentiation of the steroidogenic tissues of the adrenal and gonad.
These two factors are variably expressed in the adre- nal and gonad throughout fetal development and spa- tially correlate with the differential steroid production of these distinct tissues (10, 13, 14). Most notably, expression of Gata4 appears to positively correlate with the production of sex steroid hormones and is thus present in several gonadal cell lineages including male Leydig cells and female granulosa cells (GC) and theca cells (TC) (15). Gata4 is also present in the fetal murine adrenal cortex, which produces the androgen precursor DHEA, but is down-regulated in the postna- tal adrenal cortex, which instead produces the steroid hormones aldosterone and corticosterone in response to angiotensin-II and pituitary ACTH, respectively (16). Gata6, on the other hand, is expressed in both the fetal and adult adrenal cortex and the ovarian interstitium, although it is not prominently expressed in the testis nor in the GC or TC compartments of the ovary (17, 18).
The transcriptional control of Gata4 expression in ste- roidogenic tissues, as well as in other tissues, is poorly understood, but is likely to be highly complex based on the intricate spatial and temporal expression patterns of Gata4 throughout development and adulthood (11, 19, 20). Mechanisms that specify the initial expression of Gata4 in the gonads are unknown, although its expres- sion is clearly regulated by the pituitary gonadotropins. FSH up-regulates Gata4 expression in the testis and ovary, whereas LH and chorionic gonadotropin have dif- ferential effects, which can be both stimulatory and in- hibitory toward Gata4 expression (13, 14, 21). Receptors for these hormones are normally restricted to steroido- genic cells of the testis and ovary, and are not promi- nently expressed in the adult adrenal gland. In response to gonadotropins, Gata4 is both transcriptionally and posttranslationally induced to turn on an array of target genes necessary for cellular differentiation and steroido- genesis (12, 22, 23). Many targets of Gata4 have been identified in vivo and in vitro, and include such genes as anti-Müllerian hormone (Amh), steroidogenic acute reg- ulatory protein (Star), inhibin « (Inha), inhibin/activin ß-B (Inhbb), P450c17a (Cyp17) and P450aromatase (Cyp19) (24-27). The latter two genes are particularly important for sex steroid production because they catalyze the synthesis of sex steroids from other steroid precursors in the gonad.
Whereas Gata4 is normally absent from the adult ad- renal cortex, recent studies of adrenocortical tumors suggest that this key transcriptional regulator may be reactivated under pathological circumstances in the ad- renal gland (28, 29). Most notably, a switch from pre- dominant Gata6 expression to Gata4 expression has been reported in a subset of human and mouse adreno- cortical carcinomas (30, 31). Although the relationship between the steroidogenic phenotype of adrenocortical tumors and Gata4 expression is unclear in human stud- ies, several mouse models of adrenocortical tumorigen- esis (i.e. SF1$172 allele bearing mouse strains and inhibin « promoter-simian virus 40 T-antigen [Inha/Tag] trans- genic mice) demonstrate that induction of Gata4 in ad- renocortical tumors correlates with sex steroid produc-
tion (32-34). Gata4 misexpression in the adrenocortical tumors from these various mouse models is induced by gonadectomy and subsequent increased levels of pitu- itary gonadotropins, which occur as a result of perturbing the pituitary-gonadal axis by removing the negative feed- back loop that gonad-derived sex steroids normally ex- ert upon pituitary gonadotrope cells.
Current studies in our laboratory focus on the inhibin-a knockout mouse (Inha-/-) model of adrenocortical tu- morigenesis, in which the @ subunit of the heterodimeric TGF& family ligand inhibin has been genetically ablated (35). Like the SF1$172 and Inh/Tag mice, these mice develop both gonadal and adrenocortical tumors, which are absolutely dependent upon gonadotropins for induc- tion (36-38). Whereas adrenocortical tumors in these mice are also sex steroid-producing, the cellular identity of these tumors is not clear. Production of estrogen suggests that Inha-/- adrenocortical tumors might rep- resent a mixed gonadal identity because ovarian estro- gen biosynthesis requires the presence and distinct en- zymatic functions of both TC-which contain P450c17x and catalyze androgen synthesis from steroid precur- sors-and GC-which contain P450 aromatase and cata- lyze androgen aromatization to estrogens (39). It is not clear, however, whether this phenotype results from sim- ple activation of the steroidogenic machinery for estro- gen production in otherwise normal adrenocortical cells, or whether the Inha-/- tumor cells have adopted a different cellular fate that includes a more global gonadal transcriptional program.
To address this question, we compared the expres- sion profile of several gonad-restricted markers-both TC and GC-specific-in wild-type and Inha-/- adrenal glands before and after surgical gonadectomy to deter- mine the identity of these tumor cells. We found that several gonad-restricted markers were induced in the adrenal glands of Inha-/- mice, and unexpectedly, also in the adrenal glands of wild-type littermates after go- nadectomy. Expression of these genes correlated with the ectopic expression of the transcription factor Gata4, which is sufficient to induce both TC and GC-specific genes in adrenocortical Y1 cells. Further analysis of this observation suggests that a subset of subcapsular ad- renocortical cells retain the developmental plasticity to respond to pituitary gonadotrope stimuli that normally specify gonadal cell fates, and that these cells-like those found in the gonad-become misregulated in the absence of inhibin, thus forming hyperproliferative clus- ters of gonad-like cells in the adrenal cortex.
RESULTS
Inha-/- Adrenocortical Tumors and Adrenals from Gonadectomized Mice Express Gonad- Restricted Transcripts Consistent with a Mixed Gonadal Lineage
The adrenal cortex and gonad share in common the expression of a large subset of genes, including
Cyp11a1 and Star, consistent with their common em- bryonic lineage and identity as steroidogenic organs (3). Many of these genes are under the transcriptional control of Sf1/Nr5a1, which is required both for the proper development and steroidogenic function of the adrenal, ovary, and testes (40). The identity of these tissues can be readily discriminated from nonsteroi- dogenic organs on the basis of Sf1 expression (Fig. 1A). The inhibin-a subunit (Inha), which is a transcrip- tional target of Sf1, is also restricted to the gonads and adrenal gland (Fig. 1A). Genetic deletion of Inha in mice can be readily demonstrated by semiquantitative RT-PCR analysis of adrenal RNA because transcripts for this gene are completely undetectable. Using this method of analysis, we compared the expression pro- file of adrenal tissues before and after gonadectomy in wild-type and Inha-/- mice to nonsteroidogenic tis- sues and gonads to determine the extent to which Inha-/- adrenocortical tumors resemble gonadal tis- sue at the transcriptional level.
