Differential Requirement for Steroidogenic Factor-1 Gene ...

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Differential Requirement for Steroidogenic Factor-1 Gene ...

  1. 1. 0888-8809/04/$15.00/0 Molecular Endocrinology 18(4):941–952 Printed in U.S.A. Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2003-0333 Differential Requirement for Steroidogenic Factor-1 Gene Dosage in Adrenal Development Versus Endocrine Function MICHELLE L. BLAND, ROBERT C. FOWKES, AND HOLLY A. INGRAHAM Department of Physiology, Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, California 94143-0444 The importance of steroidogenic factor-1 (SF-1) embryos. However, later in development, medul- gene dosage in endocrine function is evidenced by lary growth was compromised in both genotypes. phenotypes associated with the heterozygous Despite the small adrenal size in SF-1 heterozy- state in mice and humans. Here we examined gotes, an unexpected elevation in steroidogenic mechanisms underlying SF-1 haploinsufficiency capacity per cell was observed in primary adult and found a striking reduction (12-fold) in SF-1 adrenocortical SF-1 / cells compared with wild- heterozygous ( / ) adrenocortical size at embry- type cells. Elevated cellular steroid output is con- onic day (E) 12. Loss of one SF-1 allele led to a sistent with the up-regulation of some SF-1 target selective decrease in adrenal precursors within the genes in SF-1 / adrenals and may partially be adrenogonadal primordium at E10.0, without af- due to an observed increase in nerve growth fecting the number of gonadal precursors, as factor-induced-B. Our findings underscore the marked by GATA-4. Beginning at E13.5, increased need for full SF-1 gene dosage early in adrenal cell proliferation in SF-1 / adrenals allows these development, but not in the adult adrenal, where organs to approach but not attain a normal size. compensatory mechanisms restore near normal Remarkably, neural crest-derived adrenomedullary function. (Molecular Endocrinology 18: 941–952, precursors migrated normally in SF-1 / and null 2004) D URING EMBRYONIC DEVELOPMENT, elabora- tion of genetic programs controlling organ growth is critical for optimal performance in the adult. Once postnatal lethality due to severe adrenal insufficiency (1–4). In humans, three distinct partial loss-of-function mutations in SF-1 are associated with XY sex reversal proper organ size is achieved, cellular function is reg- and severe adrenal insufficiency, demonstrating that ulated to meet physiological demands to maintain ho- SF-1 acts in a dose-dependent manner (5–7). Similarly meostasis. In the adult endocrine system, circulating in mice, loss of one SF-1 allele leads to adrenal insuf- peptide hormones serve as trophic signals to influence ficiency due to hypoplastic and disorganized adrenal organ size and simultaneously regulate tissue-specific glands, underscoring the importance of full SF-1 gene gene expression. This is exemplified in the adrenal, dosage during adrenal organogenesis (8). where ACTH both maintains adrenal cortex weight and Normal adrenal development is apparent at embry- stimulates steroidogenesis. In the embryo, genetic onic day (E) 9.0 when a population of cells derived pathways controlling the earliest stages of adrenal from the coelomic epithelium of the intermediate me- growth are presumed to function cell autonomously soderm begin to express SF-1; these cells form the and without input from other endocrine organs. One adrenogonadal primordium (9). Later at E11.0, cells in factor known to be essential for adrenal development the adrenogonadal primordium differentiate and give is the orphan nuclear receptor steroidogenic factor-1 rise to both the adrenal cortex and gonad. Further (SF-1, AD4BP, NR5A1). Indeed, deletion of SF-1 in development at E13.0 involves the migration and infil- mice results in adrenal and gonadal agenesis and tration of neural crest cells into the developing adrenal Abbreviations: BrdU, Bromo-deoxyuridine; 8Br-cAMP, cortex; these cells give rise to the adrenal medulla and 8-bromo-cAMP; D H-nLacz, dopamine -hydroxylase-nu- become completely enveloped by the cortex by E15.5 clear LacZ; DTT, dithiothreitol; E, embryonic day; LRH, liver (10). Although SF-1 expression is restricted to adre- receptor homolog; MC2R, melanocortin 2 receptor; NGFI-B, nocortical cells, loss of both SF-1 alleles results in nerve growth factor-induced-B; SCC, side chain cleavage; Ser, serine; SF-1, steroidogenic factor-1; SR-B1, scavenger massive apoptosis in the adrenal cortex, and conse- receptor-B1; StAR, steroidogenic acute regulatory protein; quently, agenesis of both the adrenal cortex and me- TUNEL, terminal deoxynucleotidyl transferase-mediated de- dulla (1). Whereas human and mouse genetics have oxyuridine triphosphate nick end-labeling. established a role for SF-1 in adrenal development, the Molecular Endocrinology is published monthly by The defective developmental processes contributing to Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine decreased adrenal growth in SF-1 / mice have not community. been explored. 941 Downloaded from mend.endojournals.org on December 27, 2004
