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Molecular Endocrinology, doi:10.1210/me.2002-0282
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Molecular Endocrinology 18 (10): 2553-2569
Copyright © 2004 by The Endocrine Society

Adrenocortical Tumorigenesis in Transgenic Mice Expressing the Inhibin {alpha}-Subunit Promoter/Simian Virus 40 T-Antigen Transgene: Relationship between Ectopic Expression of Luteinizing Hormone Receptor and Transcription Factor GATA-4

Nafis A. Rahman, Sanne Kiiveri, Adolfo Rivero-Müller, Jérôme Levallet, Susanna Vierre, Jukka Kero, David B. Wilson, Markku Heikinheimo and Ilpo Huhtaniemi

Department of Physiology (N.A.R., A.R.-M., J.L., S.V., J.K., I.H.), University of Turku, 20520 Turku, Finland; Program for Developmental and Reproductive Biology, Biomedicum Helsinki and Children’s Hospital (S.K., M.H.), University of Helsinki, 00029 Helsinki, Finland; Departments of Pediatrics (D.B.W., M.H.), and Pharmacology (D.B.W.), Washington University, St. Louis, Missouri 63110; and Institute of Reproductive and Developmental Biology (I.H.), Imperial College London, Faculty of Medicine, London W12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Ilpo Huhtaniemi, M.D., Ph.D., Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail: ilpo.huhtaniemi{at}imperial.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have analyzed the ontogeny and putative mechanisms of transregulation of LH receptor (LHR) and transcription factor GATA-4, coexpressed during the adrenocortical tumorigenesis of prepubertally gonadectomized transgenic (TG) mice expressing the inhibin {alpha}-subunit promoter/simian virus 40 T-antigen (inh{alpha}/Tag) transgene. The onset of adrenal LHR mRNA and protein expression coincided with that of GATA-4 at the age of 4 months and preceded the appearance of discernible adrenal tumors at about 6 months. In situ hybridization and double-immunohistochemistry demonstrated colocalization of the LHR and GATA-4 messages and proteins in the adrenal cortex. A GATA-4 expression plasmid cotransfected with a murine LHR promoter-driven luciferase reporter plasmid, containing a consensus GATA-binding site, induced a dose-dependent significant transactivation of the LHR promoter in nonsteroidogenic human embryonic kidney 293, steroidogenic murine mLTC-1 Leydig cells and in murine adrenal Y-1 cells. The C{alpha}1 cells derived from an Inh{alpha}/Tag adrenal tumor did not show this response, apparently due to their high endogenous GATA-4 expression. However, an additional link between GATA-4 and LHR in C{alpha}1 cells was provided upon the LH/human chorionic gonadotropin stimulation of LHR promoter activity; mutations or deletion of the consensus GATA-4 binding site of the LHR promoter abolished this transactivation. EMSAs further proved GATA-4 binding to the putative consensus GATA recognition site. Our results demonstrate direct interrelationship between LHR and GATA-4 expression during adrenocortical tumorigenesis of the inh{alpha}/Tag mice. There is apparently a positive and reciprocal feed-forward amplification link between LHR and GATA-4 expression. This mechanism gradually and in synergy with Tag expression leads to formation of the LH-dependent adrenocortical tumors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ADRENOCORTICAL TUMORS ARE rare malignancies with poor prognosis because they are often diagnosed late and are usually resistant to chemotherapy (1). The lack of suitable animal models for adrenocortical tumors has been a major obstacle for unraveling the molecular mechanisms involved in their pathogenesis, as well as for improving their diagnostic and therapeutic strategies. We have earlier described a gonadotropin-responsive adrenal tumor, which provides a good experimental model for the study of this malignancy (2). In this transgenic (TG) model, the mice harbor an inhibin {alpha}-subunit promoter/simian virus 40 T-antigen (inh{alpha}/Tag) fusion gene. The TG mice were originally found to develop gonadal tumors (3, 4), but when gonadectomized prepubertally, adrenocortical tumors appeared with 100% penetrance by the age of 5–6 months (2). The adrenal tumors, and a cell line (C{alpha}1) derived from them, were found to express a high level of LH receptor (LHR), and their growth and steroidogenesis were dependent on LH stimulation (5, 6). The post-castration elevation of LH levels is apparently crucial for the induction of LHR expression, which together with Tag expression triggers formation of the adrenocortical tumors (6). The tumors failed to appear if the post-castration increase in gonadotropins was blocked either by treating the mice with a GnRH antagonist or by crossbreeding them to the gonadotropin-deficient hpg genetic background (7, 8). LHR expression in the mouse adrenal gland is an exceptional condition because LHR are not normally found in wild-type (WT) mouse adrenal glands (5, 9, 10). Some recent studies have reported LHR expression in normal human adrenal glands (11). However, we have observed that, in addition to the gonadectomized inh{alpha}/Tag TG mice (5), LHR expression in the mouse adrenal gland can be induced by chronically elevated LH levels (9). In the inh{alpha}/Tag TG mice, we suggested that adrenal expression of the potent oncogene, simian virus 40 T-antigen, permitted the elevated LH levels to function as tumor copromoter (5).

GATA-4 and GATA-6 belong to the family of zinc-finger transcription factors involved in the regulation of gene expression and cell proliferation and differentiation in a variety of tissues. Within the endocrine system, GATA-4 and/or GATA-6 are expressed in the ovary (12), testis (13), adrenal (10), and in hypothalamic GnRH neurons (14). Distinct functions have been demonstrated for GATA-4 and GATA-6 in the regulation of gene expression and cellular differentiation within the testis and ovary (12, 13, 15). In the testis, abundant expression of GATA-4 has been shown in proliferating Sertoli cells (13). In the ovary, GATA-6 expression is present in late antral follicles and luteal cells, whereas GATA-4 expression is localized in granulosa cells of primary and early antral follicles (12). Gonadotropin stimulation in vitro up-regulates GATA-4 expression, but not that of GATA-6, in granulosa and Leydig cells (12, 13). It has also been shown that, in androgen resistance, GATA-4 expression in human Sertoli cells is weak or totally absent (15). On the other hand, GATA-4 and GATA-6 are abundantly expressed in human Sertoli and Leydig cell tumors as well as those of granulosa and theca cells, suggesting a role for them in tumorigenesis and tumor progression in somatic cell-derived human gonadal neoplasms (15, 16). We have recently shown abundant GATA-6 but no GATA-4 expression in adult murine adrenal glands (10). Conspicuously, upon malignization of the adrenal cortex in inh{alpha}/Tag mice, GATA-6 expression disappears, whereas that of GATA-4 is highly up-regulated (10). There are also preliminary data on a similar phenomenon with GATA-4 up-regulation in human adrenal tumorigenesis (10), whereas both factors are expressed in the fetal adrenal cortex (17).

The findings of our previous studies indicate that there appear to be two simultaneous functional changes in the inh{alpha}/Tag mouse adrenal glands during the process of their malignization, the up-regulation of LHR and GATA-4 expression. We now hypothesized that establishment of the causal relationship between these two events would increase our understanding of the molecular mechanisms involved in the pathogenesis of adrenocortical tumors of the inh{alpha}/Tag mouse model.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Appearance of Adrenal Tumors in the inh{alpha}/Tag Mice
To induce the adrenal tumorigenesis in the inh{alpha}/Tag mice, gonadectomies were undertaken at the age of 21–24 d postpartum. When tumorigenesis was followed by adrenal gland weights, a clear increase from those measured in intact and gonadectomized WT and nongonadectomized TG mice (mean weights 8.2–11.1 mg) was observed at the age of 6 months, when the adrenals of the gonadectomized TG mice weighed 290 ± 69 mg (P < 0.001 vs. all other groups), which was 20- to 30-fold higher than the adrenal weights in the other groups studied. The tumor tissue was cystic and occupied the whole adrenal gland compared with sporadic hyperplastic areas in the adrenals 3 months after gonadectomy. Before this age, no clear differences were found between adrenal weights of any of the groups, in agreement with our earlier reports (2, 5).

