Characterization of big stanniocalcin variants in mammalian adipocytes and adrenocortical cells
Mark Paciga,1
Edward R. Hirvi,1
Kathi James,1 and
Graham F. Wagner1,2
Departments of 1Physiology and Pharmacology, Faculty of Medicine and Dentistry, and 2Department of Biology, Faculty of Science, The University of Western Ontario, London, Ontario, Canada
Submitted 10 December 2004
; accepted in final form 23 February 2005
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ABSTRACT
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The hormone stanniocalcin (STC) is widely distributed, and in rodents the highest levels of expression are in the ovaries. In both cows and rodents, ovarian STC consists of three high-molecular-weight variants collectively known as big STC. In the ovary, big STC is made by theca cells and interstitial cells and is targeted to lipid storage droplets of nearby luteal cells to inhibit progesterone release. An endocrine pathway is operative during pregnancy and lactation. Whether or not big STC is made by tissues other than ovary has never been addressed. Therefore, the purpose of this study was to determine via a detailed characterization of adrenal glands and adipocytes whether big STC is present in other cells that store and release lipids. The results showed that STC was made in bovine and mouse adrenals, mainly in steroidogenic, adrenocortical cells. The majority of ligand and receptor were likewise confined to cortical zone cells. As in luteal cells, high levels of ligand and receptor were found in the adrenocortical cell lipid droplet fraction. However, adrenals made only the largest (135 kDa) of the three big STC variants. Nonetheless, adrenal STC had much greater receptor affinity than a mixture of the three big STC variants. Adipocytes contained all three big STC variants, and both ligand and receptor were heavily concentrated on the lipid droplets. Moreover, adipocyte lipid storage droplets had 50-fold more receptors than those in steroidogenic cells, indicating that big STC is heavily targeted to adipose cells. The findings collectively support the hypothesis that big STC is not unique to ovarian steroidogenic cells but is in fact common to cells with a role in lipid storage and release.
stanniocalcin receptor; adipose cells; lipid storage droplets
STANNIOCALCIN (STC) was first characterized in fish as a blood-borne regulator of calcium balance. Structurally, fish STC is an
50-kDa dimer of identical
30-kDa subunits (32). A highly homologous, 50-kDa dimeric form of STC (STC50) also exists in mammals and is widely distributed in tissues such as kidney, liver, lung, and spleen (2, 17, 30). Mammalian ovary contains the highest levels of STC gene expression (3, 30) and produces a structurally unique form of the hormone. In contrast to STC50, ovarian STC comprises three higher molecular-mass proteins (84, 112, and 135 kDa) that are collectively known as big STC (20). Big STC is synthesized in ovary by androgen-producing theca cells and interstitial cells, and its structural uniqueness is reflected by marked differences in function. Whereas STC50 is targeted to and sequestered by the mitochondria of receptor-bearing cells (12), theca cell-derived big STC is targeted to and sequestered by the cholesterol/lipid storage droplets of nearby luteal cells to inhibit progesterone release (19). For both forms of the hormone, the sequestering process appears to be receptor mediated (12, 19).
Big STC does not appear to be present in tissues such as liver and kidney, which synthesize and/or are targeted by STC50. However, it remains to be seen whether big STC is uniquely found in ovarian steroidogenic cells or is in fact produced elsewhere. For instance, adrenocortical tissue is highly steroidogenic and responsible for the production of corticosteroids, mineralocorticoids, and androgens (31). And, like ovarian luteal cells, adrenocortical cells contain cholesterol/lipid storage droplets. Moreover, STC mRNA is readily detectable in the adrenals (15, 30). This is also true in the case of mammalian adipocytes, which have recently been shown to contain both STC and STC receptors (13, 21). Therefore, the purpose of this study was to address the hypothesis that big STC is not unique to ovarian steroidogenic cells but is in fact present in other cell types that store and release lipids.
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MATERIALS AND METHODS
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Northern blot analysis.