  2. 2. 942 Mol Endocrinol, April 2004, 18(4):941–952 Bland et al. • Compensation for SF-1 Haploinsufficiency In the adult adrenal, extensive in vitro studies have increase in apoptosis, 2) a decrease in cell prolifera- suggested that SF-1 functions to coordinately regulate tion, 3) a defect in homing of medullary precursors to basal expression of steroidogenic genes such as ste- the developing adrenal cortex, and/or 4) a decrease in roidogenic acute regulatory protein (StAR), scavenger allocation of cells in the adrenogonadal primordium to receptor-B1 (SR-B1), melanocortin 2 receptor (MC2R), the adrenal. Moreover, we asked how a reduction in and the steroid hydroxylases. In addition, SF-1 is pro- SF-1 gene dosage affected the steroidogenic capacity posed to mediate ACTH-stimulated up-regulation of of adult SF-1 / adrenocortical cells. Our findings these steroidogenic genes via the cAMP-protein ki- demonstrate that SF-1 gene dosage is most critical at nase A pathway (11). The mechanism linking SF-1 with the onset of adrenal development within the adreno- cAMP- and protein kinase A-mediated increases in gonadal primordium. We propose that compensatory steroidogenic gene expression has yet to be deter- pathways deployed later in adrenal development and mined. Several groups have proposed that posttrans- in the adult allow SF-1 heterozygous adrenals to func- lational modifications of SF-1 in response to extracel- tion at high, albeit insufficient, levels in the adult lular signaling modulate its activity (12–15). Despite an mouse. abundance of in vitro evidence supporting SF-1’s cen- tral role in regulating steroidogenic gene expression, recent findings showed a paradoxical increase in SF-1 RESULTS target gene expression in SF-1 / adrenals that express low levels of SF-1 (8, 16). These results are at Early Adrenal Development Is Severely odds with SF-1’s dose-dependent activity during ad- Compromised in SF-1 Heterozygous Mice renal development and raise questions as to whether SF-1 is critical for activating steroidogenic gene ex- SF-1 functions in a dose-dependent manner in mice to pression or whether functionally redundant pathways affect adrenal function, but the precise stage of adre- are induced in SF-1 heterozygous mice. nal growth compromised in SF-1 heterozygotes re- In this study, we investigated the mechanisms that mains unknown. Consistent with our previous findings underlie SF-1 haploinsufficiency beginning at the ear- in adult mice, SF-1 / adrenals were clearly smaller liest stage of adrenal development. Specifically, we than / adrenals at late stages of embryonic devel- asked what mechanisms might account for reduced opment, E18.5 (Fig. 1A) (8). Earlier in development, at adrenal size in SF-1 heterozygotes, including: 1) an E13.5, we noted a more dramatic difference in SF-1 Fig. 1. Adrenal Size Is Decreased in SF-1 / Embryos throughout Development A, Genitourinary systems were dissected from E18.5 SF-1 / , / , and / embryos. Arrows point to adrenal glands. Bar, 1 mm; k, kidney; g, gonad; b, bladder. B, SF-1 immunoreactivity in transverse sections of E13.5 embryos shows decreased adrenal cortex size (arrows) in SF-1 heterozygotes. Bar, 250 m; sc, spinal cord; da, dorsal aorta; g, gonad. C, Adrenal size (cross section area) is decreased in heterozygous embryos ( / , black bars) compared with wild-type embryos ( / , green bars) from E12.0-E18.5, n 3–5 embryos per group; **, P 0.01 vs. / . Downloaded from mend.endojournals.org on December 27, 2004
  3. 3. Bland et al. • Compensation for SF-1 Haploinsufficiency Mol Endocrinol, April 2004, 18(4):941–952 943 / and / adrenal size (Fig. 1B). Quantification of E17.5) also showed a significant increase in BrdU la- adrenal size throughout development revealed a 12- beling and histone-3 phosphorylation in / adrenals fold decrease in size at the earliest stages of develop- when compared with / adrenals (Fig. 3, C and D). ment, whereas at late time points, heterozygous adre- Given that SF-1 / and / mice exhibit abnor- nals were only 2-fold smaller than wild-type adrenals mal adrenocortical development, we asked whether (Fig. 1C). medullary development was affected by loss of one or both alleles of SF-1 (1, 8). To examine migration of Increased Cell Proliferation at Late Stages of sympathoadrenal neural crest precursors [ -gal ( )] to SF-1 / Adrenal Development the adrenal cortex, SF-1 / mice were crossed with mice expressing LacZ under the control of the human We examined whether alterations in cell death and cell dopamine- -hydroxylase promoter (21). At E13.5, an proliferation contributed to decreased adrenal size in equivalent number of -gal ( ) cells migrated to the SF-1 / embryos. Here we confirmed the results of adrenal cortex in SF-1 / and / embryos. Sur- Luo and colleagues (1) that adrenal and gonadal agen- prisingly, the same number of -gal ( ) cells also esis in SF-1 / mice is due to programmed cell migrated to the same rostral-caudal location in SF-1 death at E12.0. However, rates of apoptosis did not null embryos, despite the lack of adrenocortical cells differ between SF-1 / and / embryos at any time point examined (Fig. 2, A and B, and data not (Fig. 4A). However, by E15.0, no tissue corresponding shown). We next asked whether a proliferation defect to an adrenal medulla was found in SF-1 / em- could account for decreased adrenal size in SF-1 / bryos. In the presence of one functional SF-1 allele, embryos and predicted that bromo-deoxyuridine growth of the adrenal medulla was diminished and no (BrdU) labeling and histone-3 phosphorylation (indices appreciable infiltration of -gal ( ) cells into the adre- of S phase and mitosis, respectively) would be de- nal was observed at E15.0 or E16.5 compared with creased in SF-1 / embryos (17–20). Unexpectedly, wild type (Fig. 4B and data not shown). although no apparent difference in BrdU labeling was Finally, we asked whether SF-1 haploinsufficiency observed between / and / embryos at E12.5, a affected the earliest stage of adrenal development, significant increase in BrdU-positive cells was de- when adrenal and gonadal precursors are found in a tected in SF-1 / adrenals by E13.5 (Fig. 3, A and B). common primordium derived from the coelomic epi- Two additional stages in development (E15.5 and thelium of the intermediate mesoderm at E9.0. This Fig. 2. Increased Apoptosis Cannot Account for Decreased SF-1 / Adrenal Size A, SF-1 immunoreactivity in genital ridges (white, left panels) and TUNEL staining (for detection of programmed cell death, right panels) in adjacent transverse sections of SF-1 / , / , and / embryos at E12.0. Arrows indicate TUNEL-positive cells in the SF-1 null embryo. Bar, 100 m; da, aorta; m, mesonephros. B, TUNEL-positive cells per genital ridge area in wild-type ( / , black bars), heterozygous ( / , gray bars), and knockout embryos ( / , white bars) from E11.0-E12.0. Rates of apoptosis did not differ between wild-type and heterozygous embryos at any time point examined, although, as expected, rates of apoptosis were increased in knockout embryos at E11.5 and E12.0 (**, P 0.01 vs. wild type, n 3–4 embryos per group). Downloaded from mend.endojournals.org on December 27, 2004
