【关键词】 function
departments of molecular and integrative physiology (h.n.w., g.d.h.) and internal medicine (g.d.h.), division of metabolism, endocrinology
diabetes, university of michigan medical school, ann arbor, michigan 48109-0678
department of molecular and cellular biology, baylor college of medicine (j.x., b.w.o.), houston, texas 77030
abstract
steroidogenic factor-1 (sf-1), has emerged as a critical nuclear receptor regulating development and differentiation at several levels of the hypothalamic-pituitary-steroidogenic axis. although many coregulatory factors have been shown to physically and functionally interact with sf-1, the relative importance of these interactions in sf-1 target tissues has not been thoroughly established. in this study we assessed roles of steroid receptor coactivator-1 (src-1) in hypothalamic-pituitary-adrenal (hpa) axis function using src-1-deficient (src-1/) mice in the absence or presence of sf-1 haploinsufficiency. surprisingly, src-1 deficiency did not alter baseline hpa axis function or the acute rise in corticosterone after acth administration and failed to exacerbate adrenocortical dysfunction in sf-1+/ mice. however, after exposure to paradigms of acute and chronic stress, src-1/ mice exhibited an elevation in serum corticosterone despite normal (nonsuppressed) acth, suggesting an increase in adrenal sensitivity as well as a concomitant defect in glucocorticoid-mediated feedback inhibition of the hpa axis. an examination of potential compensatory mechanism(s) revealed an increase in adrenal weight, selective elevation of melanocortin 2 receptor mrna, and a coincident increase in src-2 and src-3 expression in src-1/ adrenals. a reduction in blood glucose was observed in src-1/ mice after chronic stress, consistent with a generalized state of glucocorticoid resistance. dexamethasone suppression tests confirmed a weakened ability of glucocorticoids to 1) elevate serum glucose levels and induce hepatic phosphoenolpyruvate carboxykinase transcription and 2) suppress pituitary proopiomelanocortin transcript levels in src-1/ animals. collectively, these data are consistent with an indispensable role for src-1 in mediating actions of glucocorticoids in pituitary and liver.
introduction
although originally cloned as the transcription factor responsible for the camp-dependent regulation of steroid hydroxylases in the adrenal cortex, the use of gene-targeting approaches to examine the in vivo function of steroidogenic factor-1 (sf-1) have revealed additional roles for this nuclear receptor in adrenal/gonadal organogenesis, pituitary gonadotrope expression, and migration of cell populations during development of the ventromedial hypothalamus as well as its traditional role as a transcriptional coordinator of genes involved in the steroid biosynthetic pathway (1, 2, 3, 4, 5). human patients and mice harboring mutations/deletions in the sf-1 gene present with various degrees of adrenal insufficiency and/or sex reversal (6, 7, 8).
the mammalian stress response elicits a coordinated transcriptional program within the adrenal cortex through the actions of the proopiomelanocortin (pomc)-derived peptide hormone, acth. the binding of acth to its cognate g protein-coupled receptor activates a variety of signaling cascades that collectively implement acth-dependent responses, namely, the transcriptional activation of genes involved in steroid biosynthesis. sf-1 is an indispensable mediator of this process by virtue of its essential role in the transcriptional regulation of major steroidogenic enzyme genes, including cytochrome p450 cholesterol side-chain cleavage enzyme (scc) (9), 17-hydroxylase (10), 3-hydroxysteroid dehydrogenase (11), and 21-hydroxylase (12). several additional genes involved in steroid biosynthesis require sf-1 for their appropriate regulation, including the receptor for acth [melanocortin 2 receptor (mc2r)] and the steroidogenic acute regulatory protein (star), both early determinants of steroid biosynthesis in the adrenal gland (13, 14, 15, 16).