The Onset of Adrenal LHR and GATA-4 Expression Coincides with the Appearance of Adrenocortical Tumors
To assess the relationship of the onset and progression of adrenocortical tumorigenesis with GATA-4 and LHR mRNA expression, we examined RNA extracts of the adrenal glands of gonadectomized inh{alpha}/Tag mice at 2, 4, and 6 months of age as well as those of 6-month-old intact TG mice. Northern hybridization revealed that, along with the tumor formation in gonadectomized mice, there was a concomitant appearance of GATA-4 and LHR mRNA expression in the adrenal glands at the age of 6 months (Fig. 1Go). We have shown earlier LHR (5) and GATA-4 (10) expression separately in adrenal tumors of 6-month-old gonadectomized inh{alpha}/Tag mice, and we now demonstrate their concomitant appearance.



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  		Fig. 1. Northern Hybridization Analysis of GATA-4 and LHR mRNA Expression in Adrenal Tissues of Intact 6-Month-Old TG Females, and of Gonadectomized 2-, 4-, and 6-Month-Old TG Females

Each lane contains 20 µg of total RNA. The migration of the 28S rRNAs is depicted on the right of the GATA-4 panel, and the 28S and 18S rRNAs on the left side of the LHR panel. The sizes of the different LHR mRNA splice variants (in kilobase pairs) are presented on the right. Two lanes for each type of sample are depicted. A, Shows on the top the ethidium bromide (EtBr) staining of the 28S rRNA for RNA loading control. A, Middle and lower parts show Northern hybridization for GATA-4 and LHR mRNA, respectively. B, Shows the densitometric quantification of the GATA-4 band (open bars) and the longest (7.0 kb) LHR mRNA splice variant (filled bars) in arbitrary densitometric units (mean of intact 6-month-old TG mice 100%) corrected for intensity of the 28S rRNA band. Each bar represents mean ± SEM of three independent experiments in duplicates. **, P < 0.01, (6-month-old intact vs. gonadectomized TG mice).

 
Localization of GATA-4 and LHR mRNA Expression in the Adrenal Tumors
In addition to Northern hybridization, we performed in situ hybridization analyses to localize the GATA-4 and LHR messages and to confirm the timing of their appearance along with the tumorigenesis. We have previously shown that the LHR and Tag messages and proteins are coexpressed in the adrenal cortex in adrenal tumors of inh{alpha}/Tag TG mice (2, 5). The Tag immunoreactivity was localized in the nuclei, whereas that of the LHR was strongest in the periphery of the same cells (2). In situ hybridization revealed that control adrenals from 6-month-old gonadectomized mice expressed clearly the GATA-6 message, but neither LHR nor GATA-4 mRNA were detectable (Fig. 2AGo). In contrast, clear GATA-4 and LHR mRNA expression was detectable already in the adrenals of 4-month-old gonadectomized inh{alpha}/Tag mice (Fig. 2BGo), and it became clearer at 6 months of age (Fig. 2CGo) when discernible adrenal tumorigenesis was apparent. GATA-4 and LHR mRNAs were colocalized to the same regions in serial sections of the 4-month (Fig. 2BGo) and 6-month (Fig. 2CGo) gonadectomized adrenal tissue, and as shown in 6-month gonadectomized adrenal tumor tissue in these areas, GATA-6 was clearly down-regulated (Fig. 2CGo). Moreover, RT-PCR of samples of normal and tumorous adrenal tissue, separated by laser pressure microdissection and catapulting, showed colocalization of GATA-4 and LHR messages in tumor tissue, and their absence in normal adrenal cortex (results not shown).



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  		Fig. 2. In Situ Hybridization Analysis of GATA-6, GATA-4 and LHR mRNA Expressions in Adrenal Tissue of Normal and TG Mice A, RNA in situ hybridization of a representative male WT mouse adrenal 5 months after gonadectomy. In situ results demonstrate high GATA-6 mRNA levels (B), but no GATA-4 (D) or LHR (F) mRNA expression. Bright-field views with the hematoxylin and eosin staining of the same sections for GATA-6 (A), GATA-4 (C) and LHR (E) are shown. Adrenocortical zones are marked with vertical lines as follows: z.g., Zona glomerulosa; z.f., zona fasciculata; z.r., zona reticularis; m, medulla. Bar, 100 µm. B, RNA in situ hybridization from a representative TG female mouse 3 months after gonadectomy. The open frame in A shows the location of the hyperplastic area of the adrenal cortex. In the indicated area, up-regulation of GATA-4 (C) and LHR (D) mRNA expression is detected. Bright-field view of the LHR in situ section (A and B) at two magnifications is shown for comparison. Arrowheads indicate the area with most intense LHR and GATA-4 mRNA expression. Bar, 100 µm c, RNA in situ hybridization of a TG mouse with adrenocortical carcinoma 5 months after gonadectomy. A, Shows the bright-field view, and the expression patterns of GATA-6 (B), GATA-4 (C), and LHR (D) mRNA are shown in the other panels. Arrowheads point to the tumorous area with high GATA-4 mRNA expression. Note that clear LHR expression is seen only in a part of the GATA-4 positive area. *, LHR negative tumor tissue. Bar, 100 µm.

 
RT-PCR/Southern Blot Analysis of Adrenal LHR and GATA-4 mRNA Expression
The Northern blot and in situ hybridization results did not reveal clear differences in the timing of appearance of adrenal GATA-4 and LHR mRNA expression during the process of tumorigenesis. We therefore used the more sensitive RT-PCR/Southern hybridization method to clarify the order of onset of expression of the two genes. This method clearly showed that LHR and GATA-4 expression preceded the discernible adrenal tumorigenesis by about 2 months (ages 4 vs. 6 months, respectively) in the post-gonadectomy inh{alpha}/Tag adrenals (Fig. 3Go), and both messages were present simultaneously at the age of 4 months, in line with the in situ hybridization findings. However, LHR mRNA could be found in 6-month-old gonadectomized control WT mouse adrenals and in 6-month-old nongonadectomized inh{alpha}/Tag mouse adrenals (also in agreement with former reports on LHR expression by RT-PCR) (9), when GATA-4 expression was undetectable (Fig. 3Go). In the adrenals of 6-month-old bLHß-CTP females (TG mice overexpressing a bovine LHß subunit/human chorionic gonadotropin ß-subunit C- terminal fragment fusion protein) (18), with chronically elevated LH levels, both LHR and GATA-4 expression were observed (Fig. 3Go). Neither LHR nor GATA-4 mRNA expression could be found in the adrenal glands of LHR knockout (19) mice (Fig. 3Go). Hence, these data revealed situations where LHR is expressed in the absence of GATA-4 message, but not vice versa.



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  		Fig. 3. LHR and GATA-4 mRNA Expression in Mouse Adrenal Glands by RT-PCR/Southern Hybridization

The upper part of the figure shows autoradiograms for LHR and GATA-4 detection. The lower part is a table indicating the types of tissue samples analyzed and the types of animals of their origin. Testis and spleen of WT mice were used as positive and negative controls, respectively. All other samples were from adrenal glands. + and –, Indicate whether the animals were gonadectomized prepubertally. LuRKO, LHR knockout mouse (19 ); bLHß-CTP, mouse overexpressing bovine LHß/hCGß C-terminal peptide fusion protein (18 ).

 
Colocalization of GATA-4 and LHR Proteins
The colocalization of GATA-4 and LHR expression at protein levels was studied by immunohistochemistry on the same sections. In hyperplastic adrenal cortex 3 months after gonadectomy, we could find colocalization of nuclear GATA-4 and cytoplasmic LHR immunoreactivities. The LHR protein could be detected in a subset of cortical cells, without GATA-4 expression, although also some LHR negative spindle shaped GATA-4 positive cells could be seen (Fig. 4AGo). In discernible areas of the adrenal tumor tissue, where the tumor tissue was cystic and occupied the whole adrenal gland (5 months after gonadectomy), the LHR protein expression colocalized with that of GATA-4 (Fig. 4BGo).