Bovine adrenal glands were obtained at a local abattoir. The glands were dissected free of all adipose tissue and split lengthwise, and the adrenocortical zone was isolated for RNA extraction. Retroperitoneal white adipose tissue was obtained from male Wistar rats and dissected free of all associated vascular and connective tissue elements. Total RNA was isolated separately from bovine adrenocortical tissue and rat fat with TRIzol reagent (Invitrogen, Burlington, ON, Canada), resolved on 1% agarose-3% paraformaldehyde gels and subjected to Northern blot analysis. Blots were hybridized with a random primed, 32P-labeled, 742-bp bovine STC-coding region-specific cDNA, as previously described (20). To standardize the STC mRNA signal, blots were subsequently probed with a 32P-labeled 18S ribosomal rRNA gene fragment.
Immunocytochemistry, in situ hybridization, and in situ ligand-binding studies.
Immunocytochemistry was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections (5 µM) of mouse adrenals, as previously described, using a specific rabbit antiserum generated against recombinant human (h)STC (3, 16). The sites of antibody binding were visualized with biotinylated secondary antibodies and the Vectastain ABC peroxidase detection system (Vector Laboratories, Burlingame, CA). Sections were subsequently counterstained with hematoxylin, dehydrated, and mounted.
In situ hybridization was performed under high-stringency conditions using an 850-bp mouse STC cDNA as a template for sense and antisense cRNA synthesis of digoxigenin (DIG)-labeled probes, as previously described (3, 14).
In situ ligand binding was performed as previously described for the cellular localization of STC receptors (12, 19). The method employs a fusion protein of STC and human placental alkaline phosphatase (AP) known as STC·AP, which is stably expressed in MDCK cells (12). Fixed, paraffin-embedded tissue sections were equilibrated in Hanks' balanced salt solution containing 0.1% BSA (HBHA buffer), pH 7.5, and then incubated for 90 min in HBHA buffer containing 1 nM STC·AP. Control slides were incubated in either AP alone or STC·AP containing 1 µM hSTC. Slides were then washed and processed for visualization of bound AP activity.
Subcellular fractionation.
Fresh bovine adrenals were split sagitally and the cortical region was separated from the medullary zone. Nuclei, mitochondria, plasma membrane, and cytosol fractions were isolated from cortical zone cells as previously described (19). For isolation of the cholesterol/lipid droplet (CLD) fraction, bovine adrenocortical and rat adipose tissue were minced separately on ice with razor blades and homogenized in 5 volumes of hypotonic buffer (23). The homogenates were centrifuged at 27,000 g for 30 min and yielded a floating phase containing the CLDs. Proteins were extracted separately from both CLD fractions as described (23) and stored at 20°C. Protein concentrations of the resulting subcellular fractions were determined using the Bio-Rad protein assay kit. STC content of the various subcellular fractions was determined by RIA (16).
Western blot analysis.
Western blot analysis was carried out as previously described, using a well-characterized antiserum to recombinant hSTC (19, 20). The subcellular fractions isolated above were boiled in SDS sample buffer in the presence and absence of the reducing agent
-mercaptoethanol and resolved on 10% SDS-PAGE. Proteins were transferred to PVDF membrane (Roche, Montreal, QC, Canada), which was then incubated overnight with a 1:40,000 dilution of rabbit anti-hSTC antiserum, washed, and subsequently incubated in a horseradish peroxidase-linked donkey anti-rabbit secondary antiserum. STC-immunoreactive bands were then visualized with an ECL Western blotting detection kit (Amersham Pharmacia Biotech, Baie d'Urfie QC, Canada).
Receptor-binding assays.
To obtain estimates of Kd and Bmax, both the microsomal membrane fraction (100 µg protein/tube) and the intact CLD fraction (50 µg protein/tube) from bovine adrenocortical cells and rat adipocytes were subjected to receptor-binding assays. The assay has been previously employed to characterize STC receptors on kidney and liver membranes and mitochondria (12) and membranes and CLDs from bovine luteal cells (19). These assays were performed as previously described using a fusion protein of STC and human placental AP as the experimental ligand (STC·AP), and human placental AP as the control ligand (12, 19). For binding studies on membranes, the membranes were pelleted by high-speed centrifugation for the separation of bound and free ligand. The assays on intact CLDs were conducted as previously described (19), whereby the assay tubes were periodically vortexed to prevent the separation of aqueous and nonaqueous phases. Following centrifugation, free and CLD-bound STC·AP were separated by aspiration of the CLD-free infranatant with a 23-gauge needle. AP detection buffer was then added to the supernatant containing the CLD fraction, and AP activity was detected as previously described (2). Additional controls involved incubating CLD and plasma membrane fractions with STC·AP in the presence of 1 µM recombinant hSTC.