the activation of target genes by nuclear receptors requires the regulated recruitment of a variety of coregulatory factors that intrinsically possess or recruit proteins with enzymatic activities that alter the local chromatin environment in a manner that favors promoter occupancy and the subsequent recruitment of rna polymerase ii. a variety of cofactors have been implicated in the coordinated regulation of the acth-dependent transcriptional response based upon the ability of these proteins to physically and/or functionally interact with sf-1 to modulate gene transcription. included in this group of coregulators are general transcription factors [specificity protein 1, jun, transcription initiation factor iib (tfiib), nuclear factor-1, ccaat/enhancer-binding protein-, and activating protein-1] (17, 18, 19, 20, 21), transcriptional corepressors [dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the x-chromosome protein 1), dp103 (dead-box protein 103), cops2 (cop9 constitutive photomorphogenic homolog subunit 2), rip 140 (receptor-interacting protein 140), and smrt (silencing mediator of retinoic acid and thyroid hormone receptor)] (22, 23, 24, 25, 26) as well as a variety of general coactivators [e1a binding protein p300/cbp (camp response element binding protein-binding protein), trep 132 (transcriptional regulating factor 1), and p160 steroid receptor coactivator (src) family] (27, 28, 29, 30, 31). surprisingly, the in vivo significance of these interactions within the context of the hypothalamic-pituitary-steroidogenic axis has not been thoroughly assessed despite the documented involvement of coregulatory factor dysfunction in various states of hormone resistance and cancer (32, 33, 34).
each member of the src family of coregulators has been shown to serve as a coactivator for sf-1 on a variety of adrenal-specific promoters. gene-targeting approaches to ablate each member of this family in mice has revealed redundant functions as well as differential roles for each member in various endocrine systems (35). in this study, we assessed the impact of src-1 deficiency on hypothalamic-pituitary-adrenal (hpa) axis function to discern whether src-1 participates in sf-1-dependent adrenocortical function in vivo.
materials and methods
experimental animals
all experiments involving animals were performed in accordance with institutionally approved and current animal care guidelines. male study cohort mice were obtained by performing an initial intercross between inbred male src-1/ mice (c57bl/6j) and female sf-1+/ mice (c57bl/6j x 129/svj) to generate src-1+//sf-1+/ progeny. these animals were bred to produce male offspring [wild type (wt), sf-1+/, src-1/, and sf-1+//src-1/], which were examined in the current study. animals were genotyped by pcr of genomic tail dna as previously described (36, 37). mice were maintained at constant temperature (22 c) and light cycle (12-h light, 12-h dark) with food and water given ad libitum. all animals were individually housed for 24 h preceding all procedures. baseline blood samples were obtained at 0900 h by retroorbital puncture within 1 min of initial mouse handling to minimize stress-induced acth secretion.
chromatin immunoprecipitation (chip)
primary adrenocortical cells were prepared from adrenals obtained from six mice (c57bl/6j) as previously described (38). primary cells were plated in 60-mm dishes and serum deprived for 6 h in rpmi 1640 medium supplemented with 0.05% bsa. cells were treated with vehicle or acth-(124) at a final concentration of 1 x 108 m for 60 min. proteins were cross-linked by incubation in 1% paraformaldehyde at 37 c for 10 min, washed twice with ice-cold pbs, resuspended in lysis buffer [1% sodium dodecyl sulfate, 10 mm edta, 50 mm tris-hcl (ph 8.1), and 1x protease inhibitor cocktail], sonicated four times for 10 sec each time at maximum setting (sonic dismembrator, model 300, fisher scientific, pittsburgh, pa), followed by centrifugation at 4 c for 15 min. chip assays were performed essentially as previously described (39).
immunohistochemistry
adrenal glands from wt and src-1/ animals were rapidly dissected and placed in 4% paraformaldehyde for 1 h at room temperature. tissues for histochemistry (n = 4/genotype) were dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard protocols. for src-1, src-2, and src-3 immunohistochemistry, paraffin-embedded sections were rehydrated, boiled in 10 mm sodium citrate (ph 6.0) for 10 min to retrieve antigen, and blocked with blocking buffer [5% goat serum (jackson immunoresearch laboratories, inc., west grove, pa), 3% bsa (roche, indianapolis, in), and 0.5% tween-20] for 30 min at room temperature. src-1, src-2, and src-3 rabbit polyclonal antibodies (santa cruz biotechnology, inc., santa cruz, ca) and nonimmune rabbit igg (pierce chemical co., rockford, il) were diluted 1:200 in blocking buffer and incubated overnight at 4 c. sections were incubated with a biotin-conjugated goat antirabbit secondary antibody (1:250; molecular probes, eugene, or) for 30 min at room temperature, followed by incubation with streptavidin-conjugated fluorescein (1:400; molecular probes). nuclei were counterstained with propidium iodide (1:4000; kirkegaard & perry laboratories, gaithersburg, md) for 5 min at room temperature. after rinsing, coverslips were mounted with aqueous mounting medium. sections were observed using a optiphot-2 fluorescence microscope (nikon, melville, ny), and pictures were captured with spot advance software (diagnostics instruments, inc., sterling heights, mi).