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  		Fig. 4. Immunohistochemistry for GATA-4 and LHR in Adrenocortical Tumors

A, Double immunohistochemistry for GATA-4 and LHR in a TG male mouse adrenal gland 3 months after gonadectomy. GATA-4 is seen as gray nuclear staining, whereas LHR staining is brown and cytoplasmic. A, Low magnification of the adrenal section. B, GATA-4 (gray) and LHR (brown) colocalize in the hyperplastic adrenocortical tissue. In addition, LHR protein is detected in a few cortical cells with no GATA-4 staining. Some spindle shaped GATA-4 positive cells are LHR negative. C, Control staining utilizing GATA-4, but no LHR primary antibody, reveals only GATA-4 positive nuclei as expected. Bar, 50 µm. B, Immunohistochemistry of an adrenocortical carcinoma from a TG male mouse 5 months after gonadectomy. Tumor tissue was cystic and occupied the whole adrenal gland. A, Most of the tumor cells are GATA-4 positive (brown). B, The majority of the cells express also cytoplasmic LHR (brown). C, Double immunohistochemistry demonstrates colocalization of GATA-4 and LHR in tumor cells. GATA-4 positivity is seen as a gray nuclear staining and LHR positivity as a brown cytoplasmic color. In A and B, hematoxylin was used as a counterstain. Bar, 25 µm.

 
Human Chorionic Gonadotropin (hCG) Stimulation Up-Regulates GATA-4 and LHR Expression in C{alpha}1 Adrenal Tumor Cells
hCG stimulation of C{alpha}1 cells resulted in a dose- dependent increase in the expression of GATA-4 and LHR messages (statistically significant at ≥ 50 µg/liter hCG) (Fig. 5AGo). These results support earlier findings on up-regulation of GATA-4 expressions by hCG/LH in Leydig and granulosa tumor cell lines (12, 13). We also stimulated C{alpha}1 cells by transfecting them with increasing doses of a GATA-4 expression plasmid (0.1–1 µg) (12) (Fig. 5BGo). Despite the slight gradual increase in GATA-4 expression in C{alpha}1 cells, no concomitant up-regulation of the LHR was found, whereas in the murine Leydig tumor mLTC-1 cells, transfection with 0.5 µg of GATA-4 expression plasmid brought about clear up-regulation of the LHR message (Fig. 5BGo). In another murine adrenal Y-1 cell line, no endogenous GATA-4 or LHR could be found (results not shown) and transfection with 0.5 µg of GATA-4 expression plasmid induced no LHR expression (Fig. 5BGo). The poor GATA-4 and LHR mRNA responses to GATA-4 stimulation in the C{alpha}1 cells could be due to either the poor transfection efficiency of these cells, and/or their high endogenous GATA-4 mRNA expression (Fig. 5BGo). The former was demonstrated by the marginal increase of GATA-4 mRNA levels after the transfection. The latter possibility was addressed by small interfering RNA (siRNA) experiments, which tentatively showed that down-regulation of endogenous GATA-4 expression in C{alpha}1 cells allowed transfected GATA-4 expression plasmid to up-regulate LHR expression (results not shown).



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  		Fig. 5. Northern Hybridization Analysis of GATA-4 and LHR mRNA in C{alpha}1, mLTC-1, and Y-1 Cell Lines, and Effects of hCG and GATA-4 on Their Expression

A, Northern hybridization analysis of GATA-4 and LHR mRNA expression in hCG stimulated C{alpha}1 cells. Each lane contains 20 µg of total RNA from C{alpha}1 cells, cultured for 24 h in the absence (0) or presence of 1, 10, 50, and 100 µg/liter of recombinant hCG. The migration of the 28S rRNA is depicted on the right of the GATA-4 panel, and the 28S and 18S rRNAs on the left of the LHR panel. The sizes of the different LHR mRNA splice variants (in kilobase pairs) are presented on the right. Two lanes for each type of sample are depicted. A, Shows on the top the ethidium bromide (EtBr) staining of the 28S rRNA for RNA loading control. A, Middle and lower parts show Northern hybridization for GATA-4 and LHR mRNA, respectively. B, Shows densitometric quantification of the GATA-4 band (open bars) and the longest (7.0 kb) LHR mRNA splice variant (filled bars) in arbitrary densitometric units (mean of 0 hCG 100%) corrected for intensity of the 28S rRNA band. Each bar represents mean ± SEM of three independent experiments in duplicates. **, P < 0.01 vs. control 0 (0 hCG). b, Northern hybridization analysis of GATA-4 and LHR mRNA after transient transfection of GATA-4 expression plasmid into cultured C{alpha}1, Y-1 and mLTC-1 cells. Each lane contains 20 µg of total RNA. The C{alpha}1 cells with increasing amounts (0.1 to 1µg) and mLTC-1 or Y-1 cells were transfected with (0.5 µg) or without pMT2-GATA-4 expression plasmid. The migration of the 28S rRNA is depicted on the right of the GATA-4 panel, and the 28S and 18S rRNAs on the left side of the LHR panel. The sizes of the different LHR mRNA splice variants (in kb) are presented on the right. Two lanes for each type of sample are depicted. A, Shows on the top the ethidium bromide (EtBr) staining of the 28S rRNA for RNA loading control. A, Middle and lower parts show Northern hybridization for GATA-4 and LHR mRNA, respectively. The lower panel shows densitometric quantification of the GATA-4 band (open bars) and the longest (7.0 kb) LHR mRNA splice variant (filled bars) in arbitrary densitometric units (mean of expression in the absence of GATA-4 expression plasmid 100%) corrected for intensity of the 28S rRNA band. Each bar represents mean ± SEM of three independent experiments in duplicates.

 
A Consensus GATA Binding Site in the mLHR Promoter Sequence
Several in vitro studies have revealed that the basal transcriptional activity of the LHR promoter lies within the first 173/176 nucleotides upstream of the translation initiation codon in all species so far studied (20, 21, 22, 23, 24). Most experiments on the LHR promoter have used about 2-kb stretches of the 5'-flanking sequence, and very recently a study on a longer promoter sequence has been reported (23). Only one putative GATA consensus recognition site, (A/T)GATA(A/G) (25), was observed in the 2040-bp mLHR promoter region between nucleotides –1557 to –1560 (21) (data not shown).

GATA-4 Transactivates the LHR Promoter in Vitro
In addition to C{alpha}1 cells, we also investigated the GATA effects on LHR promoter activity in human embryonic kidney (HEK) 293 (nonsteroidogenic, with no endogenous GATA-4 and LHR expression), Y-1, and mLTC-1 cells. A slight but not significant increase in transactivation of the LHR promoter activity was observed in adrenal C{alpha}1 tumor cells (Fig. 6AGo), in keeping with a similar lack of response of the endogenous LHR gene (Fig. 5BGo). In contrast, cotransfection of the GATA-4 expression plasmid with the 2040-bp mLHR promoter/luciferase reporter gene into HEK 293, mLTC-1 and Y-1 cells revealed clear dose dependent transactivation of the LHR promoter (Fig. 6AGo). The LHR transactivation was observed already at a 0.3-µg dose of the pMT2-GATA-4 expression vector. At the highest 1.0 µg dose of the GATA-4 plasmid, the transactivation rates increased 4.1-, 4.5-, and 7.1-fold, in HEK 293, Y-1 and mLTC-1 cells, respectively. The transactivation of mLTC-1 cells was significantly higher than those of the other responsive cell lines (Fig. 6AGo).