As reported previously, we found that both LH re- ceptor (Lhr) and Cyp17 are highly expressed in Inha-/- adrenocortical tumors (Fig. 1B) (36). Tran- scripts for both of these molecules are only weakly detectable in the normal adult mouse adrenal, which lacks the capacity to produce androgens. In the ovary, both Lhr and Cyp17 are initially restricted to the TC compartment, although during follicle maturation Lhr is also expressed in fully differentiated GC (41). The presence of these transcripts in Inha-/- adrenocor- tical tumors suggests that a more extensive TC iden- tity might also be present.
To explore this possibility, we compared the expres- sion of several TC markers in Inha-/- adrenocortical tumors to the normal adrenal cortex (Fig 1B). These markers include the transcriptional cofactor Set, which is required for optimal Cyp17 expression, progester- one receptor (Pr), which functions as a regulator of terminal differentiation and growth in luteal TC, and the cell surface enzyme aminopeptidase-N (Anpep), whose function in follicular physiology is unclear (42- 45). We also analyzed expression of the secreted pep- tides relaxin (Rlx) and relaxin-like factor (Rlf), which play important roles in remodeling follicular matrix pro- teins and in uterine conditioning during pregnancy (46, 47). Interestingly, several of these factors are ex- pressed in the normal adrenal cortex, and are slightly increased in Inha-/- adrenocortical tumors. This find- ing is consistent with the previously suggested hy- pothesis that the adrenocortical lineage is closely re- lated to testicular Leydig cells, which are the testicular homologs of ovarian TC (8). Because the adrenal does not express significant levels of Lhr or Cyp17, how- ever, it is clear that Inha-/- adrenocortical tumors contain a population of TC-like cells that are distinct from the cells that normally populate the adrenal cortex.
In addition to containing cells with a theca-like iden- tity, it would be predicted that Inha-/- adrenocortical tumors also contain a population of cells that have the
enzymatic capability of producing estrogen by arom- atizing androgenic precursor steroids. Consistent with this prediction, the GC-specific enzyme P450 (Cyp19) is highly expressed in Inha-/- adrenocortical tumors, although absent from the adrenal gland before gonadectomy, suggesting that a granulosa-like cellu- lar identity is also present in these tumors (Fig. 1C). In the ovary, Cyp19 expression is tightly restricted to the GC compartment and is stimulated primarily by FSH- induced maturation of these cells (48). Consistent with the presence of Cyp19 expression, we found that FSH receptor (Fshr) is highly expressed in these tumors, but not in the normal adrenal cortex (Fig. 1C). This obser- vation is interesting because FSH is dispensable for gonadal tumor induction in Inha-/- mice, although its loss significantly slows tumor growth and decreases estrogen output (49). Whereas the requirement for FSH in Inha-/- adrenal tumor formation has not been explored, it is clear that Inha-/- adrenocortical tu- mors are responsive to FSH and likely require FSH signaling for maximal steroidogenic output.
To further determine the extent to which Inha-/- adrenocortical tumors contain a GC-like cellular iden- tity, we analyzed the expression of several GC-specific markers in the same tissue panel as above (Fig. 1C). In addition to Cyp19 and Fshr, we found significant ex- pression of the inhibin/activin-ß (Inhba and Inhbb) sub- units and anti-Müllerian hormone (Amh) in these tu- mors (Fig. 1). These members of the TGFß family of secreted ligands are specifically produced by GC and play critical roles in gonadal development, reproduc- tive maintenance and hormonal cyclicity (50-52). Amh is normally present in immature and differentiated GC, whereas Inha/Actv-BA and -BB are primarily produced in response to FSH-induced follicle maturation. Nota- bly, transcripts for all three of these peptide ligands are completely absent from the adrenal gland before gonadectomy.
Estrogen receptor-ß (Esr2) and oxytocin (oxt) are other GC-specific markers also present in Inha-/- adrenocortical tumors, but not the adrenal gland be- fore gonadectomy (Fig. 1C). Unlike ERa, which is ubiq- uitously expressed in a large range of tissues including the adrenal and liver, ERß is specifically induced in GC during gonadotropin-induced maturation (53). Expres- sion of this factor in GC is critical because it mediates both the differentiation and growth-promoting effects of estrogens in this compartment of the ovary and permits appropriate GC responsiveness to ovulatory surges of LH (54). The secreted hormone oxytocin is produced specifically in GC in response to gonado- tropins, although the role of gonadally derived oxyto- cin is unclear (55). Together with the expression of several TC-specific markers and other GC-specific markers, these data support the hypothesis that Inha-/- adrenocortical tumors are not simply com- posed of normal adrenal cells that express sex ste- roid-producing enzymes but are instead composed of one or more cell types that have adopted a mixed ovarian identity.
A
non-GDX
GDX
WT Inh-/- WT Inh-/- Liv Kid Ov Test
Inha
Nr5a1
Gapdh
B
non-GDX
GDX
WT Inh-/- WT Inh-/- Liv Kid Ov Test
Lhr
Cyp17
Anpep
Rlx
TC
RIf
Set
Pr
C
non-GDX
GDX
WT Inh-/- WT Inh-/- Liv Kid Ov Test
Fshr
Cyp19
Inhba
Inhbb
GC
Amh
Esr2
Oxt
Because only Inha-/- mice develop estrogen-pro- ducing adrenocortical tumors after gonadectomy, we did not expect that their wild-type counterparts would
express any gonad-restricted transcripts after gonad- ectomy. Contrary to this expectation, we found that several of these transcripts, both GC and TC specific,
were also expressed in the adrenals of wild-type mice after gonadectomy, albeit at lower levels than those observed in Inha-/- adrenocortical tumors (Fig. 1). This observation has several important implications. First, it suggests that the specification of gonadal cells in the adrenal cortex after gonadectomy is a result of chronic gonadotropin up-regulation as opposed to the loss of inhibin. This distinction is critical because it implies that the mechanistic role of inhibin in the ad- renal is not to prevent the misspecification of adrenal cells in response to elevated gonadotropins, but rather to control the growth of these cells once they are specified. Second, it suggests that the normal adult adrenal cortex contains a population of developmen- tally plastic cells that given the improper hormonal stimuli, will respond by adopting a gonadal identity.
Constitutive Overexpression of LH Is Sufficient to Specify the Thecal Lineage in the Adrenal Gland
The observation that surgical gonadectomy is suffi- cient to specify gonadal cellular identities in the normal adrenal cortex prompted us to ask whether simply elevating gonadotropin levels was sufficient to specify either the TC or GC identity as well. Because elevated LH is required for adrenal tumor formation in Inha-/- mice, we examined the adrenals of bLH-BCTP mice (LH-CTP), which display constitutively elevated serum LH levels comparable to wild-type mice after gonad- ectomy (36, 56). As previously reported in these mice, both Lhr and Cyp17 were expressed in the adrenal in absence of gonadectomy (Fig 2A) (57). All of the TC- specific markers we previously analyzed and found up-regulated in Inha-/- tumors were also up-regu- lated in LH-CTP mice (Fig 2B), consistent with the sufficiency of LH to ectopically induce the TC identity in the adrenal.