acth stimulation test
dexamethasone (sigma-aldrich corp., st. louis, mo) was reconstituted in saline and injected ip at a dose of 5 μg/g body weight at 1800 h the night before and at 0800 h the morning of the procedure. acth (peninsula laboratories, inc., belmont, ca) was reconstituted in saline and injected ip at a dose of 1 μg/g body weight at 1000 h. tail blood was collected at 0-, 15-, 30-, and 60-min intervals for determination of serum corticosterone levels using a commercially available assay (alpco diagnostics, windham, nh). each group consisted of five to 13 animals.
restraint stress
adult male mice were subjected to restraint stress by placement in a perforated 50-ml plastic conical tube. blood was collected by performing retroorbital bleeds after 30 min. serum acth and corticosterone were determined using commercially available assays (alpco diagnostics). each group consisted of nine to 13 animals.
food deprivation stress
mice were deprived of food with free access to water for 48 h, followed by decapitation and trunk blood collection within 1 min of initial handling adrenals were isolated, dissected, and snap-frozen in liquid nitrogen for subsequent protein/rna analysis. determination of serum acth, and corticosterone was performed as previously mentioned. each group consisted of six or seven animals.
dexamethasone suppression
water-soluble dexamethasone (sigma-aldrich corp.) was reconstituted in saline and injected at a dose of 2 μg/g body weight at 2300 h the evening before experimental procedures were performed. dexamethasone-suppressed and control nonsuppressed mice were killed within 1 min of initial handling. adrenals, liver, and pituitaries were isolated, dissected, and snap-frozen in liquid nitrogen for subsequent rna analysis. each group consisted of four to six animals.
quantitative rt-pcr
total rna was isolated from single mouse adrenals using the trizol reagent (invitrogen life technologies, inc., carlsbad, ca) according to the manufacturer’s suggestions. isolated rna was subjected to deoxyribonuclease treatment using the dna-free reagent (ambion, austin, tx). one microgram of mrna was used to generate cdna using the iscript cdna synthesis kit (bio-rad laboratories, inc., hercules, ca) according to the manufacturer’s directions. quantitative pcr was performed using either the quantitect sybr green pcr kit (qiagen, valencia, ca) or the platinum quantitative pcr supermix-udg reagent (invitrogen life technologies, inc.) when lux primers were employed. real-time pcr was performed on the 7500 fast real-time pcr system (applied biosystems, foster city, ca). all quantitative pcr was performed using gene-specific primers (table 1), and data were normalized using glyceraldehyde-2-phosphate dehydrogenase as an internal standard.
western blotting
protein extracts were prepared as previously described (40). briefly, adrenals were sonicated in lysis buffer [50 mm hepes (ph 7.6), 250 mm nacl, 0.5 m edta, and 0.5% nonidet p-40] supplemented with protease inhibitor mixture (sigma-aldrich corp.). samples were allowed to rotate at 4 c for 1 h, and protein contents of the high-speed supernatant were determined using the bradford reagent (bio-rad laboratories, inc.). equal quantities of protein (25 μg) lysate were resolved by sds-page and transferred to nitrocellulose for western blotting. membranes were blocked in 25 mm tris-hcl (ph 8.0), 125 mm nacl, and 0.1% tween 20 supplemented with 5% nonfat dry milk for 1 h at room temperature. membranes were incubated with primary rabbit polyclonal antibodies to src-1, src-2, and src-3 (santa cruz biotechnology, inc.) overnight at 4 c in 25 mm tris-hcl (ph 8.0), 125 mm nacl, and 0.1% tween supplemented with 2.5% nonfat dry milk. proteins were visualized using the supersignal west dura extended duration substrate (pierce chemical co.).
results
src-1 is recruited to steroidogenic enzyme gene promoters in an acth-dependent manner
chip assays were performed to investigate whether src-1 was actively recruited to steroidogenic enzyme gene promoter regions harboring sf-1 response elements that confer acth responsiveness (14, 15, 21, 41, 42). acth stimulation of primary mouse adrenocortical cells led to the robust recruitment of src-1 to the mc2r and star proximal promoter regions (fig. 1). pcr amplification of 1% input controls using the same primers verified the presence of equal quantities of chromatinized template in -src-1 immunoprecipitations. moreover, acth stimulation of the y1 adrenocortical cell line led to the active recruitment of all src family members to the mc2r promoter. interestingly, the temporal dynamics of recruitment for each src family member was distinct, suggesting a unique role(s) for each coactivator in the processes leading to transcriptional initiation (data not shown) (43). these data demonstrate that src-1 is actively recruited to the mc2r and star gene promoter regions encompassing sf-1 response elements in an acth-dependent manner, suggesting that src-1 is an in vivo component of sf-1 containing coactivator complex(s) within a chromatinized environment.