  4. 4. 944 Mol Endocrinol, April 2004, 18(4):941–952 Bland et al. • Compensation for SF-1 Haploinsufficiency Fig. 3. Cell Proliferation Is Increased in SF-1 Heterozygous Adrenals Cell proliferation in embryonic adrenals was studied by measuring BrdU incorporation and histone 3 phosphorylation (indices of S phase and mitosis, respectively). A, BrdU (red) and SF-1 (green) immunoreactivity in transverse sections of SF-1 wild-type and heterozygous embryos at E12.5 and E13.5. White dotted lines outline the embryonic adrenal cortex. Bar, 100 m; g, gonad. B, The ratio of BrdU and SF-1 double-positive cells (yellow) to the total number of SF-1 positive cells is increased in heterozygous embryos ( / , gray bars) compared with wild type ( / , black bars) at E13.5 (*, P 0.05, n 3 per group) but not at E12.5 (P 0.093, n 4 per group). C, BrdU (red) and phospho-histone 3 (green) immunoreactivity in cross sections of SF-1 wild-type and heterozygous adrenals at E17.5. Bar, 100 m. D, The number of BrdU positive cells per area of adrenal is increased in heterozygous embryos ( / , gray bars) compared with wild type ( / , black bars) at E15.5 and E17.5 (**, P 0.01, n 4 per group). Phospho-histone 3 labeling (positive cells per 104 m2) is also increased in heterozygous adrenals compared with wild type at both E15.5 ( / : 1.20 0.03; / : 4.01 0.29; P 0.01; n 4 per group) and E17.5 ( / , 0.99 0.03; / , 1.89 0.15; P 0.01; n 4 per group). precedes the stage when the adrenogonadal primor- embryos, suggesting that SF-1 gene dosage is most dium splits, and the adrenal cortex and gonad begin to critical at the onset of adrenal development (Fig. 5C). develop separately (E11.0). Although SF-1 is required for both adrenal and gonadal development, GATA-4 is SF-1 / Adrenals Have Increased Capacity for required for gonadal development but is dispensable Corticosterone Production for adrenal development (22–24). In comparing the expression patterns of GATA-4 and SF-1 at E12.0, we We have previously shown that adult SF-1 / mice found that, unlike SF-1, GATA-4 marks only gonadal exhibit blunted corticosterone secretion in response to progenitors (Fig. 5A). At E10.0, SF-1/GATA-4 double- stress. Here we asked whether this impaired glucocor- positive cells were observed in the coelomic epithe- ticoid secretion resulted from decreased adrenocorti- lium of the intermediate mesoderm, whereas SF-1 sin- cal mass or a reduced steroidogenic capacity due to gle-positive cells were observed dorsal and somewhat lower levels of SF-1. Surprisingly, SF-1 / adrenals rostral to the SF-1/GATA-4 double-positive population contain more corticosterone per milligram of adrenal (Fig. 5B). We found that the total area of the adreno- weight compared with / adrenals ( / : 70.0 ng/ gonadal primordium did not differ between / and mg, / : 115.9 ng/mg); this finding predicts that cor- / embryos (data not shown). However, the percent- ticosterone secretion per cell would be increased in age of SF-1 single-positive cells in the adrenogonadal SF-1 / adrenals. To test this hypothesis, we mea- primordium was decreased significantly in SF-1 / sured corticosterone secretion from equal numbers of Downloaded from mend.endojournals.org on December 27, 2004
  5. 5. Bland et al. • Compensation for SF-1 Haploinsufficiency Mol Endocrinol, April 2004, 18(4):941–952 945 Fig. 4. Normal Medullary Development Requires a Full Dose of SF-1 in the Cortex SF-1 heterozygous mice were crossed with D H-nLacZ transgenic mice, and embryos were subjected to -gal staining to identify neural crest cells that give rise to the adrenal medulla and sympathetic ganglia. A, Equivalent numbers of medullary precursors (blue) arrived at the adrenocortical blastema or its approximate location at E13.5 in SF-1 / , / , and / embryos. Medullary precursors appeared to infiltrate the wild-type adrenal cortex at this stage. Black dotted lines outline the edges of the adrenal cortex ( / and / embryos) or the ventral body wall ( / embryo). Bar, 100 m; da, dorsal aorta; sg, sympathetic ganglia. B, By E16.5, wild-type adrenals contained a central medulla (arrow), whereas medullary cells had not appreciably infiltrated heterozygous adrenals. In SF-1 knockout embryos, no tissue corresponding to an adrenal medulla was found. Dotted lines outline the adrenal glands. Bar, 500 m; sg, sympathetic ganglia; k, kidney. Fig. 5. GATA-4 Immunoreactivity Defines a Subset of Cells in the Adrenogonadal Primodium A, GATA-4 (red) and SF-1 (green) immunoreactivity in transverse sections of E12.0 embryos. SF-1 and GATA-4 colocalize (yellow) in the gonads but not in the adrenals (arrows). Note decreased adrenal size in the heterozygous embryo. Bar, 100 m; g, gonad; da, dorsal aorta; ce, coelomic epithelium. B, GATA-4 and SF-1 immunoreactivity in longitudinal sections of E10.0 embryos. Arrows point to SF-1-positive, GATA-4-negative cells (green) dorsal to the SF-1 and GATA-4 double-positive cells (yellow) that form the bulk of the adrenogonadal primordia. Bar, 100 m; m, mesonephros. C, The percentage of SF-1-positive, GATA-4-negative cells in the adrenogonadal primordia is decreased in SF-1 heterozygous ( / ) embryos at E10.0. The area of the adrenogonadal primordia was calculated by measuring the area of all SF-1 immunoreactive cells in 15–25 sections per embryo for each genotype, n 4 embryos per genotype. SF-1 / and / adrenocortical cells stimulated mM 8Br-cAMP compared with / adrenocortical with 8-bromo-cAMP (8Br-cAMP). SF-1 / adreno- cells (Fig. 6A). Morphological inspection of cultured cortical cells secreted significantly more corticoste- / and / adrenocortical cells revealed no major rone basally and in response to 0.1 mM, 0.5 mM, and 1 differences (Fig. 6B). Further analysis of SF-1 / Downloaded from mend.endojournals.org on December 27, 2004