Additional studies were performed to compare adrenal and HT-1080-derived big STC in their abilities to displace STC·AP in receptor binding-assays. For these studies, 100, 200, and 400 pg/ml of STC immunoreactivity (as assessed by RIA) from the adrenal CLD protein extract and immunopurified HT-1080 big STC were added to tubes containing adrenal membranes (100 µg protein/tube) and STC·AP fusion protein (500 mU/ml). Additional sets of tubes were incubated with AP alone, plus the aforementioned additions for estimates of nonspecific binding. Following a 90-min incubation at room temperature, the membranes were pelleted by centrifugation, and specific binding was determined as previously described (12, 19). The displacement assay was performed twice, each time on quadruplicate sets of tubes.
Adrenocortical cell culture.
Primary cultures of bovine adrenocortical cells and luteal cells were prepared and maintained using previously described methods (19). The objectives here were to establish whether or not adrenocortical cells and luteal cells were equally responsive to big STC. Second, we wanted to determine whether the cells were capable of secreting STC and, if so, whether or not secretion was subject to regulation.
Bovine ovaries and adrenals were obtained from a local abattoir. In the case of ovaries, the corpora lutea were dissected out of the ovary and washed several times in HBHA (Sigma). A highly pure population of dispersed large and small luteal cells was obtained by mincing the corpora lutea and subsequently digesting the tissue in an enzymatic mixture of collagenase type II (Invitrogen) and DNase I (Roche, Laval, QC, Canada), as previously described (19). Cell viability was assessed by Trypan blue exclusion. Cells were seeded at a density of 500,000 cells/ml in 24-well tissue culture plates and maintained at 37°C in UltraCulture medium (BioWhittaker) supplemented with an antibiotic-antimycotic solution (Invitrogen). The proportion of steroidogenic cells was determined by 3
-hydroxysteroid dehydrogenase enzyme biochemistry, also as previously described (19). The ability of cells to respond to hormone stimulation was assessed by measuring progesterone output in response to luteinizing hormone (obtained from Dr. A. F. Parlow, National Hormone and Pituitary Program, University of California at Los Angeles Medical Center, Torrance, CA).
Bovine adrenal glands were rinsed in 70% ethanol, and regions of the cortical zone were dissected free and finely minced with razor blades. The minced tissue was subsequently digested in an enzymatic mixture of collagenase type II and DNase I, as previously described (19). Cell viability (>90%) was assessed by Trypan blue exclusion. Adrenal cells were seeded at a density of 2 x 106 cells/ml in six-well tissue culture plates and maintained at 37°C in UltraCulture medium supplemented with an antibiotic-antimycotic solution (Invitrogen). Following attachment to the plate, cells were washed and the medium was replaced. The cells were then cultured for 24 h in the presence and absence of 2 ng/ml of immunopurified big STC derived from HT-1080 cells (19) or 1 µM forskolin, a known activator of protein kinase A (PKA), which is a major pathway governing cortisol release.
Big STC was purified from conditioned media obtained from HT-1080 cells, a human fibrosarcoma cell line that secretes big STC (20). This entailed preparing an STC immunoaffinity column by coupling the IgG fraction from a polyclonal hSTC antiserum to CNBr-Sepharose 4B (Amersham Pharmacia Biotech). HT-1080 cells were plated and maintained in DMEM-F-12 containing 10% fetal bovine serum and supplemented with an antibiotic-antimycotic solution (Invitrogen). Conditioned medium (24 h) from the cells was then recycled through the column overnight. Bound big STC was eluted with 0.1 M glycine, pH 3.0, containing 0.5 M sodium chloride. The elution profile was monitored by RIA for STC content. The relevant fractions were pooled, concentrated, and buffer exchanged on a Centricon YM-10 centrifugal filter device (Millipore, Bedford, MA). The STC content of the concentrate was then quantified by RIA before use.
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RESULTS
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Northern blot analysis.