In contrast to their ectopic adrenal expression or up-regulation of TC markers, LH-CTP mice do not
demonstrate expression of Cyp19 or Fshr (Fig. 2A), nor do they express any of the other GC markers found in the adrenals of wild-type mice post-gonadectomy (data not shown). As such, elevated FSH levels appear to be absolutely required to specify the GC identity in the mouse adrenal cortex after gonadectomy. This observation is interesting, as it suggests that TC and GC cellular fates may be induced separately by LH and FSH, respectively. In the absence of data from mice that overexpress only FSH, however, we are unable to determine whether elevated FSH is sufficient to induce the GC identity in the adrenal.
Inha-/- Adrenocortical Tumors Are Composed of Multiple, Distinct Steroidogenic Cell Types
Although both the TC and GC cellular identities are presumably specified from the same population of developmentally plastic cells in the adrenal cortex, it is not clear whether these identities are contained in a single differentiated cell or two separate cell popula- tions. Because ovarian expression of several genetic markers is compartmentalized into TC and GC popu- lations respectively, the mixed cellular phenotype of Inha-/- adrenocortical tumors suggests that two separate cell populations might be specified within the tumors. Gene expression data from LH-CTP mice sup- port this hypothesis, as they suggest that each cellular identity in the adrenal is separately specified by a different gonadotropin. Because the expression data from Inha-/- tumors are taken from whole tumors and not individual cells, however, they do not rule out the possibility that the tumors are composed of a single cellular lineage that contains the cumulative differentiating effects of LH and FSH. To address this question, we analyzed the spatial expression of Lhr and Amh to determine whether the different cellular identities were mixed in a single cell type, or specified
A
WT LH-CTP Inh-/-
B
WT LH-CTP Inh-/-
Inha
Lhr
Nr5a1
Cyp17
Fshr
GC
Anpep
Cyp19
Rlx
TC
Gapdh
Rif
Set
Pr
Semiquantitative RT-PCR was performed on cDNA libraries that were reverse-transcribed from 1.0 µg of total RNA harvested from the adrenals of nongonadectomized 10-wk-old mice. A, Adrenals from bLH-BCTP (LH-CTP) mice express normal levels of Nr5a1 and Inha, although they lack any expression of GC markers including Fshr and Cyp19. B, In contrast to the lack of GC markers, theca markers are prominently expressed or up-regulated in LH-CTP mice. Notably, aminopeptidase-N (Anpep) and Lhr are completely absent from both wild-type (WT) and Inha-/- animals but are prominently expressed in LH-CTP mice, suggesting that constitutively elevated LH is sufficient to induce a thecal lineage of differentiation in the adrenal.
as two distinct cellular populations within adrenocor- tical tumors.
Immunofluorescent detection of Lhr and Amh in nor- mal ovarian follicles clearly demonstrates that Lhr and Amh specifically mark TC and GC, respectively, as the staining for these markers is mutually exclusive (Fig. 3A). Interestingly, small adrenocortical tumors from Inha-/- mice, which have an adenomatous structure reminiscent of ovarian follicles, also show distinct staining for Lhr and Amh. Lhr expression is restricted to the outer part of tumor nodules, whereas Amh is expressed in a more central pattern (Fig. 3B). As these tumors become increasingly larger, they appear to lose this organization, and become more structurally heterogeneous. In the larger tumors, Lhr-positive and Amh-positive regions of cells are mixed together, al- though staining for the two markers in cells of these tumors still appears to be mutually exclusive (Fig. 3C). Together, these data suggest that Inha-/- adreno- cortical tumors are cellularly heterogenous and com- posed of at least two cell types that appear to be directed toward either a TC or GC fate in a mutually exclusive fashion.
The Specification of Gonadal Cellular Identities in the Adrenal Correlates with Ectopic Expression of Gata4
Within the ovary, both GC and TC express the tran- scription factor Gata4, which is directly involved in the
differentiation and steroidogenic function of these cells in mammalian reproduction (12). Several of the gonad-specific transcripts that we analyzed in Inha-/- tumors are direct transcriptional targets of Gata4, including Cyp17, Cyp19, Amh, Inhbb, and Inha itself (27). As predicted by these data, Gata4 can be readily detected at both the mRNA and protein levels in Inha-/- adrenocortical tumors, but not in the adre- nals of Inha-/- or wild-type mice before gonadec- tomy (Fig. 4, A and C). Gata4 transcripts are also detectable in the wild-type adrenal gland after gonad- ectomy and in the adrenals of LH-CTP mice before gonadectomy, although only at the mRNA level (Fig 4A). As such, Gata4 expression in the adrenal clearly correlates with elevated levels of gonadotropins and the specification of gonadal cells after gonadectomy.
Due to the fact that gonadectomized female Inha-/- mice develop adrenocortical tumors more rapidly than their male counterparts, we chose to focus the bulk of our studies on female Inha-/- adrenocortical tumors. Because adrenocortical tumors from both sexes primar- ily produce estrogens, it would be predicted that male adrenocortical tumors are phenotypically similar to fe- male tumors at the molecular level, and therefore more representative of ovarian cells rather than testicular cells. To ensure that this is true and that the conclusions drawn from these studies are applicable to both sexes of Inha-/- mice, we screened Inha-/- tumors from both male and female mice for expression of several markers
anti-Lhr
anti-Amh
merge
A
WT ovary
B
G
Inh-/- adrenal tumor (early)
To
C
Inh-/- adrenal tumor (late)
A
non-GDX
GDX
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WT LH-CTP Inh-/- WT LH-CTP Inh-/-
Percent Expression
30
Gata4
25
Gata6
20
Mc2r
Gata6
15
Gata4
Cyp21a1
10
Cyp11b2
5
Gapdh
WT
Inh-/-
C
non-GDX
GDX
D
female adrenal
male adrenal
ovary
testis
WT LH-CTP Inh-/-
WT LH-CTP Inh-/-
Inh +/+ -/- +/+ -/- +/+ -/- +/+ -/-
Gata6
Lhr
Gata4
Gata4
ß-actin
P450
aromatase
Sry
ß-actin
Expression of Gata4 and Gata6 was measured in adrenal and/or adrenal tumor tissue from WT, LH-CTP, and Inha-/- mice. A, Semiquantitative RT-PCR performed from adrenal cDNA libraries shows that Gata6 is prominently expressed in the adrenal gland across genotypes but is decreased in Inha-/- adrenocortical tumors. Gata4 is not normally expressed in the adrenal gland before gonadectomy, except in LH-CTP mice, which have elevated serum LH. After gonadectomy, Gata4 is found in the adrenal across genotypes but is most highly expressed in Inha-/- tumors. Up-regulation of Gata4 and loss of Gata6 expression is concomitant with loss of other adrenocortical-specific markers including the melanocortin-2 receptor (Mc2r), 21-hydroxylase (Cyp21a1), and aldosterone synthase (Cyp11b2). B, Quantitative analysis of Gata4 and Gata6 transcript abundance was performed in WT adrenals after gonade- comty and Inha-/- adrenocortical tumors. Levels of abundance are displayed as a percentage of Arbp, with error being calculated by the standard deviation of triplicate measurements from four separate tissue samples for each genotype. Inha-/- mice display both an increase in Gata4 expression and a decrease in Gata6 expression compared with WT mice, consistent with the semiquantitative data for these tissues. C, Protein levels of Gata4 and Gata6 were measured by immunoblotting from adrenal and adrenal tumor lysates. Whereas Gata6 is broadly expressed in adrenal tissue across genotypes, it is either absent or weakly detectable in Inha-/- adrenocortical tumors. Gata4 protein is exclusively detected in Inha-/- adrenocortical tumors and is not found at measurable levels in any adrenal lysates. D, Expression of ovary and testis-specific markers were analyzed by immunoblotting to determine the identity of male Inha-/- adrenocortical tumors. These tumors express high levels of Gata4, Lhr and P450aromatase, and lack expression of Sry, consistent with an ovarian phenotype in both male and female adrenocortical tumors.