  6. 6. 946 Mol Endocrinol, April 2004, 18(4):941–952 Bland et al. • Compensation for SF-1 Haploinsufficiency Fig. 6. SF-1 / Adrenocortical Cells Have Increased Steroidogenic Capacity A, Wild-type and heterozygous cortical cells (20,000 cells per tube, two tubes per treatment) were incubated for 90 min with vehicle or 8Br-cAMP at the concentration indicated. Corticosterone secreted into the media was measured by RIA. Heterozygous cells ( / , gray bars) secreted more corticosterone than wild-type cells ( / , black bars) in response to 0.1 mM 8Br-cAMP, *, P 0.05. Differences in corticosterone secretion at 0.5 mM and 1 mM 8Br-cAMP did not reach statistical significance due to variability (P 0.12 and P 0.11, respectively). B, SF-1 wild-type and heterozygous adrenocortical cells in primary culture show similar morphology (oil red O staining, gray; nuclei, black). Bar, 25 m. C, Western blot analyses of SF-1, StAR, SCC, and actin levels in wild-type and heterozygous primary adrenocortical cells. adrenocortical primary cells revealed reduced expres- maintains high expression levels of SF-1 target genes sion of SF-1, whereas expression of two rate-limiting in SF-1 / adrenals. proteins in steroidogenesis StAR and side chain cleav- Given that SF-1 is a primary regulator of basal and age (SCC), was increased (Fig. 6C); these results are cAMP-stimulated steroidogenic gene expression in identical with those obtained from whole / adrenals the adrenal cortex, increased SF-1 target gene ex- (8, 16). Collectively, these data suggest that factors pression and steroidogenic capacity observed in distinct from SF-1 drive increased expression of ste- SF-1 / adrenals are paradoxical with reduced roidogenic genes, and thus, would account for the SF-1 levels. Therefore, we explored potential mech- increased cellular function observed in SF-1 / ad- anisms that would increase steroidogenic gene ex- renocortical cells. pression downstream of ACTH signaling. One pos- sibility is that phosphorylation of SF-1 on serine Mechanisms Underlying Increased SF-1 Target (Ser) 203 may be increased in SF-1 / adrenals Gene Expression in SF-1 / Adrenals because this posttranslational event is known to enhance SF-1’s ability to recruit coactivators (13). Given that ACTH is the primary regulator of steroido- Using an antibody directed against phospho-Ser genic gene expression and adrenal mass (25), ele- 203 to supershift SF-1 bound to its response ele- vated basal ACTH levels observed in SF-1 / mice ment in gel shift assays (15), we found that the basal most likely drive the increased function observed in phosphorylation state of SF-1 was not significantly SF-1 / adrenals (8). Indeed, inhibiting pituitary different between wild-type and heterozygous adre- ACTH secretion with dexamethasone treatment re- nals (Fig. 8A and data in legend). Other potential sulted in significant decreases in both wild-type and mechanisms may include altered expression of tran- heterozygous adrenal weight due to decreased corti- scription factors that positively regulate steroido- cal cell size (Fig. 7, A and B, and data in legend) (26). genic genes or negatively regulate SF-1 in heterozy- Dexamethasone treatment resulted in a marked re- gous adrenals. One such factor is the orphan duction in the levels of StAR, SCC, and SR-B1, with nuclear receptor liver receptor homolog-1 (LRH-1). equivalent, low levels observed in wild-type and het- LRH-1 shares high identity with SF-1 and thus could erozygous adrenals (Fig. 7, C and D). By contrast, potentially regulate SF-1 target genes (27–29). How- SF-1 levels were unaltered after hormone treatment in ever, no appreciable levels of LRH-1 transcripts either genotype. Finally, dexamethasone treatment were detected in either / or / adrenals using also suppressed corticosterone secretion and led to Northern and RT-PCR analyses (data not shown). high, equivalent levels of CD4 CD8 thymocyte pro- Another candidate factor that binds to similar DNA grammed cell death in both genotypes (data not binding sites as SF-1 is the orphan nuclear receptor shown) (8). These results demonstrate that ACTH nerve growth factor-induced-B (NGFI-B) (30). More- Downloaded from mend.endojournals.org on December 27, 2004
  7. 7. Bland et al. • Compensation for SF-1 Haploinsufficiency Mol Endocrinol, April 2004, 18(4):941–952 947 Fig. 7. Dexamethasone Treatment Normalizes Cellular Hypertrophy and Steroidogenic Gene Expression in SF-1 / Adrenals Adrenal histology and gene expression were assessed in SF-1 wild-type and heterozygous mice treated with vehicle or dexamethasone for 3 d. Dexamethasone treatment inhibited corticosterone secretion after 10 min of restraint stress in wild-type and heterozygous mice (vehicle: / : 24.7 1.7 g/dl, / : 18.3 2.9 g/dl; dexamethasone: / : 0.8 0.2 g/dl, / : 1.3 0.4 g/dl; P 0.01 vs. vehicle treated). A, Toluidine blue staining of adrenal cross sections showed that in vehicle-treated mice, SF-1 heterozygous adrenocortical cells are significantly larger than wild-type cells (cells per 0.01 mm2: / , 98.4 0.4; / , 73.2 0.3; P 0.01 vs. wild type). Dexamethasone treatment reversed SF-1 / adrenocortical cellular hypertrophy and normalized differences in cell size between wild-type and heterozygous adrenals (cells per 0.01 mm2: / , 100.7 0.6; / , 108.3 0.6, P 0.06 vs. wild type). Bar, 100 m. B, Dexamethasone treatment (dex, gray bars) led to decreased adrenal weight in wild-type and heterozygous mice compared with vehicle treatment (veh, black bars), *, P 0.05 vs. vehicle. SF-1 heterozygous adrenals