src family members are expressed throughout the definitive zone of the adrenal gland
although src-1 expression has been documented in the adrenal gland, specific localization of src-1 expression to different anatomical and functional regions has not been assessed (44, 45, 46, 47). immunohistochemistry performed on sections from wt adrenals using -src-1 antibodies confirmed that src-1 expression is predominantly nuclear and localized to the glomerulosa and fasciculata as well as the subcapsular region of the adrenal cortex (fig. 2). nuclear src-1 localization was also observed throughout the adrenal medulla. appreciable staining was not observed when similar immunohistochemical analysis was performed on src-1-deficient adrenals (see supplemental fig. 1, published as supplyemental data on the endocrine society’s journals online web site at http://endo.endojournals.org). similar patterns of expression were observed for src-2 and src-3. immunohistochemistry performed in parallel with nonimmune igg revealed the presence of low, nonspecific background immunofluorescence (data not shown). the anatomical expression pattern observed for all p160 src family members in the adrenal cortex correlates with their proposed involvement in acth-mediated transcription of steroidogenic enzyme genes.
src-1 deficiency does not alter steady-state hpa axis function or acute acth responsiveness
we next examined the baseline hormone levels of the hypothalamic-pituitary-adrenal axis in wt, sf-1+/, src-1/, and sf-1+//src-1/ mice to assess whether src-1 deficiency alters hpa axis dynamics and whether the primary adrenocortical dysfunction manifest by sf-1 haploinsufficiency would exacerbate underlying src-1-dependent defects. as previously reported, sf-1+/ mice exhibited a substantial increase in acth at baseline compared with wt animals (410.81 ± 52.40 vs. 260.53 ± 12.96 pg/ml; p < 0.01) due to primary adrenal insufficiency (fig. 3a) (37, 48). acth levels in sf-1+//src-1/ animals paralleled those observed in sf-1+/ mice compared with wt controls (375.183 ± 61.24 vs. 260.53 ± 12.96 pg/ml; p < 0.05) suggesting that src-1 deficiency has little or no impact on sf-1-dependent, steady-state hpa axis function. aldosterone (data not shown) and corticosterone levels at baseline were similar for all groups studied (fig. 3b). to more carefully address any functional role of src-1 in adrenal responsiveness, acth stimulation tests were performed on dexamethasone-suppressed wt, sf-1+/, src-1/, and sf-1+//src-1/ mice. after ip administration of acth, wt and src-1/ animals exhibited similar time-dependent increases in serum corticosterone over the 1-h time course (fig. 3c). as expected, sf-1+/ mice exhibited a blunted response, with significantly lower serum corticosterone levels at 20 and 40 min (37). src-1 deficiency did not exacerbate the response observed in sf-1+/ mice, because sf-1+//src-1/ mice exhibited a response similar to that observed in sf-1+/ mice. collectively, these data suggest that src-1 deficiency does not dramatically alter baseline or acute adrenal responsiveness to the actions of acth.
src-1-deficient mice elicit an elevated corticosterone response after restraint stress
mice were subjected to restraint stress for 30 min, followed by measurement of plasma acth and corticosterone. after restraint stress, sf-1+/ mice exhibited a slightly higher, nonsignificant increase in plasma acth compared with wt controls (1669.36 ± 212.45 vs. 1204.96 ± 77.65 pg/ml) with a concomitantly lower corticosterone level compared with wt littermates (265.05 ± 19.77 vs. 390.14 ± 29.17 ng/ml; p < 0.01) as previously described (fig. 4) (37). interestingly, src-1/ mice had a significantly elevated corticosterone response (508.03 ± 28.08 vs. 390.14 ± 29.17 ng/ml; p < 0.01) despite normal (nonsuppressed) acth values compared with wt mice (1265.17 ± 128.79 vs. 1204.96 ± 77.65 pg/ml). the combined findings that 1) acth was not lower in src-1/ mice after restraint; and 2) acute acth administration did not result in an increased corticosterone response in src-1/ mice suggests the possibility that in addition to a primary increase in adrenocortical responsiveness during stress, an altered sensitivity to glucocorticoid dependent feedback inhibition of pomc gene transcription may contribute to this exaggerated corticosterone response. as expected, the underlying adrenal insufficiency imposed by sf-1 haploinsufficiency precluded any potential increase in corticosteroids in compound sf-1+//src-1/ littermates.