Although STC mRNA is readily detectable in mouse adrenal (30), Northern blot analysis was performed on bovine adrenals to confirm that STC gene expression in this tissue was not species specific. Fig. 1 shows the results of this analysis, in which a 4-kb STC transcript was detectable in as little as 10 µg of total RNA. Rat adipose tissue contained barely detectable levels of a 4-kb transcript (results not shown).

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Fig. 1. Northern blot analysis of stanniocalcin (STC) in bovine adrenals. Total RNA was extracted from bovine adrenals, and 10-, 20-, and 40-µg aliquots were subjected to Northern blot analysis using a bovine STC cDNA probe, as described in MATERIALS AND METHODS. A single 4-kb STC transcript was evident. As a loading control the blot was reprobed with an 18S cDNA.
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Immunocytochemistry, in situ hybridization, and in situ ligand-binding studies.
The histological staining results using mouse adrenal glands are shown in Fig. 2. Immunocytochemistry revealed that the highest levels of STC immunoreactivity (STCir) were found in the three layers of cells comprising the steroidogenic cortical zone, the outermost glomerulosa layer, the intervening fasciculata layer, and the inner reticularis layer. Figure 2A is a low-power micrograph of mouse adrenal gland and shows that the majority of STCir was present in the cortex (C). By comparison, the medullary zone chromaffin cells were weakly stained. The results of the in situ hybridization analysis are shown in Fig. 2B. As in the case of the immunocytochemical findings, the majority of transcript was confined to the three layers of the cortical region. Cells of the glomerulosa zone sometimes exhibited a stronger hybridization signal, as evidenced by the intense purple staining.. In comparison, much less STC transcript was evident in the medullary chromaffin cells in the center of the gland. The positive signal obtained by in situ hybridization was specific to antisense probes as the use of a DIG-labeled sense probe yielded little or no signal (Fig. 2B, inset). In situ ligand-binding analysis revealed that the highest numbers of STC binding sites were likewise present in the three cortical zone layers, as evidenced by the dark brown staining. However, reduced levels of ligand binding activity were clearly evident in medullary zone chromaffin cells (Fig. 2C). Binding activity in both zones was confined to the cell cytoplasm. Furthermore, ligand binding in both zones was also specific, as the addition of hSTC as a competing ligand abolished the majority of ligand binding (Fig. 2C, inset).

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Fig. 2. Histological localization of STC protein, mRNA, and receptor binding sites in the adult mouse adrenal. AC: presence of STC ligand (A; immunocytochemistry), mRNA (B; in situ hybridization) and binding sites (C; in situ ligand binding) in adjacent sections of male mouse adrenal gland. A: immunocytochemistry revealed that the majority of STC immunoreactivity (STCir) was confined to steroidogenic cells in the 3 cortical zones (G, glomerulosa; F, fasciculata; R, reticularis). Medullary zone (M) cells were weakly immunoreactive compared with cortical zone cells (C). B: in situ hybridization revealed that the majority of STC transcript was confined to the steroid-producing cortical zone cells, although a weak signal was also evident in medullary zone cells. Inset: extent of hybridization signal obtained with sense probe. C: in situ ligand binding revealed that STC receptors were most abundant in the cortical zone cells, although medullary zone cells also contained significant binding activity. Inset: absence of ligand binding obtained in the presence of 1 µM hSTC. D: higher magnification of box D in A shows close association between STCir and CLDs in fasciculata zone cells. The same pattern was also evident, albeit to a lesser degree, in glomerulosa zone cells. Nuclear staining is also evident in some cells in both zones. E: higher magnification of box E in A showing the adrenal cortical-medullary junction. Compared with cortical zone cells, medullary zone cells were weakly stained for STC throughout the cytoplasm. Within the cortex, STCir cells of the reticularis zone were smaller and much more intensely stained than those in the fasciculata. F: immunocytochemical staining for STC in female mouse adrenal also reveals high ligand levels in the cortex and lower levels in the medulla. Surrounding adipose tissue (A) also contained high levels of STC and, as was the case in steroidogenic cells, much of the STC appeared to surround the lipid droplets (red arrows in box A at bottom right). Inset, top right: staining was abolished when the antiserum was preincubated with 1 µM hSTC.