that discriminate between the male and female gonad (Fig. 4D). Whereas both Gata4 and Lhr are normally found in the testis and ovary, they are more highly ex- pressed in the latter. Male Inha-/- testicular and adre- nocortical tumors show increased expression of both of these proteins, consistent with a switch toward a more ovarian phenotype. An even more specific ovarian marker, P450 aromatase’ is also abundant in male Inha-/- tumors, which is expected given their high level of es- trogen production. In contrast to these markers, the Sox- family transcription factor Sry is only found in the male testis, and is normally absent from both the ovary and adrenal cortex. Both testicular and adrenocortical tu- mors from male Inha-/- mice lack expression of Sry,
confirming that these tumors adopt an ovarian cellular identity as opposed to a testicular identity. These find- ings are interesting as they suggest a role for inhibin not only in the regulation of cellular proliferation, but also in the cell fate determination of bipotential gonadal progen- itor cells.
Expression of Gata4 in the Adrenal Cortex Is Limited to the Subcapsular Zone
Our inability to detect significant levels of Gata4 protein by immunoblotting in any adrenal tissue besides Inha-/- tumors (Fig. 4C), even when mRNA transcripts are present (Fig. 4A), suggests either that very little pro-
tein is being produced, or that its production is limited to a very small subset of cells in the adrenal that represent only a fraction of the total cellular mass. To differentiate between these two possibilities, we performed immuno- histochemical staining for Gata4 on paraffin sections of adrenals from wild-type, LH-CTP and Inha-/- mice be- fore and after gonadectomy. Consistent with our RT- PCR analysis, Gata4 expression was not detected by immunohistochemistry in the adrenal cortex of wild-type or Inha-/- mice before gonadectomy, although it was weakly expressed in a narrow rim of subcapsular adre- nocortical cells in LH-CTP mice (Fig. 5A). Gata4 expres- sion was more prominent in all three genotypes after gonadectomy and was similarly restricted to the subcap- sular zone of the adrenal (Fig. 5A). Because this popu- lation of cells composes only a fraction of the entire
cortex, it is likely that we cannot detect Gata4 protein in the adrenal by immunoblotting because it is diluted be- yond the limit of detection in the total protein mass. Interestingly, cellular proliferation in the adrenal cortex is restricted to the same subcapsular zone where Gata4 expression was detected, suggesting that the adreno- cortical progenitor cells that reside in this region may be the cellular targets of gonadotropin signaling, which eventually become Gata4 positive under chronic stimu- lation (58, 59).
Gata6 Is Excluded from the Adrenal Tumor Tissue of Inha-/- Mice
Previous reports of Gata4 expression in adrenocortical tumors have indicated that activation of Gata4 occurs
A
pre-GDX
WT
LH-CTP
Inh-/-
pst-GDX
WT
LH-CTP
Inh-/-
B
anti-Gata4
anti-Gata6
1
TC
IC
WT ovary
GC
GC
Inh-/- adrenal tumor
P
P
S
S
9
coincident with loss of Gata6 expression, which is normally found throughout the adult adrenal cortex (16, 29). To determine whether this was also true of Inha-/- adrenocortical tumors, we performed immu- nofluorescent staining for Gata6 and Gata4 in adreno- cortical tumors. Similar to studies in other tumor mod- els, we found that Gata6 was excluded from the parenchymal tissue of Inha-/- adrenocortical tu- mors, although it appeared to be retained in the ad- renal-derived stromal tissue surrounding these clus- ters of proliferative tumor cells (Fig. 5B). Parenchymal tumor cells that were Gata6 negative instead ex- pressed Gata4, whereas Gata6-expressing stomal cells did not. These data are supported by quantitative RT-PCR and immunoblot analyses comparing tumor tissue to wild-type adrenal tissue, which show de- creases in both Gata6 mRNA and Gata6 protein in tumors, and dramatic increases in the presence of both Gata4 mRNA and protein (Fig. 4, A-C). These data imply that the conserved phenotype among the several mouse models of adrenal tumorigenesis is commonly induced by gonadectomy, despite the dis- parate genetic mechanisms by which the tumors arise.
The expression pattern of Gata4 and Gata6 may also be relevant with regards to the cellular identity of Inha-/- adrenocortical tumors because the rodent ovary displays cell-specific staining for these factors. In the growing follicle, both GC and TC broadly ex- press nuclear Gata4, which can be seen from immu- nufluorescent staining in ovarian tissue sections, whereas Gata6 expression appears to be tightly re- stricted to ovarian interstitial cells (Fig. 5B). The fact that the majority of follicular cells express only Gata4-and not Gata6-suggests that not only have Inha-/- adrenal tumor cells adopted a mixed follicu- lar identity, but that they have done this at the expense of adopting an adrenocortical identity. This change in cellular identity is additionally demonstrated by the decrease or loss of adrenal-specific gene expression from Inha-/- adrenocortical tumors, including the melanocortin-2 receptor (Mc2r), 21-hydroxylase (Cyp21a1), and aldosterone synthase (Cyp11b2) (Fig. 4A). Gata4/Gata6 switching therefore suggests a com- plete identity change of adrenocortical progenitors to- ward a gonadal cell fate as opposed to a superim- posement of the gonadal fate onto adrenocortical cells.