weighed less than wild-type adrenals regardless of vehicle or dexamethasone treatment (**P 0.01 vs. wild type; n 4 per group). C, Western blot analyses of SF-1, SCC, SR-B1, StAR, and actin levels in adrenals from vehicle- and dexamethasone- treated SF-1 wild-type and heterozygous mice (n 2–3 per group). D, SF-1, SCC, SR-B1, and StAR levels were normalized to actin levels. Protein levels in vehicle-treated heterozygous mice (gray bars), dexamethasone-treated wild-type mice (black, stippled bars), and dexamethasone-treated heterozygous mice (gray, stippled bars) are expressed as fold increases or decreases relative to vehicle-treated wild-type mice (black bars) (n 3–4 per group). over, NGFI-B expression is induced by ACTH, and NGFI-B protein levels (2.3-fold on average) ob- NGFI-B, in turn, is thought to participate in ACTH- served in SF-1 / adrenals compared with / induced up-regulation of 21-hydroxylase (31–33). adrenals. It should be noted that the broadly migrat- Northern blot analysis showed a 1.4-fold up-regula- ing NGFI-B signal consists of multiple bands due to tion of NGFI-B in adult heterozygous adrenals com- hyperphosphorylation, as previously reported (Fig. pared with wild type, whereas decreased SF-1 ex- 8D) (34). Finally, we excluded that diminished levels pression was observed in / adrenals (Fig. 8, B of a putative repressor of SF-1, Dax1, might account and C). Up-regulation of NGFI-B expression was for the up-regulation of SF-1 target genes. Instead, confirmed by Western blot analysis, with elevated we find equivalent levels of Dax1 protein in wild-type Downloaded from mend.endojournals.org on December 27, 2004
  8. 8. 948 Mol Endocrinol, April 2004, 18(4):941–952 Bland et al. • Compensation for SF-1 Haploinsufficiency Fig. 8. Up-Regulation of NGFI-B in SF-1 / Adrenal Glands A, EMSA of SF-1 from wild-type and heterozygous adrenal nuclear extracts binding to the gonadotrope-specific element (GSE) from the -glycoprotein subunit promoter. The positions of the SF-1-DNA complex and free probe are indicated (arrows). Increasing amounts of phospho-Ser 203 antisera resulted in the formation of a larger protein complex (arrowhead). Quantitation of results of three separate experiments revealed that, at the highest antibody concentration used, the ratio of shifted to total SF-1 did not differ between genotypes ( / , 52 2%; / , 58 7%; P 0.68). B, Northern blot analyses of NGFI-B, SF-1, and Actin mRNA levels in SF-1 wild-type and heterozygous adrenals. C, NGFI-B was up-regulated 1.4-fold, and SF-1 was expressed at 1.8-fold lower levels in heterozygous adrenals compared with wild-type adrenals. D, NGFI-B was up-regulated in adult SF-1 heterozygous adrenals compared with wild-type (n 6 per genotype). NGFI-B migrates as a broad band due to hyperphos- phorylation. The fold induction of NGFI-B in each sample (relative to the lowest level observed) is indicated above each lane. E, Dax1 levels were equivalent in SF-1 wild-type and heterozygous adrenals from mice treated with vehicle or dexamethasone. and heterozygous adrenals, with or without dexa- erozygous adrenals leads to a marked increase methasone treatment (Fig. 8E). rather than a decrease in steroidogenic capacity due to elevated steroidogenic proteins. Indeed, on a per cell basis, SF-1 / adrenocortical cells produce DISCUSSION more corticosterone per dose of cAMP than / adrenocortical cells. Although these cellular Our results demonstrate that loss of one SF-1 allele changes permit SF-1 heterozygotes to maintain rel- results in a dramatic reduction of adrenocortical atively normal basal glucocorticoid secretion, their precursors, which ultimately leads to adrenal insuf- ability to secrete sufficient glucocorticoids during ficiency in the adult. However, partial compensation severe stress remains limited by the overall loss of for decreased adrenal mass occurs both during late adrenal precursors during development (8). adrenal development and in the adult adrenal. The SF-1 gene dosage is most critical during the earliest early growth defect in the SF-1 / adrenocortical stages of adrenal development as evidenced by the primordium is met with increased cell proliferation severe reduction of adrenocortical precursors in SF-1 later in development that allows / adrenals to / embryos at E12.0. Whereas our immunocyto- approach but not attain / adrenal size. Unex- chemical analysis of GATA-4 and SF-1-positive cells pectedly, the loss of SF-1 protein observed in het- at E10.0 revealed that the total size of the adrenogo- Downloaded from mend.endojournals.org on December 27, 2004
  9. 9. Bland et al. • Compensation for SF-1 Haploinsufficiency Mol Endocrinol, April 2004, 18(4):941–952 949 nadal primordium does not differ in / and / iological responses to stress, but elevated ACTH embryos, our findings indicated that fewer cells are levels in / mice stimulate steroidogenic gene ex- dedicated to the adrenal (SF-1 positive, GATA-4 neg- pression and raise steroidogenic capacity per cell, ative) in SF-1 heterozygotes at this stage of develop- ensuring relatively normal basal corticosterone secre- ment. Our data suggest that SF-1 is important for tion (1, 8). Are heterozygous levels of SF-1 sufficient to expansion of adrenal progenitors in the adrenogo- maintain SF-1 target gene expression at supernormal nadal primordium. Separation of adrenal and gonadal levels in / adrenals or are other mechanisms such precursors may also rely on SF-1. At this step, SF-1 as posttranslational modification, ligand availability, or could act in concert with the signaling molecule Wnt4, up-regulation of other transcription factors employed? which was recently shown to repress migration of Although it is known that phosphorylation of SF-1 on adrenal precursors into the developing gonad (35). Ser 203 promotes SF-1 transcriptional activity, we did Delineating SF-1’s role in the earliest steps of adrenal not detect significant