adrenal glands from src-1-deficient mice appear histologically normal but are significantly larger than wt adrenals
histological examination of hematoxylin- and eosin-stained adrenal sections from wt and src-1/ animals revealed no apparent anatomical differences in src-1-deficient animals (fig. 5). although body weights of src-1/ mice were not significantly different from those of wild-type animals as previously reported (data not shown) (36), adrenals of src-1-deficient mice (1.94 ± 0.04 mg) weighed significantly more than those of their age-matched, wt littermate controls (1.78 ± 0.02 mg; p < 0.01; fig. 5, bottom panel). these data indicate that although src-1 is not required for normal adrenocortical growth and development, src-1 deficiency does alter the dynamics of adrenal growth maintenance, resulting in a surprising increase in adrenal weight.
expression of the mc2r is elevated in src-1-deficient animals
we next examined potential mechanisms for the observed increase in adrenal responsiveness to stress in src-1/ mice. because of the lack of a significant hpa axis defect at baseline, we first evaluated potential mechanisms of adrenal compensation by measuring transcript levels of genes involved in steroid biosynthesis, including mc2r, scc, and star. interestingly, although scc and star transcript levels were unchanged, mc2r mrna levels were significantly increased in src-1/ adrenals at baseline compared with littermate controls (1.77 ± 0.25- vs. 1.00 ± 0.11-fold of wt; p < 0.05; fig. 6a), suggesting a potential mechanism to normalize adrenal sensitivity to the actions of acth. a more dramatic increase was observed after a 48-h fast, with src-1/ mice exhibiting a significant increase in mc2r mrna compared with wt controls (2.34 ± 0.33- vs. 1.00 ± 0.14-fold of wt; p < 0.01; fig. 6b). this increase in mc2r expression may account for the enhanced acth responsiveness observed in src-1/ mice after stress.
src-2 and src-3 are specifically up-regulated in the adrenals of src-1/ mice
next, we determined whether src-1 deficiency altered the expression levels of src-2 and src-3, a phenomenon that has been previously reported in studies evaluating endocrine systems in src-1/ mice (36). we performed immunoblot analysis and quantitative rt-pcr to assess the relative expression levels of src-2 and src-3 in src-1-deficient adrenal glands. western blot analysis of lysates prepared from adrenals isolated at baseline demonstrated a considerable up-regulation of src-2 and src-3 protein levels in src-1/ mice (fig. 7a). in addition, baseline transcript levels for src-2 (2.37 ± 0.59- vs. 1.00 ± 0.09-fold of wt; p < 0.05) and src-3 (3.79 ± 1.00- vs. 1.00 ± 0.15-fold of wt; p < 0.05) were significantly elevated (2- to 2.5-fold) over wt levels in src-1/ mice (fig. 7b, top panel). similar increases in src-2 (2.59 ± 0.69- vs. 1.00 ± 0.11-fold of wt; p < 0.05) and src-3 (3.75 ± 2.11- vs. 1.00 ± 0.35-fold of wt; p < 0.05) were observed after 48 h of food deprivation (fig. 7b, bottom panel). the transcript levels of various members of the nurr77/nerve growth factor ib (ngfi-b; nurr 77, nurr1, and nor-1) and chicken ovalbumin upstream promoter-transcription factor-1 and -2 families of nuclear receptors were also examined. these studies failed to demonstrate significant alterations in the expression profiles of these factors in src-1-deficient adrenals compared with wt littermate controls (data not shown). collectively, these data suggest that the up-regulation of src-2 and src-3 may be sufficient to maintain baseline adrenocortical function within the normal physiological range, potentially through a mechanism involving the selective up-regulation of mc2r expression leading to an enhancement of adrenocortical responsiveness to stress in src-1/ mice.