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  		Fig. 6. Dose-Response of mLHR Promoter Activity to Transiently Transfected GATA-4 (A) or GATA-6 (B) Expression Plasmids in Cultured C{alpha}1, HEK 293, mLTC-1, and Y-1 Cells

The cells were transiently transfected with a mLHR promoter/luciferase reporter gene construct (pBL-2040Luc) and increasing amounts (0.1 to 1µg) of either pMT2-GATA-4 (A) or pCDNA3-GATA-6 (B) expression plasmid. pCMV-ß-Galactocidase plasmid was cotransfected to control for transfection efficiency. The results shown are luciferase/ß-galactocidase ratios in relation to luciferase activity in cells transfected with the empty pMT2 plasmid, taken as 100%. Each bar represents mean ± SEM of three independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01 vs. activity in the absence of GATA-4 plasmid (–). C{alpha}1 (gray bars), HEK 293 (white bars), mLTC-1 (black bars) and Y-1 (cross-hatched bars). In addition, the luciferase activity measured in the mLTC-1 cells in the presence of 0.3–1.0 µg of GATA-4 plasmid was significantly higher that the respective responses of the other two cell lines (P at least < 0.05).

 
No Effect of GATA-6 on mLHR Promoter Transactivation
As GATA-4 and GATA-6 are the two major gonad-specific members of the GATA family (10, 12, 13), involved in endocrine systems and candidates for LHR transactivation, we also assessed the possible effect of GATA-6 on mLHR promoter activity. Cotransfection of the GATA-6 expression plasmid (pCDNA3-GATA-6; Ref. 26) at increasing concentrations with the murine LHR promoter-driven luciferase reporter gene revealed no transactivation in C{alpha}1, HEK 293, Y-1, and mLTC-1 cells (Fig. 6BGo).

The Consensus GATA Binding Site Present in the mLHR Promoter Sequence Is Functional
We used previously described mLHR/luciferase reporter gene deletion constructs (21, 22), namely pBL-2040, –1600, –715, –173 and –55 (numbers indicate the length of the 5'-untranslated region, 173 being the basal mLHR promoter); pBL-0Luc was used as negative control. The cells were stimulated with 0.5 µg of the GATA-4 expression plasmid or empty pMT2 control vector. Deletion of region –1600/–700 bp caused a profound 3.5- to 6.2 -fold reduction in the effect of GATA-4 on the promoter activity in HEK 293, Y-1 and mLTC-1 cells, compared with the –2040 bp maximum activity (Fig. 7Go). The GATA-4 activator domain thus seems to be located in the distal part of the LHR promoter, between nucleotide –715 to –1600 bp upstream of the translation initiation site. Mutations of the GATA binding site of the mLHR promoter fully abolished the transactivation in HEK 293, Y-1 and mLTC-1 cells, proving the specificity of the LHR promoter activation by the GATA-4 protein (Fig. 8Go).



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  		Fig. 7. mLHR Promoter Activity after Transient Transfection of the GATA-4 Expression Plasmid or pM2 Control Plasmid into Cultured Y-1, HEK 293 and mLTC-1 Cells, Expressing the Luciferase Reporter Gene under Control of Different Fragments of the mLHR Promoter

The cells were transiently transfected with mLHR promoter/luciferase reporter gene constructs (pBL-2040Luc, pBL01600, pBL-715, pBL-173, pBL-0Luc) and 0.5 µg of the pMT2-GATA-4 expression plasmid. pCMV-ß-Galactocidase plasmid was cotransfected to monitor transfection efficiency. The results shown are luciferase/ß-galactocidase activity ratios as compared with activity of the construct carrying promoter pBL-2040 Luc, assigned a mean value of 100%. The molar amounts of transfected DNA were equalized using the empty pMT2 control vector. Each bar represents mean ± SEM of three independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01 vs. control pBL-2040Luc (mean 100%).

 


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  		Fig. 8. mLHR Promoter Activity after Transient Transfection of the GATA-4 Expression Plasmid with Different pBL-2040 mLHR Promoter Elements into Cultured HEK 293, mLTC-1, and Y-1 Cells, Either Intact or Carrying Mutations (mut-1 and mut-2) in the GATA Consensus Recognition Sequence

The cells were transiently transfected with 0.5 µg of pMT2-GATA-4 expression plasmid, the mLHR promoter/luciferase reporter gene construct (pBL-2040Luc), or the latter carrying mutations of the GATA biding sites (first mut-1 and second mut-2 or mut-1 and mut-2 together). The empty pMT2 control plasmid was used as control. pCMV-ß-Galactocidase plasmid was cotransfected to monitor transfection efficiency. The results shown are luciferase/ß-galactocidase activity ratios related to activity measured with the empty pMT2 control plasmid assigned a mean value of 100%. The molar amounts of the transfected DNAs were equalized using empty pMT2 vector. Each bar represents mean ± SEM of three independent experiments performed in triplicate. **, P < 0.01 vs. pMT2 control.

 
The Stimulatory Effect of hCG on LHR Promoter Function in Ca1 Cells Requires a Functional GATA Binding Site
Interestingly, we found in the C{alpha}1 cells that hCG stimulation was able to up-regulate LHR expression (Fig. 5AGo). This is a unique finding because in other similar experiments using other endocrine cell types, acute hCG/cAMP stimulation brings about LHR down-regulation (22, 27). The stimulatory effect was found when a longer LHR promoter containing the GATA recognition site was used (Fig. 9Go). However, when this site was either deleted, using a shorter promoter fragment, or mutated, the stimulatory hCG effect was totally lost. However, no change in the basal promoter activity was found.



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  		Fig. 9. Effect of the GATA Consensus Sequence in LHR Promoter on hCG (A) and 8-Bromo-cAMP (B) Stimulated Activity of the LHR/Luciferase Reported Construct in C{alpha}1 Cells

Activity of the transfected LHR/luciferase reporter gene in C{alpha}1 cells was measured after a 24-h stimulation with control medium or 50 µg/liter of rec hCG or 10 µM of 8Br-cAMP. Before stimulation, the cells were transienly transfected with the pBL-1600 reporter vector (with GATA site) or with pBL-715 (without GATA site) and variants of this vector with mutated GATA sites (Mut-1, Mut-2; see Table 1Go).

 
GATA-4 Is Able to Bind to the GATA Consensus Sequence in the mLHR Promoter
Using EMSA, we examined whether GATA-4 was able to bind to the putative consensus GATA recognition motif located at nucleotide (nt) –1557 to –1560 of the LHR promoter. Nuclear extracts were prepared from mLTC-1, C{alpha}1, and HEK 293 cells transfected with 0.5 µg of GATA-4 expression plasmid, and incubated with specific radiolabeled double-stranded (ds) oligonucleotides containing the putative GATA consensus recognition sequence. The GATA site formed a single protein/DNA complex with extracts of mLTC-1, C{alpha}1 and HEK 293 cells transfected with 0.5 µg of GATA-4 expression plasmid, and its intensity could be reduced partially or completely by 50- or 200-fold molar excesses of the corresponding WT ds oligonucleotides used as competitors (Fig. 10Go). Mutated GATA-4 ds oligonucleotides were unable to compete for the protein/DNA complexes. To ensure that the protein identified by EMSA was GATA-4, we tested the effect of a rabbit polyclonal antibody directed against full-length bovine GATA-4 protein on the binding reaction. The antiserum at a dilution of 1:100 reduced the intensity, whereas a higher concentration of 1:10 totally abolished the protein/DNA complex formation (Fig. 10Go). Nonimmunized rabbit serum was not able to displace the complex (data not shown). This suggests that the GATA consensus motif on the mLHR promoter has high binding affinity for GATA-4.