Gata4 Is Sufficient to Activate the Transcription of Both Theca and Granulosa-Specific Markers in Adrenocortical Cells
The mutually exclusive pattern of gene expression found for Gata4 and Gata6 in the gonad and adrenal suggests that these two closely related transcription factors may interact with one another at the transcrip- tional level by means of direct or indirect repression. To address this possibility, we stably expressed mouse Gata4 in the mouse Y1 adrenocortical cell line, which does not express endogenous Gata4 at any
appreciable level (Fig. 6A). After screening several monoclonal lines of stably transfected cells, we chose a single monoclonal line designated Y1-G4 for further studies. Y1-G4 cells show a dramatic increase in Gata4 mRNA, and display significant levels of Gata4 protein as well (Fig. 6A). Interestingly, these cells also show a 2- to 3-fold decrease in Gata6 mRNA com- pared with normal Y1 cells, suggesting that Gata4 may repress Gata6 expression, either directly or indirectly. Because the transcriptional repression was minimal and did not grossly affect Gata6 protein levels, how- ever, it is likely that other genetic mechanisms than direct transcriptional repression of the Gata6 promoter account for the mutually exclusive expression of Gata4 and Gata6 in the ovary and in Inha-/- adre- nocortical tumors.
Y1 cells represent an exclusively adrenocortical cell lineage and are therefore not expected to express transcripts that specifically characterize steroidogenic cells of the gonad (60). Because Gata4 is coordinately expressed with several GC and TC-specific genes in Inha-/- adrenocortical tumors and is known to reg- ulate expression of several of these genes in the con- text of gonadal cells, we asked whether overexpres- sion of Gata4 is sufficient to activate expression of these genes in the context of an adrenocortical cell. To answer this question, we compared the expression of genes that are exclusively found in the ovary between Y1 and Y1-G4 cells.
In their endogenous state, Y1 cells fail to express Cyp17, although-like the normal adrenal cortex- they do express other TC-specific genes including Set. Y1-G4 cells express comparable levels of Set, but unlike Y1 cells, they also express Cyp17 (Fig. 6B). All of the GC-specific genes we tested-including Cyp19, Esr2, Amh, and Inhbb-follow the same pattern as Cyp17 and are absent from Y1 cells, but are expressed in Y1-G4 cells (Fig. 6B). Expression of both sets of genes in this system is clearly independent of gonad- otropins because neither Lhr nor Fshr can be detected in Y1 or Y1-G4 cells. These data indicate that Gata4 expression likely lies downstream of gonadotropins and is sufficient to drive expression of several ovary- specific genes in an adrenocortical cell line, most im- portantly those required to stimulate estrogen biosyn- thesis. In contrast to the loss of adrenal markers in Inha-/- adrenocortical tumors, which demonstrate a complete switch in Gata6/Gata4 expression, the Y1-G4 cells retain expression of Gata6 (Fig. 6A) and adrenal-specific markers such as Mc2r (data not shown). This limited data set therefore implies that both the loss of Gata6 and concomitant gain of Gata4 are required for phenotypic switching between adrenal and gonadal cellular identity.
DISCUSSION
Previous reports have demonstrated that adrenocor- tical tumors from Inha-/- mice produce high levels of
A
Y1 Y1-G4
B
Y1
Y1-G4
+
+
RT
Gata6
Gata4
Inha
Gata4
Nr5a1
B-actin
Gapdh
Lhr
Percent Expression
25
Cyp17
20
TC
15
Gata4
Set
10
Gata6
Fshr
5
Cyp19
Y1
Y1-G4
Inhbb
GC
Amh
Esr2
estrogen, and that these tumors both respond to and require pituitary gonadotropins for their initiation and subsequent growth (37, 38). In this study, we show that the cellular phenotype of these tumors is not limited to the expression of estrogenic enzymes and includes an array of genes that are normally restricted to the gonad. This array includes genes that are spe- cific markers of distinct somatic cell types of the ovar- ian follicle, suggesting that a global change in cellular identity has occurred in Inha-/- adrenal tumor cells. The global change in cell identity from an adrenocor- tical to gonadal phenotype in tumors is centrally char- acterized by loss of Gata6 expression and transcrip- tional activation of Gata4 expression, which is normally restricted to steroid-producing cells of the gonad. Gata4 not only characterizes the aberrant specification of gonadal cells in the adrenal but is also a vital mediator of this process, as evidenced by the
sufficiency of Gata4 to drive expression of a large panel of gonad-restricted genes in an adrenocortical cell line.
Both the expression of Gata4 and the broader go- nadal phenotype of Gata4-positive cells in the adrenal cortex have important implications for the mechanism of inhibin action in the adrenal. The inhibin-a gene- which is genetically disrupted in Inha-/- mice-con- tains specific Gata4 binding sites within its promoter, and has been shown to be directly activated by Gata4 in several in vitro studies (27). This transcriptional in- teraction is critical in the context of ovarian follicles, where Gata4 mediates a negative feedback loop that is initiated by gonadotropins and ends with the pro- duction of bioactive inhibin from follicular GC. Release of inhibin from the follicle serves two critical roles in the control of follicular growth: 1) endocrine negative feedback to the pituitary gonadotropes to down-reg-
ulate activin-stimulated production of FSH, and 2) au- tocrine/paracrine feedback to follicular GC to directly inhibit cellular growth (61). Whereas the first of these two roles has been well understood for some time, the second role is more recently inferred from mouse ge- netic models, which have shown that simple overex- pression of FSH is not sufficient to stimulate gonadal tumorigenesis (49). Precisely how inhibin prevents cel- lular growth in the gonad is unclear at this time, al- though recent studies have indicated that inhibin can interfere with multiple TGFß family signaling pathways, suggesting that the mechanism may be more complex than previously thought (62, 63). Whatever the mech- anism may be, however, it is clear that inhibin is ab- solutely required to directly prevent overgrowth of fol- licular cells. The fact that Inha-/- adrenal tumor cells have essentially adopted the identity of follicular cells strongly suggests that the tumor suppressive mecha- nism inhibin employs is conserved between these tissues.