src-1-deficient mice exhibit marked elevation in corticosterone after a 48-h fast despite normal acth levels
to evaluate the integrity of the entire hpa axis after stress, we used the chronic stress paradigm of 48-h food deprivation. once again, src-1/ mice exhibited a statistically significant elevation of plasma corticosterone compared with wt littermates (498.64 ± 95.33 vs. 231.06 ± 58.28 ng/ml; p < 0.05) despite the presence of nonsuppressed acth values, consistent with both an increase in adrenocortical responsiveness to acth and a defect in glucocorticoid-mediated negative feedback inhibition of the hpa axis (fig. 8). as expected, a significant increase in plasma acth (466.89 ± 26.25 vs. 401.16 ± 9.46 pg/ml; p < 0.05) and a concomitant decrease in corticosterone levels (181.27 ± 27.20 vs. 231.06 ± 58.28 ng/ml; p < 0.05) were observed in sf-1+/ animals compared with wt littermates. in addition, sf-1+//src-1/ mice displayed a significant increase in plasma acth (620.80 ± 78.81 vs. 401.16 ± 9.46 pg/ml; p < 0.05) without alterations in plasma corticosterone (226.73 ± 36.52 vs. 231.06 ± 58.28 ng/ml) compared with wt controls, most likely the result of the combined effects of adrenal insufficiency and a blunted feedback inhibition imposed by sf-1 haploinsufficiency and src-1 deficiency, respectively.
src-1/ mice exhibit generalized resistance to the action of glucocorticoids
the above results are indicative of a blunting of hypothalamic/pituitary glucocorticoid responsiveness. to examine whether the observed phenotype in src-1/ mice was due to generalized glucocorticoid resistance, glucose levels were measured in serum obtained from 48-h fasted wt, sf-1+/, src-1/, and sf-1+//src-1/ mice (fig. 9a). significantly lower serum glucose levels were observed for src-1/ mice compared with wt controls (123.25 ± 7.40 vs. 149.90 ± 5.58 mg/ml; p < 0.05), suggesting the presence of hepatic glucocorticoid resistance. in addition, serum glucose and transcript levels were evaluated in wt and src-1/ animals after administration of the synthetic glucocorticoid agonist, dexamethasone. src-1/ animals exhibited a blunted increase in blood glucose compared with wt controls (154.20 ± 3.61 vs. 173.76 ± 7.12 mg/ml; p < 0.05), consistent with a defect in the ability of glucocorticoids to increase the relative rate of hepatic glucose production (fig. 9b). to evaluate this proposed mechanism, hepatic phosphoenolpyruvate carboxykinase (pepck) mrna levels were measured in dexamethasone-suppressed mice. as anticipated, the administered dose of dexamethasone failed to stimulate transcription of the pepck gene to a similar extent in src-1/ mice compared with littermate controls (60.82 ± 9.65% vs. 100.00 ± 11.58% of wt; p < 0.05), suggesting the presence of hepatic resistance to the actions of glucocorticoids (fig. 9c).
although baseline pituitary pomc transcript levels were comparable between src-1/ and wt mice (100 ± 4.7% vs. 105 ± 5.5% of wt), a dramatic failure of src-1/ mice to suppress pomc gene expression after dexamethasone treatment (2 μg/g body weight) was observed (66.9% vs. 19.9% suppression relative to wt baseline; p < 0.01; fig. 10). collectively, these data suggest the presence of glucocorticoid resistance in src-1/ mice at the level of the pituitary that manifests as a lack of feedback inhibition of pomc gene transcription as well as hepatic resistance, which is suggested by statistically significant lower blood glucose. evaluation of the transcript levels of chicken ovalbumin upstream promoter-transcription factor-1 and -2 and nurr77/ngfi-b family members as well as the pituitary-specific t box factor (tpit) in the pituitaries of src-1-deficient and wt mice failed to demonstrate significant alterations in mrna levels (data not shown).
discussion
the mammalian stress response is initiated by activation of a concerted neuroendocrine response involving the hypothalamic secretion of crh and arginine vasopressin, which stimulate the synthesis and secretion of a variety of pomc-derived peptides from the pituitary corticotroph. pomc was originally cloned as the prohormone cleaved to acth, the primary peptide hormone responsible for the synthesis and secretion of glucocorticoids from adrenocortical cells within the adrenal cortex (49). glucocorticoids are critical endocrine hormones that exert steady-state and stress-related regulatory control over physiological processes, including carbohydrate, protein, and fat metabolism; salt and water balance; inflammation; and the immune response, as well as effects on cellular proliferation and differentiation (50). to achieve homeostasis, glucocorticoids suppress hpa axis drive through classical feedback inhibition of hypothalamic crh and pituitary pomc synthesis and secretion (51, 52, 53, 54, 55). therefore, the appropriate regulation of glucocorticoid synthesis is dependent upon a physiologically appropriate adrenal responsiveness to the actions of acth and a suitable degree of glucocorticoid-mediated feedback inhibition of the hpa axis.