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Figure 2, D and E, are higher magnifications of insets D and E in Fig. 2A, and are meant to highlight the immunocytochemical staining in the different types of cortical zone cells. Here, it can be seen that the cells in all three layers were homogeneously stained for STC. However, in the cells of the fasciculata zone, the staining was honeycombed in appearance, presumably due to the higher concentrations of STCir around individual lipid droplets (Fig. 2, D and E). Figure 2F illustrates a second adrenal gland after immunocytochemical staining with STC antiserum that was surrounded by a significant amount of adipose tissue. The staining patterns within the cortical and medullary zones of this adrenal were similar to those in Fig. 2A. However, substantial amounts of STCir were also evident in the fat. At higher magnification, it was apparent that much of this STCir was concentrated in a halo-like manner around the lipid droplets (Fig. 2F, inset A, bottom right). Figure 2F, top right inset, is an immunocytochemical staining control to show that the majority of antibody staining was STC specific.
Subcellular fractionation.
Having established the cellular distribution patterns of STC ligand, transcript, and receptor at the histological level in the mouse, bovine adrenocortical tissue was fractionated and the ensuing mitochondrial, plasma membrane, nuclear, and CLD fractions were analyzed by RIA for STC content. The results of this analysis are shown in Fig. 3, where it can be seen that the highest concentrations of STC per mg protein were found in the CLD fraction compared with the nuclear, mitochondrial, and plasma membrane fractions. In contrast, the cytosolic fraction contained no immunoassayable STC after removal of the lipid droplets (i.e., no statistically significant displacement of tracer hormone by RIA), indicating that little STC was associated with the aqueous phase of the cytoplasm.

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Fig. 3. Immunoassayable STC content of adrenocortical subcellular fractions. Fractions shown were isolated as described and assayed for total protein and total STC content by RIA. The lipid droplet fraction contained the highest STC content per mg total protein. CLD, cholesterol/lipid droplet.
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Western blot analysis.
The Western blot analyses of adrenocortical cell CLD proteins are shown in Fig. 4A. The results revealed no STCir bands in the 50-kDa range. Instead, there was one higher-molecular-mass STCir protein with an estimated size of 135 kDa (Fig. 4A). Moreover, this presumptive big STC variant was entirely resistant to chemical reduction with
-mercaptoethanol. These findings were in marked contrast to those obtained in ovary, where big STC comprises three high-molecular-mass species of 84, 112, and 135 kDa, all of which collapse into an
45-kDa band after chemical reduction (20). The Western blot analyses of proteins extracted from the adipocyte CLD fraction are shown in Fig. 4B and proved to be more typical. Unlike the results in adrenal, all three big STC variants were detected in adipocytes (84, 112, and 135 kDa). Furthermore, as in the case of ovary, all three proteins collapsed into a
45-kDa band following chemical reduction with
-mercaptoethanol.
Receptor-binding assays.
The results of the receptor binding assays using bovine adrenals are shown in Fig. 5. Figure 5A shows that there were saturable, high-affinity binding sites on adrenocortical cell membranes. On the basis of three separate saturation binding assays, the mean estimates of Kd and Bmax were 4.5 ± 1.2 nM and 6.6 ± 1.8 pmol/mg protein, respectively (means ± SE). However, because adrenocortical cell CLD yields were so much lower than those obtained from luteal cells (19), it was not possible to perform saturation binding assays on this fraction. Instead, estimates of specific binding were done on fixed amounts of this fraction using STC·AP or STC·AP containing 1 µM recombinant hSTC. The results revealed that adrenocortical CLDs contained measurable binding activity, a significant proportion of which was displaceable with recombinant hSTC (Fig. 5B).

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Fig. 5. Receptor-binding studies on bovine adrenal subcellular fractions. A: typical saturation binding curve obtained from bovine adrenal membranes. Specific binding was obtained by subtracting alkaline phosphatase (AP) binding (nonspecific binding) from total STC·AP binding. Binding of STC·AP to the membrane fraction proved to be saturable (Bmax = 6.6 ± 1.8 pmol/mg protein) and had a calculated Kd of 4.5 ± 1.2 nM (n = 3 determinations). B: STC·AP also proved capable of binding to the CLD fraction from adrenocortical cells. Because of the low yield of lipid droplets from the adrenals, it was not possible to conduct saturation binding assays for estimated Kd and Bmax. Total binding is represented by binding in the presence of STC·AP alone. Nonspecific binding is represented by the binding obtained with STC·AP in the presence of 1 µM recombinant hSTC. The difference between the 2 is indicative of specific binding. C: protein extracts derived from adrenocortical CLDs proved to be more effective than HT-1080-derived big STC in displacing STC·AP from adrenocortical cell membranes in receptor-binding studies. Aliquots of adrenocortical microsomal membrane (100 µg/tube) were incubated with STC·AP alone or together with 100400 pg/ml of radioimmunoassayable big STC and processed as described in the MATERIALS AND METHODS section. Adrenal extract caused significantly greater displacement of STC·AP than HT-1080-derived big STC at all concentrations tested (P < 0.001, Student's t-test).