Within the adrenal cortex, Gata4 expression occurs only in the context of chronic gonadotropin signaling and is restricted to a narrow subset of cells. The spatial localization of Gata4 to the subcapsular region of the adrenal cortex by immunofluorescence is of particular interest because all cellular proliferation in the adrenal cortex is restricted to the same subcapsular zone. Because the adrenal cortex continuously renews all of its distinct zonal compartments from subcapsular tis- sue, it is commonly thought that this zone contains a distinct population of cells that are both pluripotent and self-renewing-key hallmarks of a somatic stem cell (58, 59). Although it is unclear from our data pre- cisely what happens to Gata4-expressing cells in the wild-type adrenal cortex, their spatial restriction to the subcapsular zone suggests two possibilities: 1) These cells represent a transient cell type that is respecified as a normal adrenal cell upon migration out of the subcapsular zone, or 2) they are eliminated by apo- ptosis to prevent improper respecification of the ad- renal cortex. We favor the latter hypothesis, as the analogous Gata4 expressing cells of the ovarian folli- cle are eventually eliminated by apoptosis during atre- sia or after complete luteinization, although more stud- ies are needed to confirm this hypothesis.
In contrast to wild-type adrenal cells, Inha-/- ad- renal cells that express Gata4 grow and proliferate outside of the subcapsular zone, forming adrenocor- tical tumors with a distinct ovarian identity. Because Gata4 expression and adrenal tumorigenesis parallel one another in Inha-/- mice, it seems likely that tu- mor cells initially arise as Gata4 expressing cells in the subcapsular zone-as in the normal adrenal-but fail to be eliminated or properly respecified upon exit from the subcapsular zone due to the absence of inhibin. The improper specification and survival of these cells therefore requires two convergent events in a single cell type: 1) chronic exposure to elevated levels of gonadotropins and 2) the loss of inhibin. Because the gonadal phenotype of adrenocortical tumor cells re-
quires that they must arise from a developmentally plastic adrenocortical cell, these data suggest the ex- citing possibility that Inha-/- tumors are actually composed of improperly specified adrenocortical stem cells that proliferate continuously due to loss of growth control.
With respect to tumor phenotype, the Inha-/- mouse adrenocortical tumor model demonstrates striking similarities to other mouse models of adreno- cortical tumorigenesis, including strains of mice that bear the SF1S172 allele (e.g. CE, DBA/2J, C3H, and BALB/c) and inhibin-« promoter/ simian virus 40 T- antigen mice (Inha/Tag) (32-34). All of these models share a common requirement for gonadectomy-in- duced tumor initiation, as well aberrant expression of Gata4 and production of sex steroids-specifically es- trogens. A closer examination, however, shows that these adrenocortical tumor models can be further sep- arated into two distinct groups, namely 1) those that bear the SF1172 allele do not. In addition, mice bearing the SF1$172 allele display a distinct pattern of adrenal tumor progression that is not reproduced in the Inha-/- and Inha/Tag mice. In this pattern, adreno- cortical tumors develop from a distinct population of subcapsular, nonsteroidogenic, spindle-shaped cells (A-cells) that subsequently give rise to large, lipid-filled steroidogenic cells (B-cells) that compose the actual tumor (33). Tumors in Inha-/- and Inha/Tag mice appear to be histologically distinct from either A- or B-cells and arise as a single population of epitheloid cells that most resemble sex steroid-producing cells of the human fetal adrenal or adrenocortical blastomas (64). Whereas adrenocortical tumors in the Inha-/- model were not previously associated with the sub- capsular zone of the adrenal, we provide evidence here that cells from this zone give rise to the tumors in Inha-/- mice based on their common expression of Gata4. These data support the notion that all sex- steroid producing adrenocortical tumors in the various models discussed above arise from a common pluri- potent progenitor cell in the subcapsular region of the cortex, despite the divergence in molecular events that lead to loss of cellular growth regulation in these tumors.
We propose that the many similarities between Inha-/- and Inha/Tag mice exist because loss of cellular growth regulation occurs at the same time during cellular differentiation in these two models. In both cases, adrenal tumor onset is stochastic after gonadectomy and likely requires the ability of adrenal stem/progenitor cells to respond to LH. Because LH has been previously reported to drive the expression of its own receptor (LHR), it is possible that respon- siveness of adrenal stem/progenitor cells to LH in- creases progressively under chronic LH stimulation (57). As these cells gradually increase LHR expression,
they may eventually reach a threshold at which key transcriptional regulators of the gonadal fate-includ- ing Gata4-are induced. Although FSH is normally the key regulator of Gata4 expression, our data above show that LH can activate Gata4 expression in re- sponsive cells, albeit more weakly. This assertion is supported by genetic data from Inha-/- mice crossed to FSH-deficient mice, which still develop tumors, although more slowly than Inha-/- mice (49). Once Gata4 is induced, one of its transcriptional tar- gets is the inhibin & promoter. As discussed above, inhibin acts as a suppressor of cellular growth by both direct and indirect mechanisms, and in its absence, improperly specified adrenal stem/progenitor cells progress to form tumors. Although Inha/Tag mice con- tain normal expression of inhibin &, they also begin to drive expression of tumorigenic T-antigen at the same time inhibin & expression is activated (34). Studies with cells derived from transgenic mice that express tem- perature-sensitive T-antigen (the H-2Kb-tsA58 immor- tomouse) show that activation of T-antigen essentially immortalizes cells in their present differentiated state, and therefore the Inha/Tag adrenal cells become im- mortalized in the same state of abnormal differentia- tion as the cells that do not express inhibin (65). As such, we would suggest that the Inha-/- and Inha/ Tag mice represent complementary models of the same tumorigenic process, driven by disparate mech- anisms of cellular growth control.
The suggestion that adrenocortical tumors in Inha-/- and Inha/Tag mice arise from improperly specified adrenocortical stem/progenitor cells is also supported by analogous human conditions. Several cases of thecal metaplasia of the adrenal (adrenal luteomas) have been reported in post-menopausal women who, due to decreased ovarian estrogen out- put, have chronically elevated levels of gonadotropins (66-68). Similar to the tumors found in Inha-/- mice, these tumors depend on LH for growth and mainte- nance, and produce sex steroids rather than other adrenal hormones. In addition, a subset of human adrenocortical tumors has been reported to demon- strate a genetic switch from the predominant expres- sion of Gata6, which is normally found in the cortex, to Gata4, which is normally absent from the cortex (29). Although this report did not correlate this switch to steroidogenic output or tumor phenotype, the pres- ence of Gata4 strongly suggests that these tumors may have adopted a more gonadal fate. Other reports detail gonadotropin-responsive adrenocortical tumors that illicitly express LHR, although in these cases ini- tiation of the tumor was not likely due to elevated gonadotropins and resulted in tumors that produced normal adrenal steroids and not estrogens (69-71).