the acth-dependent regulation of glucocorticoid production requires the precisely coordinated transcription of a variety of genes involved in numerous aspects of steroid biosynthesis within the adrenal cortex. the nuclear receptor sf-1 represents a critical mediator of this transcriptional response due to the requirement for sf-1 to transcriptionally regulate a variety of steroid biosynthetic enzymes genes in an acth-dependent manner (56). given the critical role of sf-1 in the regulation of steroidogenesis, an active area of research has been the identification of transcriptional coregulatory proteins that modulate sf-1 action (56). unfortunately, although a variety of transcriptional coregulatory proteins have been demonstrated to physically and/or functionally interact with sf-1 on adrenal-specific promoters, the significance of these interactions has not been thoroughly established in vivo. specifically, although the entire p160/src family of transcriptional coactivators is expressed within the adrenal cortex and has been demonstrated to interact physically and/or functionally with sf-1, the relative importance of sf-1 interactions with individual src family members in vivo remains unresolved (27, 28, 31).
in the present study we evaluated the hpa axis in src-1-deficient mice to ascertain whether src-1 is required for normal hpa axis function. we demonstrate that src-1 is actively recruited to sf-1 response elements in the mc2r and star promoters in response to acth stimulation, establishing src-1 as a bona fide participant in sf-1-mediated transcriptional activation of these genes. additionally, we demonstrate that all three p160/src family members exhibit anatomical distribution consistent with their proposed roles as coregulators of sf-1.
analysis of hpa axis function at baseline and after stimulation with exogenous acth did not reveal the presence of appreciable deficits in src-1/ mice. in contrast, after restraint and the chronic stress imposed by 48 h of food deprivation, src-1/ mice exhibited a surprising increase in serum corticosterone despite having normal (nonsuppressed) serum acth levels, suggesting both an increased adrenal responsiveness to acth and a concomitant blunted feedback inhibition of the hpa axis.
a comparison of adrenal weights between src-1/ and wt littermates revealed a significant increase in src-1/ adrenal weights. based upon our findings that src-2 and src-3 are up-regulated in the adrenals of src-1-deficient mice and the documented roles of these family members in growth and tumorigenesis, we speculate that the growth dynamics of the adrenal gland may be altered through a mechanism(s) involving increased growth factor sensitivity (33, 57, 58). in support of this hypothesis, adrenals of src-1-deficient mice exhibited a selective increase in mc2r mrna, a change that may represent a mechanism that 1) compensates for a primary adrenal defect at baseline, but 2) results in accentuated corticosterone output after chronic stress-induced acth elevation.
the expression profile of src family members has been evaluated in the pituitary (47, 59, 60), and their putative roles in pituitary function have been partially dissected in vivo through the use of gene-targeting approaches. all family members exhibit expression profiles consistent with roles in pituitary-specific transcription. to evaluate the pituitary-specific response to glucocorticoids in src-1-deficient mice, we performed dexamethasone suppression tests and assessed the ability of exogenous glucocorticoids to suppress pituitary pomc mrna levels. the relative failure of dexamethasone to suppress pomc transcript levels in src-1/ mice indicates the presence of a glucocorticoid-dependent pituitary defect. the presence of statistically significant lower serum glucose levels after a 48-h fast and dexamethasone administration is consistent with an additional defect in the ability of glucocorticoids to activate gluconeogenesis in src-1/ mice. indeed, an evaluation of dexamethasone-suppressed hepatic pepck levels revealed a blunted stimulation of glucocorticoid-dependent pepck gene transcription.