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  		Fig. 10. EMSA Analysis of the GATA-Binding Site of the mLHR Promoter

Oligonucleotides (ds), containing putative GATA binding consensus sequences, were radiolabeled with [32P]. Binding of the labeled oligonucleotides to nuclear proteins from HEK 293 cells transfected with 0.5 µg of GATA-4 or adrenal tumor tissue as positive control, C{alpha}1 and mLTC-1 cells, were analyzed with or without 50- or 200-fold molar excesses of the original or mutated (only 200-fold) GATA-4 oligonucleotides or with affinity-purified goat polyclonal GATA-4 antiserum (dilutions 1:10 and 1:100). The positions of free probe and the GATA-4 specific complexes are indicated on the left. The oligonucleotides used as probes or competitors are listed in Table 1Go.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
GATA-4 and LHR Are Coordinately Up-Regulated upon Adrenal Tumorigenesis of the inh{alpha}/Tag Mice
The inh{alpha}/Tag TG mice develop adrenocortical tumors after gonadectomy, a process that is dependent on the persistently elevated post-gonadectomy levels of gonadotropins and appearance of LHR in the adrenal tumors upon the malignization process (2, 5, 6). Conversely, suppression of gonadotropin secretion after gonadectomy prevents the formation of adrenal tumors (7). Because there is considerable evidence about the relationship of gonadotropin action and GATA-4 expression in gonadal cell models (12, 13), we hypothesized that GATA expression might also be connected to the ectopic appearance of adrenocortical LHR expression in the inh{alpha}/Tag mice. Further exploration of the molecular pathways leading to LHR appearance in the adrenocortical tumors has also clinical relevance because LHR expression has been detected in normal and malignant human adrenal tissue (11, 28, 29) and in conditions of adrenal hyperfunction, i.e. LH-dependent and pregnancy-associated forms of Cushing’s syndrome (30). The expression of LHR in human adrenals has been shown to be related to high dehydroepiandrosterone sulfate production by the adrenal cortex and to high LH levels, and it may explain higher dehydroepiandrosterone sulfate levels in chronic anovulatory women with normal ACTH and elevated LH levels (31, 32). Cushing’s syndrome during pregnancy is frequently due to LH/hCG-responsive adrenal neoplasms (30, 33).

We have shown earlier that GATA-4 is expressed in murine Sertoli and Leydig cells throughout the postnatal testicular development (13). Gonadotropin stimulation of Sertoli and Leydig tumor cell lines increases their GATA-4 expression (12, 13), and treatments decreasing LHR expression also down-regulate that of GATA-4 (13). GATA-4 expression has also been shown to be linked to adrenocortical carcinomas (10) and esophageal/gastric adenocarcinomas (34) In the present study, the onset of discernible adrenocortical tumor growth at the age of 6 months in the prepubertally gonadectomized inh{alpha}/Tag mice was accompanied by abundant expression of both GATA-4 and LHR, connecting them to tumor formation and/or progression. In keeping with the current findings, we have recently reported overlapping expression of GATA-4 and LHR in adrenocortical adenomas of gonadectomized DBA/2J mice, suggesting a more general role for them in adrenal tumorigenesis (35).

Evidence for Spatial and Temporal Coexpression of GATA-4 and LHR Genes in the Adrenocortical Tumor Cells
The first prerequisite for our hypothesis of coordinate regulation of the GATA-4 and LHR genes is that their expression occurs in the same cells. We provide here several pieces of evidence to support this contention. In addition, the expression of GATA-6, a close family member of GATA-4, was distinct of that of GATA-4 and LHR in normal and malignant adrenocortical cells. In the murine ovary and testis, GATA-6 has overlapping but distinct expression with that of GATA-4 (12, 13), and whereas abundant GATA-6 expression was seen in normal adult mouse adrenals, that of GATA-4 was absent (Ref. 17 and this study). Similar to GATA-4, LHR expression could not be detected in normal adult adrenals by in situ hybridization. At the age of 4 months in the gonadectomized inh{alpha}/Tag mice, GATA-4 expression was evident in the adrenals and the same areas were positive for LHR. These findings became more obvious during advanced tumorigenesis at 6 months, and GATA-4 and LHR coexpression were clearly seen in the tumor plaques. All in all, our data demonstrate the colocalization of GATA-4 and LHR, and the absence of GATA-6 mRNA in the GATA-4 positive areas, as well as the opposite findings with surrounding normal tissue. Immunohistochemical analyses confirmed these findings at protein level. Another piece of evidence for coexpression of GATA-4 and LHR in the same malignant adrenocortical cells was provided by similar findings on the C{alpha}1 cell line originating from one of the first adrenal tumors detected in the inh{alpha}/Tag mice (2).

RT-PCR provides the most sensitive method for detecting the temporal sequence of appearance of GATA-4 and LHR expression in the adrenal tumors. Although it does not reveal the spatial localization the messages, this information is available from the in situ hybridization and immunohistochemistry studies and from findings on the C{alpha}1 cells. Taken together, the data from RT-PCR analyses demonstrated that LHR expression was detected in some samples in the absence of GATA-4 expression, i.e. 6-month-old gonadectomized WT mice and 6-month-old nongonadectomized TG mice. LHR expression may thus precede that of GATA-4 even during adrenal tumorigenesis and trigger the self-perpetuating cycle leading to reciprocal up-regulation of both messages. However, the data do not either exclude the possibility that the expression of these two genes starts simultaneously. A connection of GATA-4 expression with that of Tag is unlikely because GATA-4 and LHR were coexpressed in the hyperplastic adrenals of bLHß-CTP mice, not expressing Tag (9). Furthermore the results obtained in the latter model along with LHR knockout mice demonstrated that LH action is a prerequisite for the expression of GATA-4 and LHR.

Reciprocal Regulation of GATA-4 and LHR Expression
The next question was whether the spatio-temporal correlation of GATA-4 and LHR expression in the adrenocortical tumor cells represents a coincidental phenomenon or whether a mechanistic link can be found between the two responses. Stimulation of C{alpha}1 cells with recombinant hCG resulted in dose-dependent up-regulation of both LHR and GATA-4 messages. The up-regulation of LHR expression is a unique finding because receptor down-regulation has always been found in other cell models (22, 27, 36, 37). The reason for this may be the ectopic nature of LHR expression in the C{alpha}1 cells, which make therefore an interesting model for further exploration of the molecular mechanisms of LHR expression.

The next experiments were carried out to demonstrate whether the reciprocal response, i.e. up-regulation of LHR expression by GATA-4, could be observed. To this end, C{alpha}1, Y-1, and mLTC-1 cells were transfected with a GATA-4 expression plasmid. Although no significant LHR or GATA-4 up-regulation was found in the C{alpha}1 cells, a clear response was found in the mLTC-1 cells. No response of LHR expression was found in Y-1 cells that were devoid of endogenous GATA-4 or LHR expression. The unexpected negative finding with C{alpha}1 cells, in contrast to the response seen in mLTC-1 cells, has several possible explanations. One explanation is technical, due to resistance of the C{alpha}1 cells to transfection, and, in fact, a very small increase in GATA-4 message level was found after transfection with GATA-4 expression vector. The other explanation is that endogenous GATA-4 expression in these cells is so high that the low-efficiency transfection may not increase this level enough to bring about significant up-regulation of LHR expression. The high GATA-4 expression may also have saturated this response. The mLTC-1 cells are easy to transfect, and therefore their moderate level of endogenous GATA-4 expression allows an increase in LHR mRNA after efficient transfection with GATA-4 expression vector. In the Y-1 cells the total lack of endogenous GATA-4 and LHR could explain the absence of up-regulation or induction of LHR by GATA-4. This negative finding suggests that additional endogenous coregulators, present in mLTC-1 but absent Y-1 cells, are needed for the induction of LHR expression. However, as discussed in the next paragraph, inactivation of the LHR gene in Y-1 cells is a more likely explanation.

We next used LHR promoter/luciferase reporter gene constructs to monitor the response to GATA-4 stimulation. We found a clear dose-dependent luciferase response to cotransfected GATA-4 expression plasmid in HEK 293, Y-1, and mLTC-1 cells, but none in C{alpha}1 cells. Interestingly, the exogenous LHR promoter was responsive to GATA-4 stimulation in Y-1 cells, which suggests that the endogenous LHR gene in these cells is inactive, possibly through methylation. Because the C{alpha}1 cells showed no response of the LHR promoter/luciferase reporter gene to exogenous GATA-4 transfection, an attempt was made to reduce endogenous GATA-4 expression using the siRNA technique. These preliminary experiments brought about significant transactivation of the LHR promoter by coexpressed GATA-4 in C{alpha}1 cells, providing further support to the explanation that the high endogenous GATA-4 expression, together with low transfection efficiency of these cells, did not allow significant transactivation of the LHR promoter by transfected GATA-4 in the native C{alpha}1 cells.