Although the human cases noted above support the presence of a plastic cell population in the adrenal, case reports on patients affected with congenital ad- renal hyperplasia suggest that than an analogous, plu- ripotent population of cells might also exist in the gonad. Patients suffering from congenital adrenal hy-
perplasia lack the ability to produce adrenal cortisol, and therefore have chronically elevated ACTH levels due to the lack of negative feedback regulation on pituitary corticotrope cells. Years of exposure to ele- vated levels of ACTH in these patients often results in the acquisition of hormonally active adrenal tissue in the gonad termed adrenal rests (72, 73). Although some studies have attributed the existence of these rests to adrenocortical metastasis, this seems unlikely because the adrenal overgrowth in these patients does not represent transformed cancer tissue, but rather benign hyperplasia. We suggest instead that the gonad also contains a population of plastic somatic cells, which are able to adopt an ectopic adrenocor- tical identity under chronic stimulation from pituitary ACTH. The possibility that both the adrenal and gonad contain populations of cells able to transdifferentiate into cells of the opposite tissue is particularly credible given their common developmental origin in the uro- genital ridge. Formal identification and isolation of these hypothetical cells has not yet been accom- plished, however, and will likely be an area of increas- ing interest given the therapeutic potential these cells hold for various degenerative conditions of the adrenal and gonad.
MATERIALS AND METHODS
Experimental Animals
All experiments involving animals were performed in accor- dance with institutionally approved and current animal care guidelines from University of Michigan Committee on Use and Care of Animals. Generation and genotyping of mice with a targeted deletion of the @-inhibin gene (Inha-/-) and mice harboring the bLH-BCTP transgene (LH-CTP) under the con- trol of the bovine LH a-subunit promoter have been de- scribed previously (35, 56). Surgical gonadectomies were performed on mice at the age of 3-5 wk and mice were euthanized at 20 wk or more after gonadectomy as indicated. Only female mice were used for this study because gona- dectomized female Inha-/- mice develop adrenocortical tu- mors more rapidly than their male counterparts.
RT-PCR and Real-Time PCR
Tissues were removed, cleaned, and snap frozen as above. Frozen tissues were lysed in Trizol reagent using an electric tissue homogenizer, and total RNA was collected according to the manufacturer’s recommended protocol. In the case of cultured cells, the total cell pellet was washed once with PBS, and then directly harvested in Trizol reagent with vigorous pipetting to homogenize the cellular lysates. Total RNA was treated with deoxyribonuclease (Ambion, Austin, TX) to re- move any residual genomic DNA, and was quantified by UV spectrometry. One microgram of total RNA was used to synthesize cDNA using the iScript kit (Bio-Rad, Hercules, CA) according to the manufacturer’s recommended protocol. The final cDNA product was purified and eluted in 50 ul of Tris- EDTA buffer using PCR purification columns (QIAGEN, Va- lencia, CA). One microliter of this product was used as tem- plate for all subsequent semiquantitative PCRs. Primer sequences and thermocycling conditions for each gene are shown in Table 1. Amplified products were resolved on 1-2%
| Gene/Transcript Name | Abbreviation | Sense Primer | Antisense Primer | Annealing Temperature (C) | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Inhibin-a | Inha | GGT GAA | GGC | TCT | ATT CCT AGA TG | GAT GAT | AGC | ACC | AGA | AGA TCT AG | 56 |
| Steroidogenic factor-1 | Nr5a1 | GCA TTA CAC GTG | CAC CGA G | GGC TCT | AGT | TGC AGC AGC TG | 60 | ||||
| Glyceraldehyde | Gapdh | GTG GCC CCT GGT | GAG TGG | CAG GCT | ATG | GCT TGA TGA GG | 60 | ||||
| phosphate dehydrogenase | |||||||||||
| LH receptor | Lhr | CTG CTG TGC | TTT | CAG GAA TTT GCC | ACC CTA | AGG | AAG GCA | TAG CCC ATA | 60 | ||
| P450c17@[p] | Cyp17 | CAA CTG CAG TGA | TTG TCG GTC | GGT CTG | TAT | GGT AGT CAG TAT CG | 55 | ||||
| Progesterone receptor | Pr | TGT CAC TAT GGC | GTG CTT ACC TGT | TCA TGC | CCT | GCA TAG ACC ACA TCA | 60 | ||||
| Se. translocation | Set | AAG CTG GAG GAC | AAG TCG GC | CAA GCA | GTG | CAG ACA CTT GTG GAT | 60 | ||||
| Relaxin-like Factor | Rif | TGA CAG TCT GGG | TTC TGT GAG TGT | GAA TTG | CTG | TGA GTT GGA GGC AGT | 60 | ||||
| Aminopeptidase-N | Anpep | CAA GAA AGT GGT | GGC TAC AAC GCA | ACC ATT | GGC | TGA GAC GGA GCT TAT | 60 | ||||
| Relaxin | Rlx | AGT GGA TGG ACG | GAT TCA TTC GGA | AAC ACA | GGT | GCG TGT TGT AGC TCT | 64 | ||||
| FSH receptor | Fshr | AAG GTC TAT TCC | CTG CCC AAC CAT | CTG GGT | TCA | TCA TCT ACG AGA GAG | 57 | ||||
| P450aromatase | Cyp19 | GAC ACA TCA TGC | TGG ACA CC | CAA AGC | CAA | AAG GCT GAA AG | 55 | ||||
| Inhibin/activin-BA | Inhba | GAG ATC GTA GAG | GCT GTC AAG | CAC TTC | TGC | ACG CTC CAC TAC | 56 | ||||
| Inhibin/activin-BB | Inhbb | GGT GAA GAG | ACA | CAT CTT GAG C | CCT GTT | CTT | GGA AGT ACA CCT TG | 56 | |||
| Mullerian-inhibiting substance | Amh | GGT CTG AAC | AGC | TAT GAG TAT GCC | CTC TGC | TTG | GTT GAA GGG TTA AG | 56 | |||
| Estrogen receptor-ß | Esr2 | TGA ACT ACA | GTG | TTC CCA GCA GCA | ATA ATC | ACT | GCA GAC | GGC GCA GAA | 60 | ||
| Oxytocin | Oxt | TTG CTG CCT GCT | TGG CTT ACT | AAG GCA | GGT | AGT TCT CCT CCT GG | 64 | ||||
| GATA binding protein-4 | Gata4 | ATT CTA GTT | CTT | CTC TGC CTC GT | TGC CTT | CTG | AGA AGT CAT CAA AC | 60 | |||
| GATA binding protein-6 | Gata6 | TGT ACC AGA | CCC | TCG CC | CAG ACC | AGG | GCC AGA GC | 56 | |||
| Melanocortin-2 receptor | Mc2r | TTC TCA GCA | CCA | CAA ATG ATT C | TCT TTG | TGT | GGA AGG ATC TGG | 60 | |||
| 21-hydroxylase | Cyp21a1 | CTG CTT CAC CAC | CCT GAG A | AGC TGC | ATT | CGG TTC CTG T | 60 | ||||
| Aldosterone synthase | Cyp11b2 | AGA AGT TGC ACC | AGG TGG AGA GTA | TGG TGA | CAG | CAC ATT TCG GTT CAG | 60 | ||||
| GATA binding protein-4 (q-PCR) | Gata4 | AGA TGG GAC | GGG | ACA CTA CCT | ACA GCG | TGG | TGG TGG TAG TCT | 60 | |||
| GATA binding protein-6 (q-PCR) | Gata6 | CAA AAG CTT | GCT | CCG GTA ACA | GGT CGC | TTG | TGT | AGA | AGG AGA AG | 60 | |
| Acidic ribosomal phosphoprotein (q-PCR) | Arbp | CGT GAT GCC | CAG | GGA AGA | TCC CAC | AAT | GAA | GCA | TTT TGG | 60 | |
q-PCR, Quantitative PCR.
agarose gels and detected with ethidium bromide staining on a UV lamp.