differences in the ratio of phos- development will require identification of SF-1’s em- phorylated to total SF-1 in / and / adrenals. bryonic target genes. Increased ligand availability is also an unlikely mech- A consequence of impaired adrenocortical develop- anism because neither an exogenous nor an endoge- ment in SF-1 / and / embryos is disrupted nous obligatory ligand has been identified for SF-1, adrenomedullary development. We found that the ini- consistent with the fact that SF-1 is constitutively ac- tial migration of neural crest cells to the adrenal cortex tive in a variety of steroidogenic and nonsteroidogenic (or its approximate location) occurs normally regard- cell lines (40). Furthermore, biophysical and structural less of SF-1 dosage, showing that neural crest cells do evidence suggests that members of nuclear receptor not rely on signals from adrenocortical cells for proper subfamily V (SF-1 and LRH-1) adopt an active confor- homing. However, at later stages of development, mation in the apparent absence of ligand (15, 41). medullary cells are lost in SF-1 / embryos and the Increased or decreased activity of other transcrip- medulla fails to infiltrate the small SF-1 / adrenal tion factors may underlie the paradoxical increases in cortex. Our data show that normal growth and survival SF-1 target gene expression in SF-1 / adrenals. of the adrenal medulla depends on SF-1 function in the For example, the orphan nuclear receptor Dax1 re- adrenal cortex. It will be of interest to determine presses SF-1 activity in vitro (42–44). However, loss of whether the requirement for SF-1 is indirect or direct. Dax1 does not lead to further increases in SF-1 target For instance, normal medullary growth may simply gene expression in SF-1 / adrenals (16). Indeed, require sufficient adrenocortical mass. Alternatively, we found that Dax1 levels were similar in SF-1 / SF-1 might directly regulate genes that serve as para- and / adrenals, regardless of circulating ACTH lev- crine growth factors for medullary cells. This occurs in els. Our data show that NGFI-B levels are significantly skin, where target-derived growth factors support elevated in SF-1 / adrenals, supporting a compen- expansion and correct localization of neural crest- satory role for NGFI-B in the regulation of steroido- derived melanocyte precursors (36). genic gene expression. However, the normal adrenal How can the early growth defects in SF-1 / responses to stress in NGFI-B null mice suggest that adrenals be reconciled with their impressive increase NGFI-B does not normally regulate steroidogenic in cell proliferation later in development? Perhaps de- genes, including 21-hydroxylase (32). Taken together, creased adrenal mass is somehow sensed at E13.5 these data indicate that NGFI-B does not serve as the and leads to increased adrenal growth factor levels primary regulator of steroidogenic genes but may as- that drive cell proliferation in SF-1 heterozygotes. This sume a more important role when SF-1 dosage is strategy would parallel the compensatory response to reduced. SF-1 haploinsufficiency in the adult adrenal in which Although our data confirm and extend the essen- elevated ACTH levels lead to increased steroidogenic tial role of SF-1 in early embryonic adrenal develop- capacity per cell. Another anterior pituitary hormone, ment, they also lead us to question SF-1’s precise pro- -MSH, stimulates adrenal cell proliferation after role in late adrenal development and in the adult undergoing local proteolysis (carried out by adrenal adrenal gland. The increase in steroidogenic capac- secretory protease) that produces a shorter form with ity in SF-1 / adrenocortical cells is particularly mitogenic activity (26, 37, 38). This signaling pathway unexpected given the central role that SF-1 has is thought to account for the compensatory adrenal been thought to play in coordination of adrenal ste- growth response after removal of one adrenal. The roidogenesis. Our study strongly suggests that al- lack of this growth response in SF-1 / mice might ternative molecular mechanisms exist to increase suggest that SF-1 participates in pro- -MSH signaling expression of many SF-1 target genes and illus- (39). It will be of interest to determine the interplay trates the differential requirement for transcription between SF-1, ACTH, and/or pro- -MSH signaling factor function in development and the adult. Future pathways during early adrenal development. experiments aimed at generating a temporal- Ultimately, increased cell proliferation in SF-1 / specific SF-1 deletion in the adult will be essential adrenals cannot compensate for the early deficits in for dissecting SF-1’s well-established role in adrenal adrenal development. SF-1 / adrenals do not se- development from its less-defined role in regulating crete enough corticosterone to support normal phys- adult adrenal steroidogenesis. 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  10. 10. 950 Mol Endocrinol, April 2004, 18(4):941–952 Bland et al. • Compensation for SF-1 Haploinsufficiency MATERIALS AND METHODS Histological Analysis Detection of -gal in D H-nLacZ transgenic embryos was Animal Experiments performed as described previously (21). Adrenal sections (10 m) were stained with toluidine blue O. Cellular hypertrophy SF-1 / and / mice obtained from The Jackson Labora- was assessed by counting Hoechst-stained nuclei per 0.01 tory (Bar Harbor, ME), were maintained on a C57BL/6J FVB mm2 in four sections per / and / adrenal (n 4 background, and cared for in accordance with National Insti- adrenals per group). tutes of Health (NIH) guidelines. Experimental procedures were approved by the University of California, San Francisco Labo- ratory Animal Research Committee. Mice were kept on a 12-h Western and Northern Analysis light, 12-h dark cycle (lights on 0600–1800 h) and were given food and water ad libitum. Male mice 6–8 wk old were used