Compelling evidence for the involvement of GATA-4 in LHR expression of C{alpha}1 cells was provided by the experiments where the GATA recognition site was deleted or mutated in the LHR promoter. These alterations brought about total abolition of the hCG-stimulated up-regulation of LHR activity. However, no effect on basal LHR activity was found, which indicates that the constitutive and stimulated components of LHR expression are under differential regulation.

All in all, our data implicate GATA-4 as an important component in the cascade leading to LHR promoter activation. Of the endocrine GATA factors, this effect appears specific for GATA-4 given that GATA-6 was unable to stimulate LHR promoter activity in any cells studied. Although hCG/LH leads to up-regulation of GATA-4, this may not be the only mechanism inducing the LH/hCG-GATA-4-LHR activation cascade. This is supported by numerous recent findings that GATA-4 expression and activity are under a multitude of additional regulatory mechanisms (38, 39, 40, 41, 42, 43).

A Functional GATA Response Element in the LHR Promoter
Finally, the remaining question was to identify a functional GATA-4 response element in the LHR promoter. Such a consensus sequence was identified between nucleotides –1557 and –1560 (25) in the murine LHR promoter (21, 22). The same sequence can also be identified in the human LHR promoter (24, 44), providing further evidence for the importance of this site. When a series of LHR promoter deletion mutants (22), driving the luciferase reporter gene, were cotransfected with GATA-4 expression plasmid, clear evidence was obtained for functionality of the –715 to –1600 sequence in the positive GATA-4 response. When this sequence was deleted the stimulatory effect of GATA-4 coexpression on reporter gene activity was totally abolished. The same loss of GATA-4 effect was found when two different nucleotides either separately or together were mutated in the GATA recognition site of the LHR promoter sequence. Finally, EMSA data clearly demonstrated that the GATA-4 protein is able to bind to the ds DNA sequence containing the GATA recognition sequence of the LHR promoter, and was competed out in expected fashion by GATA-4 antiserum and excess of DNA, but not by mutated DNA. These data thus provide strong evidence that GATA-4 binding to the GATA consensus sequence in the LHR promoter can explain the molecular mechanism of GATA-4 mediated up-regulation of LHR expression.

In conclusion, we have demonstrated in the present study a direct interrelationship between LHR and GATA-4 expression upon adrenocortical tumorigenesis of the inh{alpha}/Tag mice. There is apparently a positive feed-forward amplification link between LHR and GATA-4 expression, LHR action up-regulating the GATA-4 message, and vice versa. However, additional studies are needed to confirm the role of GATA-4 in the activation of the LHR promoter in vivo. Based on the present results, we can hypothesize that these two factors, in synergy with Tag expression, induced the formation of LH-dependent adrenocortical tumors in the mouse model studied. It will be of importance to explore whether similar mechanisms can be identified in human gonadal and adrenal tumors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals and Treatments
The TG mice used here harbor a fusion gene of a 6-kb murine inhibin-{alpha} promoter sequence, fused with DNA encoding the simian virus 40 T antigen (inh{alpha}/Tag), as described in more detail elsewhere (3, 4). The female IT6-F inh{alpha}/Tag founder mouse was bred with C57Bl/6J (The Jackson Laboratory, Bar Harbor, ME) male mice to create and maintain the IT6-F TG line. The current experiments were carried out on IT6-F male and females, and non-TG littermates as controls. Genotyping of the mice was performed from tail DNA by PCR as earlier described (3). Avertin anesthesia was used during the surgical operations (gonadectomy between d 21 and 24 postpartum), and buprenorphine (3 µg/mouse, ip) was given as postoperative analgesia. The gonadectomized mice were killed at 2, 4, and 6 months of age (n = 5–6 per age group) by cervical dislocation. The adrenals were dissected out, weighed, and snap-frozen in liquid nitrogen, or fixed either in Bouin’s solution or in 4% paraformaldehyde. The mice were kept in specific pathogen-free conditions, two to four per cage, in controlled conditions of light (12-h light, 12-h dark) and temperature (21 ± 1 C). They were fed with mouse chow Special Diet Service RM-3 (E, soy free; Whitham, Essex, UK) and tap water ad libitum. The University of Turku Ethical Committee on Use and Care of Animals approved all procedures of the current experiments.

Cell Culture and Hormonal Stimulation of the Cell Lines
The mouse adrenal tumor cell line (C{alpha}1) (2, 5), Y-1 adrenal cell line and HEK 293 were cultured in HEPES (20 mm)-buffered DMEM/Ham’s F-12 1:1 medium (Sigma Chemical Co., St. Louis, MO), supplemented with 10% heat-inactivated fetal calf serum (iFCS) 50 IU/liter penicillin and 0.5 g/liter streptomycin (Sigma). The cells were allowed to grow on 9.6-cm diameter Petri dishes (Greiner, Labortechnik, Frickenhausen, Germany) or on six-well plates to 70–80% confluency under humidified atmosphere of 95% and 5% CO2 at 37 C. mLTC-1 cells were cultured in HEPES (20 mM)-buffered Waymouth’s medium (Sigma), supplemented with 9% heat-inactivated horse serum (Invitrogen Life Technologies, Paisley, Scotland, UK) and 4.5% iFCS (Bioclear, Wokingham, Berks, UK) containing 0.1 g/liter gentamycin (Invitrogen LIfe Technologies, Gaithersburg, MD).

C{alpha}1 cells (2) were plated 1 d before stimulation on 9.6-cm Petri dishes (Greiner) at a density of 8 x 106 cells per plate in 8 ml of the culture medium as mentioned above and incubated for 24 h. The C{alpha}1 cells were stimulated in the absence or presence of 1, 10, 50, and 100 µg/liter of rechCG (N.V. Organon, Oss, The Netherlands) or 0, 0.1, 0.3, 0.5, and 1.0 µg of a GATA-4 expression plasmid (pMT2-GATA-4) (12, 45), for 48 h. Thereafter, the cells were collected for RNA isolation and Northern blot hybridization (see below).

Northern Hybridization Analysis
Total RNA was isolated from cells using the single step guanidinium thiocyanate-phenol-chloroform extraction method (46). Twenty micrograms of RNA per lane were resolved on 1.2% denaturing agarose gel and transferred onto Hybond-XL nylon membranes (Amersham, Amersham International plc, Aylesbury, Buckinghamshire, UK). Membranes were prehybridized overnight at 65 C in a solution containing 5x saline sodium citrate (SSC), 5x Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), 50% formamide and 5 g/liter of denatured calf thymus DNA. A complementary RNA probe for the rat LHR generated from a fragment of the LHR cDNA, spanning nucleotides 441–849 of its extracellular domain, subcloned into the pGEM-4Z plasmid was used for hybridization (47). The [32P] -dUTP (800 Ci/mmol, Amersham) labeled probe was generated using a Riboprobe system II kit (Promega, Madison, WI). The probes were purified with NickColumns (Pharmacia Biotech, Uppsala, Sweden). Hybridization was carried out at 65 C for 20 h in the same prehybridization solution after addition of labeled probe. After hybridization, the membranes were washed twice in 2x SSC and 0.1% SDS at room temperature for 10 min, followed by two washes in 0.1x SSC and 0.1% SDS at 65 C to remove most of the background. For GATA-4, the membranes were prehybridized overnight at 42 C in a solution containing 5x SSC, 5x Denhardt’s, 0.5% SDS, 50% formamide, and 5 g/liter of denatured calf thymus DNA. The GATA-4 cDNA probes were cut out from pMT2-GATA4 (12, 45), using EcoRI/PstI and SmaI restriction enzymes, respectively, labeled with Prime-a-Gene kit (Promega) using [{alpha}-P32]-dCTP during 4 h at 37 C and purified with NickColumns. Hybridization was carried out at 42 C for 20 h in the same pre-hybridization solution after addition of the denatured labeled cDNA probe. After hybridization, the membranes were washed twice in 2x SSC and 0.1% SDS at room temperature for 10 min, followed by two washes in 0.1x SSC and 0.1% SDS at 42 C to remove most of the background. Finally, the membranes were exposed to Kodak x-ray films (Kodak XAR-5; Eastman Kodak, Rochester, NY) at –70 C for 4 to 7 d or to phosphor-imager (Fujifilm BAS-5000, Fujifilm I{alpha}I, Tokyo, Japan) for 4–24 h. The intensities of specific bands were quantified using the Tina software (Raytest, Staubenhardt, Germany), and related to those of the 28S ribosomal RNA in the gel stained with ethidium bromide. The molecular sizes of the mRNA species were estimated by comparison with mobilities of the 18S and 28S ribosomal RNAs.