For quantitative, real-time PCR analysis of mRNA tran- script abundance, PCRs were made up using a 2× SYBR Green PCR mastermix (Applied Biosystems, Foster City, CA) along with gene-specific primers, and thermocycling was performed in the ABI 7300 thermocycler system (Applied Biosystems). Each quantitative measurement was normal- ized to Rox dye as an internal standard and performed in triplicate. Transcript abundance was normalized to the aver- age cycle threshold value of mouse acidic ribosomal phos- phoprotein (Arbp) for each sample. For mRNA quantitation directly from adrenal or tumor tissue, measurements were performed with an n ≥4, with n being the number of individual tissue samples from different animals.
Immunoblotting
Ovaries and adrenals were removed, cleaned of surrounding adipose and connective tissue under a microscope in PBS, and immediately snap frozen in liquid nitrogen. Protein ly- sates were collected by briefly sonicating frozen tissues in lysis buffer (40 mm HEPES, 120 mm sodium chloride, 10 mm sodium pyrophosphate, 10 mm sodium glycerophosphate, 1 mM EDTA, 50 mm sodium fluoride, 0.5 mm sodium orthovana- date, 1% Triton X-100) containing protease inhibitor cocktail (Sigma, St. Louis, MO), followed by 1-h rotation at 4 C to
solublize proteins. Soluble protein was collected from cen- trifuged total lysates and quantified by Bradford assay. SDS- PAGE was performed on 9-10% polyacrylamide Tris-glycine gels loaded with 20-40 µg of protein per sample, and sepa- rated proteins were transferred to nitrocellulose membranes using a semidry transfer unit. After transfer, membranes were blocked 1 h in 4% nonfat dry milk in TBS/0.1% Tween 20 (TBST), and then incubated overnight at 4 C with primary rabbit antibodies against Gata4 (1:1000; Santa Cruz Biotech- nology, Inc., Santa Cruz, CA), Gata6 (1:1000; Santa Cruz), P450aromatase (1:1000; Abcam, Cambridge, MA), or Lhr (1: 1000; Sigma), or primary mouse antibodies against ß-actin (1:5000; Sigma) or Sry (Abcam; 1:1000). The next day, mem- branes were washed three times for 5-10 min in TBST, and then incubated with horseradish peroxidase-labeled goat an- tirabbit or antimouse secondary antibody for 1 h at room temperature. After three more washes in TBST, membranes were incubated for 2-5 min in West Dura ECL reagent (Pierce, Rockford, IL), and then exposed to film for detection.
Immunohistochemistry
Ovaries and adrenals were collected at indicated ages and treated as prepared for staining as above, except that final rehydration steps were performed in Tris-buffered saline (TBS, pH 7.5) instead of PBS. Antigen retrieval was per- formed by boiling rehydrated sections in 1 mm EDTA or 10
mM sodium citrate for 15 min, followed by two washes in TBS at room temperature. Tissue sections were then blocked in antibody diluent solution (TBS, 5% BSA, 0.1% Tween 20) containing 5% normal goat serum for 30 min, and incubated with anti-LHR (1:200; Sigma), anti-MIS (1:50; Abcam), anti- Gata4 or anti-Gata6 primary antibodies (1:50; Santa Cruz) overnight at 4 C in a hydration chamber. The next day, sections were washed three times for 5 min in TBS and then exposed to biotinylated secondary antibodies for 30 min at room temperature. When costaining for multiple epitopes was performed, direct fluorophore-coupled secondary anti- bodies were used. After three wash steps, sections were incubated with streptavidin-fluorescein isothiocyanate or streptavidin-rhodamine for 15 min at room temperature to fluorescently label stained tissue. Sections were finally washed three times more with TBS, nuclear costained with 4’,6-diamidino-2-phenylindole, and coverslipped with aque- ous mounting media (Biomeda, Foster City, CA). Immunohis- tochemistry performed on adrenal sections was detected using the Vectastain ABC kit (Vector Laboratories, Burlin- game, CA) and diaminobenzidine staining due to the high autofluorescent background of the adrenal cortex. Bright- field and fluorescent images were captured with an RT-Slider digital camera and Spot (version 4.6) software (Diagnostic Instruments, Sterling Heights, MI).
Y1 Cell Culture and Stable Transfection
Y1 mouse adrenocortical cells obtained from ATCC (Manas- sas, VA) were maintained in DMEM supplemented with 7.5% horse serum, 2.5% bovine serum, penicillin, streptomycin, and fungizone (Invitrogen, Carlsbad, CA). The coding se- quence for mouse Gata4 was subcloned into pCDNA3, lin- earized by restriction digest, and transfected by calcium phosphate precipitation into Y1 cells. Stable transfectants were selected with G418 for 2-4 wk and then subcultured individually to 24-well plates. Stable monoclonal lines that were successfully expanded to 10-cm plates were screened for Gata4 expression by immunoblotting as described above. A single high expressing clone designated Y1-G4 was used for subsequent experiments to analyze the expression of various gonadal transcripts by RT-PCR as described above.
Acknowledgments
The Inha-/- strain of mice was generated and provided by Dr. Martin Matzuk (Baylor College of Medicine, Houston, TX), whereas the bLH-BCTP mice were generated and pro- vided by Dr. John Nilson (Washington State University, Pull- man, WA). The expression construct for mouse Gata4 was provided by Dr. Jeff Molkentin (Children’s Hospital, Cincin- nati, OH).
Received April 28, 2006. Accepted July 11, 2006.
Address all correspondence and requests for reprints to: Gary D. Hammer, M.D., Ph.D., BSRB 1502, 109 Zina Pitcher Place, Ann Arbor, Michigan 48109-2200. E-mail: ghammer@umich.edughammer@umich.edu.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health Grant RO1 DK62027 (to G.D.H.) and American Cancer Society Grant RSG-04-236-01-DDC (to G.D.H.).
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