for Western analyses were carried out as described previously all experiments unless otherwise noted. Dopamine -hydroxy- (8). For Western analysis, each lane represents a separate lase-nuclear LacZ (D H-nLacz) transgenic mice (from Dr. R. individual, n 3–6 per genotype. Additional antibodies used Kapur, University of Washington) were crossed with SF-1 / in this study were: rabbit anti-SR-B1 (1:20,000, Novus Bio- mice. Animals were genotyped as described previously (45). For logicals, Littleton, CO), mouse anti-NGFI-B [1:10,000, a kind dexamethasone experiments, / and / mice were injected gift of Dr. J. Milbrandt (Washington University, St. Louis, ip with saline or dexamethasone sodium phosphate (0.5 mg/kg, MO)], and goat antimouse horseradish peroxidase (1:10,000, Sigma, St. Louis, MO), twice per day (0900 and 1730 h) for 3 d Bio-Rad, Hercules, CA). For quantification of protein levels, (n 5 per group). On the fourth day beginning at 0800 h, mice scanning densitometry was performed on blots developed were exposed to 10 min of restraint stress and decapitated. with chemiluminescence (ECL, Amersham Biosciences, Pis- Thymus cells from vehicle- and dexamethasone-treated mice cataway, NJ). These levels were confirmed by quantifying radioactive signals from Western blots performed with a ra- were analyzed by flow cytometry as described previously (8). diolabeled goat antirabbit secondary antibody (NEN, Boston, Plasma and adrenal corticosterone were measured using a MA). For Northern analyses, total RNA (20 g) prepared from commercially available (ICN Pharmaceuticals, Costa Mesa, SF-1 / and / adrenals was separated by formalde- CA) kit. hyde-gel electrophoresis, transferred to nylon membranes, and hybridized overnight at 42 C with random-primed, la- Measurements of Cellular Proliferation and Cell Death beled DNA probes for fragments of the mouse SF-1, mouse LRH, rat NGFI-B, and rat actin cDNAs. Membranes were washed at medium stringency (0.2 sodium chloride sodium For BrdU labeling, timed-pregnant mice received an ip injec- citrate, 0.1% sodium dodecyl sulfate at 42 C) and exposed to tion of BrdU (50 mg/kg, Sigma). After a 1-h pulse, whole X-OMAT film (Kodak, Rochester, NY). For Northern blot and embryos or fetal adrenals were collected, fixed overnight in EMSA (see EMSA) experiments, radioactive signals were 4% paraformaldehyde, cryoprotected in 30% sucrose, and quantified with ImageQuant Mac software (Amersham Bio- frozen in OCT compound (Tissue Tek Sakura, Torrance, CA). sciences after exposure to a phosphorimager screen (Storm, Cryostat sections (10 m) were treated with 2 N HCl at 37 C Amersham Biosciences). for 20 min to denature DNA, blocked in 10% normal goat serum, incubated overnight at 4 C with rat anti-BrdU antisera (1:10, Harlan Sera-Lab) and either rabbit anti-SF-1 (1:1000) or Primary Cell Culture rabbit antiphosphorylated histone 3 (phospho-His3, 1:1000, Upstate Biotechnologies, Waltham, MA), washed, and incu- Adrenals from female mice were dissected free of fat, bated for 2 h at room temperature with goat antirabbit Alexa minced, and washed in culture medium (M-199 with 4 mg/ml 488 and goat antirat Alexa 546 secondary antibodies (1:200 BSA plus penicillin and streptomycin). Cells were incubated each, Molecular Probes, Eugene, OR). Images were collected in dispersal medium (M-199 containing 20 mg/ml BSA plus with a confocal microscope, and adrenal cross-section area penicillin and streptomycin, 2.5 mg/ml type I collagenase and the number of BrdU-positive ( ) and phospho-His3 ( ) (Invitrogen, Carlsbad, CA), and 10 g/ml deoxyribonuclease cells per section were measured with the NIH Image pro- I) for 30 min at 37 C with shaking and were dissociated by gram. The number of digitally counted BrdU ( ) and phos- repeated pipetting every 10 min, filtered over 70- m nylon pho-His3 ( ) cells was confirmed by visual assessment to mesh, washed twice by centrifugation, and resuspended in ensure appropriate parameter settings. Apoptosis was de- culture medium. Equivalent numbers of SF-1 / and / tected using an in-house terminal deoxynucleotidyl trans- cortical cells were incubated in 950 l culture medium at 37 ferase-mediated deoxyuridine triphosphate nick end-labeling C with 5% CO2. After 1 h, H2O or 8-bromo-cAMP (Sigma) at (TUNEL) assay as described previously (45). For each sec- final concentrations of 0.1, 0.5, and 1 mM were added in a tion, the number of TUNEL ( ) cells was divided by the area volume of 50 l. Cells were incubated for 90 min, pelleted by of SF-1 immunoreactivity. For SF-1 / embryos, the aver- centrifugation, and the supernatant was removed for corti- age number of TUNEL ( ) cells per section was divided by costerone measurements. Cells were lysed with 2% sodium the average area of SF-1 immunoreactivity in SF-1 / dodecyl sulfate, 100 mM dithiothreitol (DTT), and 60 mM Tris- genital ridges. HCl (pH 6.8), and Western blot analyses was carried out as For measurement of adrenogonadal primordia size, em- described above. Small aliquots of / and / cells were bryos were staged by counting somites. Embryos were fixed plated on tissue culture slides coated with collagen and in- cubated overnight at 37 C, 5% CO2 in culture medium plus as described above. Cryostat sections (10 m) were blocked 10% fetal calf serum. The next day, cells were fixed with 4% for 30 min in 10% normal donkey serum, and incubated paraformaldehyde and stained with oil red O. overnight at 4 C with rabbit anti-SF-1 (1:200) and goat anti- GATA-4 (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The next day, sections were washed, incubated for 2 h EMSA at room temperature with donkey antirabbit Alexa 488 (1:200, Molecular Probes) and donkey antigoat Cy3 (1:200, Jackson Nuclear extracts were prepared from adrenal glands collected ImmunoResearch, West Grove, PA). Images were collected under basal conditions at 1730 h. Adrenals were cleaned of fat, with a confocal microscope and the total areas of all SF-1 ( ) homogenized in cold PBS, and centrifuged at 4000 rpm for 5 cells and SF-1 ( ), GATA-4 ( ) cells were measured with NIH min. Cells from eight to 12 adrenals were resuspended in 400 l Image. buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 Downloaded from mend.endojournals.org on December 27, 2004