In Situ Hybridization
The adrenal glands were washed briefly in PBS and then frozen in O.C.T. cryo-preservation solution (Tissue Tek, Miles Inc., Elkhart, IN) or fixed in 4% paraformaldehyde and embedded in paraffin. Frozen sections (10 µm) were fixed with 4% paraformaldehyde in PBS and paraffin sections (10 µm) were deparaffinized, and both were subjected to in situ hybridization. Tissue sections were incubated with 1 x 106 cpm [33P]-labeled (1000–3000 Ci/mmol, Amersham, Life Technologies, Arlington Heights, IL) antisense or sense riboprobes in a total volume of 80 µl. The antisense and sense riboprobes for GATA-4 and GATA-6 (used as control) were prepared as described elsewhere (12, 45, 48). Antisense and sense riboprobes for LHR were prepared similarly and the hybridization method was carried out as described earlier (9) using the same sequence of the LHR probe as in Northern hybridization.

RT-PCR and Southern Hybridization
One microgram of total RNA was reverse-transcribed using 20 IU of avian myeloma virus Reverse Transcriptase (RT) (Promega), 50 IU of RNase Inhibitor (Promega), 25 pmol of random primers (Promega), 10 mmol of each deoxynucleotide triphosphate (dNTP) in 25 µl final volume for 1 h at room temperature. For amplification of LHR and GATA-4 cDNA, specific primers were used as earlier described (9, 49). PCR was performed in a 25-µl final volume, 4 µl of RT solutions were mixed with 2 IU of the DyNAzyme II DNA polymerase (Finnzymes Oy, Espoo, Finland), 10 pmol of each primer, and 10 mmol of each dNTP (50). The RT and PCRs were carried out sequentially in the same assay tube. First the RT reaction was carried out (50 C for 10 min), followed by a denaturation period of 3 min at 97 C. Thereafter, a PCR with 40 cycles (96 C for 1.5 min, 57 C for 1.5 min, and 72 C for 3 min, with a final extension period of 10 min at 72 C) was performed. The sense primer for LHR corresponded to nucleotides 176–195 (5'-CTCTCACCTATCTCCCTGTC-3'), and the antisense primer to nucleotides 878–858 (5'-TCTTTCTTCGGCAAATTCCTG-3') of the mouse LHR cDNA. The sense primer for GATA-4 corresponded to sense nucleotides 1527–1550 (5'-AAACGGAAGCCCAAGAACCTGAAT) and the antisense primer to 1935–1953 (5'-GGCCCCCACGTCCCAAGTC) (expected size: 427 bp) of mouse GATA-4 cDNA (49). As control for RNA quality, a 395-bp fragment of the L19 ribosomal protein gene was coamplified with each sample (sense primer, 5'-GAAATCGCCAATGCCAACTC-3'; antisense primer, 5'-TCTTAGACCTGCGAGCCTCA-3'). As a negative control, kidney RNA was used. After RT-PCR, 20-µl aliquots of the reaction mixtures were loaded on 1% agarose gel containing ethidium bromide (0.4 mg/liter), to identify the amplified DNA fragments. Southern hybridization was used, according to standard techniques, to confirm the specificity of these PCR products. Hybridization was carried out with the 5'-end labeled oligonucleotide 5'-TGGAGAAGATGCACAGTGGA-3', corresponding to nucleotides 641–660 of LHR cDNA and for GATA-4 with the 5'-AGTGGCACGTAGACGGGCGAGGAC- 3', corresponding to nucleotides 676–699. The membranes were washed according to the manufacturer’s instructions, then exposed to Kodak x-ray film (XAR 5).

Immunohistochemistry
Paraformaldehyde fixed paraffin sections (5 µm) of representative mouse adrenal gland tumors 3 and 5 months after gonadectomy were dewaxed, rehydrated and boiled in 10 mM citric acid (pH 6.0) for antigen retrieval. 3% H2O2 in water was used to block endogenous peroxidases and then slides were incubated with 1% normal serum in 0.1% Tween 20 (Fluka, Buchs SG, Switzerland) in PBS. Serial sections of adrenal glands and testes as positive control, were subjected to immunohistochemistry with LHR antibody (dilution 1:1000, a rabbit polyclonal antiserum directed against human peptide sequence KKLPSRETFVNLLEA) or/and commercial polyclonal goat GATA-4 IgG (dilution 1:200, catalog no. sc-1237, Santa Cruz Biotechnology Inc., Santa Cruz, CA). The slides were incubated with LHR antibody at 4 C O/N or 37 C for 1 h (double immunohistochemistry). The avidin-biotin immunoperoxidase system was used to visualize bound antibody (Vectastain Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA) with 3,3'-diaminobenzedine (Sigma) as a substrate. In double immunohistochemistry sections were washed in PBS and incubated in 4 C overnight with GATA-4 antibody. The avidin-biotin immunoperoxidase system was again used to visualize bound antibody and Vector SG substrate kit (catalog no. SK-4700, Vector Laboratories, Inc.) was used as a second substrate. As a control for the LHR antibody, adjacent sections were incubated with 1% normal serum in PBS as primary antibody and antirabbit antibody as a secondary antibody to differentiate unspecific staining in comparison. GATA-4 antibody was added as above also to the control sections.

Transient Transfection of Cell Lines
The transient transfection method was optimized for each cell line using pCMV-ß-galactosidase as the marker of transfection efficiency. Cells were seeded on six-well plates at density of 1 x 106 (HEK293 and C{alpha}1 cells), or 0.5 x 106 cells/well (mLTC-1 cells), 16–20 h preceding transfection to achieve 60–70% confluency. Cells were transfected with 1.5 µg of the LHR promoter construct (22), with increasing concentrations of the pMT2-GATA-4 expression vector (12, 45) or GATA-6 expression vector (pCDNA3-GATA-6) (26) and 0.3 µg of pCMV-ß-galactosidase as control for transfection efficiency. The HEK 293 cells were transiently transfected by LipofectAMINE transfection reagents (Invitrogen Life Technologies), according to instructions of the manufacturer. mLTC-1 cells were transfected with FuGene (Roche Molecular Biochemicals, Mannheim, Germany) according to instructions of the manufacturer. C{alpha}1 cells were very difficult to transfect transiently. We tested FuGene, Lipofectamine, CaCl2 and electroporation methods to transfect them, but all with rather poor efficiency (among them FuGene had the highest efficiency, but still it was only 30% of that of the HEK 293 or mLTC-1 cells). Finally, GenePORTER (Gene Therapy Systems, Inc., San Diego, CA), which uses direct hydrophilic conjugation methodology, according to instructions of the manufacturer, gave satisfactory results, although the transfection efficiency still remained at 45% of that of the HEK 293 or mLTC-1 cells, as assessed by ß-galactosidase expression. The transfection solution was removed after 5 h incubation at 37 C and replaced by 2 ml of complete DMEM/Ham’s F-12 for a further 24 h culture.

For cotransfection experiments, we used plasmids expressing mLHR promoter constructs pBL-2040Luc, pBL-1600Luc, pBL-715Luc, pBL-173Luc, pBL-55Luc as reported earlier (22), the expression vector pMT2-GATA-4 (12, 45), and pBL-0Luc was used as the negative control. The amount of transfected DNA was equalized using the empty pMT2 vector (vector without transactivator). The volume of LipofectAMINE or GenePORTER reagents was increased and adjusted to the DNA amount according to the manufacturers’ instructions.