glucocorticoids exert their effects by binding their cognate nuclear receptors (grs), which classically serve as dna-dependent transcription factors capable of enhancing (i.e. gluconeogenic genes) or repressing (pomc- and immune-modulating genes) transcription of glucocorticoid-responsive genes. src-1-deficient mice exhibit relative hypoglycemia in the face of elevated corticosterone and pomc levels, consistent with defects in glucocorticoid-dependent transcriptional activation and transcriptional repression in liver and pituitary, respectively. src-1 has been shown to functionally and physically interact with gr to both stimulate and inhibit transcriptional activation in a variety of cellular and promoter contexts (61, 62). with respect to the hepatic defect, our data clearly indicate that src-1-deficient mice exhibit aberrant transcriptional regulation of the glucocorticoid-responsive pepck gene (63, 64), which is known to be synergistically activated by src-1 and gr (61, 65). in contrast, the transcriptional repression of the pomc gene is very complex and involves 1) direct gr binding as a homotrimer to a proximal negative glucocorticoid response element; 2) gr sequestration of coregulatory proteins, including corticosterone-binding protein/p300; and 3) gr-mediated transrepression of transcription factors required for crh-dependent transcriptional activation (66, 67). gr-mediated transrepression has been shown to antagonize the transcriptional activation of many genes through dna-independent, protein-protein interactions with a variety of factors, including nuclear factor-b, nur77, and activating protein-1 (63, 64, 68, 69). the in vivo significance of dna-dependent and -independent mechanisms of gr repression was evaluated in a mouse model engineered with a point mutation that specifically inhibits gr dimerization. in contrast to gr-deficient mice, these mice were viable, highlighting the importance of dna-independent mechanisms of gr action (70). moreover, although these mice exhibited a severe impairment in gr-dependent activation of target genes, transrepression of genes by gr was retained, suggesting a relatively important role for dna-independent gr action. importantly, these mutant mice had normal serum acth levels despite a significant elevation in plasma corticosterone, a phenotype similar to that observed in src-1-deficient mice in the present study, suggesting a likely effect of src-1 deficiency on dna-independent gr repression of pomc. indeed, the loss of src-1 in tumors that constitutively express pomc has been shown to correlate with an inability of glucocorticoids to induce gr-mediated repression of pomc (71).
in addition to the role of src-1 in processes leading to the transrepression of the pomc gene observed in the current study, the crh-dependent transcriptional activation of the pomc gene is dependent upon the combined actions of tpit and ngfi-b, which recruit src-2 to obtain a maximal transcription response (72). to exclude the possibility that altered expression of these factors potentially contributed to the observed phenotype, tpit and ngfi-b mrna levels were evaluated in the pituitaries of control and src-1/ mice. as anticipated, this analysis failed to demonstrate a significant alteration in tpit or ngfi-b expression (data not shown). therefore, the defect observed in src-1/ mice cannot be simply explained by an alteration in the expression of factors responsible for transcriptional activation of the pomc gene.
similar modes of gr-mediated transcriptional repression have highlighted the specific roles of p160/src family members in this regulatory process. for example, glucocorticoid-mediated repression of the collagenase-3 gene involves a physical interaction between gr and src-2, an established coactivator for the thyroid hormone and grs (73). a role for src-1 in nuclear receptor-dependent repression is also evident from studies examining thyroid hormone action in src-1-deficient mice, which exhibit thyroid hormone resistance and elevated pituitary tsh levels secondary to the loss of src-1-facilitated thyroid hormone receptor-mediated repression of tsh transcription (74, 75). in most cases, the precise outcome of interactions between gr and p160/src family members seems to be highly dependent upon the context in which these interactions occur (76). indeed, the functional outcome of src-2 and gr interactions on promoters regulated in a positive or negative fashion by glucocorticoids involves the promoter-specific interaction of gr with distinct surfaces on src-2 (76). presumably, these specific interactions alter the structure of the src-associated complex, ultimately dictating whether the functional outcome of the interaction will be activation or repression of gene transcription. understanding the distinct roles of other sf-1 coregulatory proteins, specifically src-2 and src-3, in the regulation of hpa axis function will be an important area of continuing research.
footnotes
this work was supported by the national institute of diabetes and digestive and kidney diseases, national institutes of health (grant r01-dk-62027; to g.d.h.) and the national institute of dental and craniofacial research (grant t32-de-007057; to j.n.w.).
first published online december 8, 2005
abbreviations: chip, chromatin immunoprecipitation; gr, glucocorticoid receptor; hpa, hypothalamic-pituitary-adrenal; mc2r, melanocortin 2 receptor; ngfi-b, nerve growth factor ib; pepck, phosphoenolpyruvate carboxykinase; pomc, proopiomelanocortin; scc, cytochrome p450 cholesterol side-chain cleavage enzyme; sf-1, steroidogenic factor-1; src-1, steroid receptor coactivator-1; star, steroidogenic acute regulatory protein; tpit, pituitary-specific t box factor; wt, wild type.
accepted for publication november 28, 2005.
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