  11. 11. Bland et al. • Compensation for SF-1 Haploinsufficiency Mol Endocrinol, April 2004, 18(4):941–952 951 mM EGTA, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride] have normal embryonic serum levels of corticosteroids. and incubated on ice for 15 min before the addition of 25 l of Proc Natl Acad Sci USA 92:10939–10943 0.1% Nonidet P-40 in buffer A. Nuclei were vortexed for 10 sec 4. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba and centrifuged at 11,000 rpm for 30 min. Nuclei were resus- H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Morohashi pended in 50 l buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 K-I, Li E. 1995 Developmental defects of the ventrome- mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsul- dial hypothalamic nucleus and pituitary gonadotroph in fonyl fluoride], rotated at 4 C for 15 min and then centrifuged for the Ftz-F1 disrupted mice. Dev Dyn 204:22–29 5 min at 11,000 rpm. The supernatant was subjected to EMSAs 5. Achermann JC, Ito M, Hindmarsh PC, Jameson JL 1999 as follows: oligonucleotides encoding the SF-1 response ele- A mutation in the gene encoding steroidogenic factor-1 ment in the human glycoprotein hormone -subunit promoter causes XY sex reversal and adrenal failure in humans. (forward: 5 -GCTGACCTTGTCGTCAC-3 , reverse: 5 -GTGAC- Nat Genet 22:125–126 GACAAGGTCAGC-3 ) were annealed and radiolabeled as de- 6. Achermann JC, Ozisik G, Ito M, Orun UA, Harmanci K, scribed (14). In each binding reaction, 1–3 g of adrenal nuclear Gurakan B, Jameson JL 2002 Gonadal determination protein extracts were mixed with the labeled probes in 20 l and adrenal development are regulated by the orphan volume of 20 mM Tris (pH 8.0), 60 mM KCl, 2 mM MgCl2, 1.2 mM nuclear receptor steroidogenic factor-1, in a dose-de- DTT, 12% glycerol, 2.5 g poly (deoxyinosine-deoxycytosine), pendent manner. J Clin Endocrinol Metab 87:1829–1833 1% (wt/vol) BSA, incubated at room temperature for 5 min 7. Biason-Lauber A, Schoenle EJ 2000 Apparently normal before the addition of 2 l of probe (200,000 cpm) and incuba- ovarian differentiation in a prepubertal girl with transcrip- tion for 15 min at 30 C. Typically, 8 l of the reaction mixture tionally inactive steroidogenic factor 1 (NR5A1/SF-1) and were resolved on a 5% native acrylamide gel, dried and visual- adrenocortical insufficiency. Am J Hum Genet 67: ized by autoradiography. For all antibody gel-shift experiments, 1563–1568 0.1–3.0 l of anti-phospho-SF-1 antiserum was added to the 8. Bland ML, Jamieson CA, Akana SF, Bornstein SR, Eisen- reaction minus probe and incubated on ice for 60 min. hofer G, Dallman MF, Ingraham HA 2000 Haploinsuffi- ciency of steroidogenic factor-1 in mice disrupts adrenal Statistical Analysis development leading to an impaired stress response. Proc Natl Acad Sci USA 97:14488–14493 9. Ikeda Y, Shen WH, Ingraham HA, Parker KL 1994 Devel- Data are presented as means SEM. Unpaired two-tailed t opmental expression of mouse steroidogenic factor-1, tests and ANOVA were used to determine statistical an essential regulator of the steroid hydroxylases. Mol significance. Endocrinol 8:654–662 10. Morohashi K 1997 The ontogenesis of the steroidogenic Acknowledgments tissues. Genes Cells 2:95–106 11. Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a We wish to acknowledge Drs. Mary Dallman and Marion key determinant of endocrine development and function. Desclozeaux [University of California, San Francisco (UCSF)] Endocr Rev 18:361–377 for discussions. We are especially grateful to Dr. C. Jamieson 12. Sewer MB, Waterman MR 2002 Adrenocorticotropin/ (University of California, Los Angeles) for measurement of cyclic adenosine 3 ,5 -monophosphate-mediated tran- thymocyte apoptosis and Dr. R. Kapur (University of Wash- scription of the human CYP17 gene in the adrenal cortex ington, Seattle, WA) for the generous gift of the D H-nLacZ is dependent on phosphatase activity. Endocrinology transgenic mice. We thank Drs. W. Miller (UCSF) for StAR and 143:1769–1777 SCC antibodies, K. Morohashi (National Institute for Basic 13. Hammer GD, Krylova I, Zhang Y, Darimont BD, Simpson Biology, Okazaki, Japan) for the SF-1 antibody, and J. Mil- K, Weigel NL, Ingraham HA 1999 Phosphorylation of the brandt (Washington University, St. Louis, MO) for the NGFI-B nuclear receptor SF-1 modulates cofactor recruitment: antibody. integration of hormone signaling in reproduction and stress. Mol Cell 3:521–526 14. Fowkes RC, Desclozeaux M, Patel MV, Aylwin SJ, King P, Ingraham HA, Burrin JM 2003 Steroidogenic factor-1 Received August 29, 2003. Accepted January 8, 2004. (SF-1) and the gonadotrope-specific element (GSE) en- Address all correspondence and requests for reprints to: hance basal and pituitary adenylate cyclase-activating Holly A. Ingraham, Department of Physiology, Box 0444, polypeptide (PACAP)-stimulated transcription of the hu- University of California, San Francisco, San Francisco, Cali- man glycoprotein hormone -subunit gene ( GSU) in fornia 94143-0444. E-mail: hollyi@itsa.ucsf.edu. gonadotropes. Mol Endocrinol 17:2177–2188 This work was supported by the American Heart Associ- 15. Desclozeaux M, Krylova IN, Horn F, Fletterick RJ, Ingra- ation (Predoctoral Fellowship to M.L.B.) and by National In- ham HA 2002 Phosphorylation and intramolecular stabi- stitutes of Health-National Institute of Diabetes and Digestive lization of the ligand binding domain in the nuclear and Kidney Diseases (RO1 to H.A.I.). receptor steroidogenic factor 1. Mol Cell Biol 22: 7193–7203 16. 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