After 24 h, the transfection media were replaced by 100 µl of cell lysis buffer [12.5 mm Tris-HCl (pH 7.8), 10 mm NaCl, 0.4 mm EDTA, 0.2 mm MgSO4, 1 mm dithiothreitol (DTT) and 0.2% Triton X-100] for 5 min. The cells were scraped off and span for 1 min at room temperature. Luciferase activity was measured from 10 µl of the lysate in a Victor multilabel counter (Wallac Oy, Turku, Finland) by adding 100 µl of luciferase assay buffer [40 mm Tris-HCl (pH 7.8), 0.5 mm ATP, 10 mm MgSO4, 0.5 mm EDTA, 10 mm DTT, 0.5 mm coenzyme A, 0.5 mm luciferin]. The ß-galactosidase activity was assessed from the same lysate in 100 mm phosphate buffer (pH 7.0) supplemented with 10 mm KCl, 1 mm MgSO4 and 50 mm ß-mercaptoethanol and incubated for 30 min at 37 C in the presence of ONPG at 0.8 g/liter final concentration. Luciferase activity was normalized for transfection efficiency by dividing its activity by that of ß-galactosidase. All transfection experiments were carried out at least three times to ensure reproducibility.

Reduction of GATA-4 Expression Using siRNA and Culture of Stably siRNA Construct Transfected C{alpha}1 Cells
We used the mU6pro vector methodology with the mouse U6 promoter (RNA polymerase III) with a Bbs1/Xba1 cloning sites, to allow insertion of siRNA template sequences after recently published methodology (51). The sequence and methodology of the mU6pro vector is available at: http://sitemaker.umich.edu/dlturner.vectors. Briefly, two hairpin siRNA templates for GATA-4 were constructed by oligos containing 27 (G5) or 22 nt (G4) antisense sequences of the central region of the GATA-4 mRNA, a 3-nt loop and 27 or 22 nt of the same sense sequence flanked by a polyT tail, annealed with their complementary oligos forming overhangs for Bbs1/Xba1 restriction sites. The ds DNA was subcloned into the Bbs1 and Xba1 restriction sites of the mU6pro vector. The hairpin siRNA oligos (Tag, Copenhagen, Denmark) are shown in Table 1Go. Vectors containing the siRNA were sequence verified using the M13R2 reverse primer (Table 1Go) (51). Thirty colonies were screened by sequencing for incorrect nucleotides by sequencing. Two vectors with more than five mispriming nucleotides (see Table 1Go) were used as negative controls (G2–G3).


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  		Table 1. The Oligonucleotides Used in the Study

 
To make a stably transfected cell line, C{alpha}1 cells from passage number 12 were allowed to grow on 9.6-cm diameter petri dishes (Greiner) to 70–80% confluency under humidified atmosphere of 95% and 5% CO2 at 37 C with the complete DMEM/F12 media as mentioned above. To block the endogenous GATA-4 expression using the siRNA, cells were then cotransfected with 6.0 µg of linearized longer (G5) hairpin siRNA template for GATA-4, where ampicillin resistance cassette was exchanged to neomycin/kanamycine cassette from pPGK-neo-loxP (Exchange of selection marker gene kit, Gene Bridges GmbH, Dresden, Germany) by Red/ET-recombination (52) (Table 1Go), and 3.0 µg of ß-galactosidase expression vector to control the transfection efficiency. For stable transfection, the cells were trypsinized and split into selection medium after 72 h of transfection into selection medium containing additionally 1000 mg/liter of G418 (active form of Neomycin antibiotic analog). The cells were maintained in the same medium for about 3 wk with changing the medium three to four times per week. Resistant colonies were isolated, expanded, and screened. The clonal line of choice (cl. 21) was subsequently cultured for further studies in medium containing 300 mg/liter of G418.

Site-Directed Mutagenesis
We mutated in the conserved consensus GATA binding site (A/T)GATA(A/G) (25) of the LHR promoter (–1557 to –1560), G to T and A to G (mut 1), or G to A and T to G (mut 2) (Table 1Go), using QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. One hundred nanograms of the plasmid DNA template were incubated with 125 ng of appropriate primers, 25 mM dNTPs and 50 µl of 1x reaction buffer in the presence of 2.5 IU of Pfu DNA polymerase. The PCR conditions included 16 cycles with denaturing step at 95 C for 30 sec, annealing at 55 C for 1 min and extension at 68 C for 13 min. The parental DNA template was digested by adding 10 IU of DpnI restriction endonuclease for 1 h at 37 C. One to 5 µl of PCR were used to transform XL-1 Blue Supercompetent cells. The mutations were verified by restriction mapping as described earlier (22).

EMSA
Nuclear extracts were prepared from confluent cell cultures of control mLTC-1 and GATA-4 transfected HEK 293 cells as previously described (53). Complementary oligonucleotides, containing the respective GATA-4 sequences, and competitors (Table 1Go) were annealed in 10 mm Tris-HCl (pH 7.5), 1 mm EDTA, 25 mm NaCl, 10 mm MgCl2, and 1 mm DTT. 5'-GG overhangs present in the ds oligonucleotide were filled with [{alpha}-P32]-dCTP for 2 h at 30 C using the Klenow DNA polymerase. The labeled oligonucleotide probes were purified in Nick Columns. Nuclear extracts (10 µg) were incubated with 6 fmol (around 40,000 cpm) radiolabeled ds oligonucleotides for 1 h at 4 C in a reaction buffer containing 12 mm HEPES (pH 7.9), 4 mm Tris-HCl, 12% glycerol, 1 mm EDTA, 60 mm KCl, 1 mm DTT, and 300 mg/liter BSA in the presence of 2 µg poly (deoxyinosine:deoxycytosine). For competition experiments, the protein extract was first incubated on ice for 30 min, either with a 50x or 200x molar excess of competitor DNA or affinity-purified goat polyclonal antibody, raised against a peptide corresponding to an amino acid sequence of the carboxy terminus of murine GATA-4 (GATA-4, C-20 for gel supershift studies) (Santa Cruz Biotechnology, Inc., catalog no. sc-1237 X) (1:10 and 1:100) before addition of [{alpha}-P32]-dCTP radiolabelled DNA. After binding reaction, the protein-DNA complexes were resolved on 5% nondenaturing polyacrylamide gels using 0.25x Tris-borate-EDTA buffer, and the gels were dried and exposed to Kodak x-ray film at –50 C for 1 to 3 d.

Statistical Analysis
All results presented here are from two to six independent experiments in triplicate, unless otherwise specified. The data are expressed as mean ± SEM. Statistically significant differences between groups were determined by one-way ANOVA, followed by Duncan’s test; P < 0.05 was considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank Dr. T. Evans (Albert Einstein College of Medicine, Bronx, NY) for the pCDNA3-GATA-6 expression plasmid, Dr. D. Turner (University of Michigan, Ann Arbor, MI) for the mU6pro siRNA vector, and Dr. A.T. Fazleabas (University of Illinois at Chicago, Chicago, IL) for the human LHR antiserum. We also thank Ms. Johanna Vesa for technical assistance.


   FOOTNOTES
 
This work was supported by grants from The Academy of Finland (to I.H.), The Finnish Cancer Societies (to I.H.), The Ahokas Foundation (to M.H.), The Finnish Pediatric Foundation (to M.H. and S.K.), Helsinki University Central Hospital (to M.H. and S.K.), The Sigrid Jusélius Foundation (to D.B.W., M.H., and I.H.), and The Wellcome Trust (to I.H.).

Abbreviations: dNTP, Deoxynucleotide triphosphate; ds, double-stranded; DTT, dithiothreitol; hCG, human chorionic gonadotropin; HEK, human embryonic kidney; iFCS, inactivated fetal calf serum; inh{alpha}, inhibin {alpha}-subunit promoter; LHR, LH receptor; nt, nucleotide; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; SSC, saline sodium citrate; TG, transgenic; WT, wild-type.

Received for publication August 14, 2002. Accepted for publication July 6, 2004.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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