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The STC extracted from adrenocortical CLDs also displayed competitive binding properties in receptor assays. Both CLD-derived and immunopurified HT-1080 big STC caused significant displacement of STC·AP from adrenocortical membranes (Fig. 5C). However, the adrenal CLD-derived hormone was clearly the more effective of the two at all concentrations employed (P < 0.010.001, Student's t-test).
The results of the receptor-binding assays on rat adipose cell membranes and the lipid droplet fraction are shown in Fig. 6, A and B, respectively. In both cases, binding was saturable and of high affinity. Remarkably, however, whereas adipocyte membranes and lipid droplets had typical values of Kd, both fractions exhibited extraordinarily high capacities for ligand binding. The membrane receptor had a Kd of 7.9 ± 2.2 nM and a Bmax of 64.1 ± 3.8 pmol/mg protein (means ± SE), the latter of which was 10-fold higher than the Bmax estimate for adrenocortical membranes. In comparison, the binding site on adipocyte lipid droplet membranes had an estimated Kd of 6.75 ± 2.8 nM and a Bmax of 1,234 ± 108 pmol/mg protein (n = 3; means ± SE).

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Fig. 6. Receptor-binding studies on rat adipocyte subcellular fractions. A: typical saturation binding curve obtained from rat adipocyte membranes. Specific binding was obtained by subtracting AP binding (nonspecific binding) from total STC·AP binding. Binding of STC·AP to membranes was saturable (Bmax = 64.1 ± 3.8 pmol/mg protein) and had a calculated Kd of 7.9 ± 2.2 nM (n = 3 determinations). B: typical saturation binding curve obtained from CLD fraction (white adipose tissue). Specific binding was calculated as described in A. Binding of STC·AP to membranes was saturable (Bmax = 1234 ± 108 pmol/mg protein) and had a calculated Kd of 6.75 ± 2.8 nM (n = 3 determinations).
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Adrenocortical and luteal cell cultures.
Given the fact that adrenocortical cells synthesized significant amounts of STC, studies were carried out on cultured bovine cells to determine whether additions of big STC would have an effect on cortisol output. Parallel cultures of luteal cells were treated with the same amounts of big STC to compare the responses of the two cell types. The results of these studies are shown in Fig. 7. Figure 7A shows that big STC caused a marked inhibition of progesterone output (P < 0.001) by bovine luteal cells as previously shown (19). In contrast, Fig. 7B shows that big STC was much less effective in blocking cortisol output by adrenocortical cells. Cortisol secretion was inhibited by big STC treatment (P < 0.05), but the effect was marginal. Figure 7C shows that adrenocortical cells release high levels of cortisol over 24 h under basal conditions and that output was increased fourfold in response to forskolin. Figure 7D shows that the same cells released low but measurable amounts of STC. Moreover, secretion was increased threefold by forskolin treatment (P < 0.001), indicating that STC release was subject to regulation by the PKA pathway.

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Fig. 7. Comparative secretory responses of adrenocortical and corpus luteal cells to big STC. A: luteal cells released high levels of progesterone over 24 h and responded to big STC (2 ng/ml) with a marked inhibition of progesterone output (P < 0.001, Student's t-test). B: big STC also inhibited cortisol output by adrenocortical cells, but the effect was much reduced compared with luteal cells (P < 0.05, Student's t-test). C: adrenocortical cells released high levels of cortisol over 24 h basally, and this was increased 4-fold in response to forskolin. D: the same cells released low but measurable amounts of STC, and secretion was increased 3-fold by forskolin treatment (P < 0.001).
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DISCUSSION
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In support of the stated hypothesis, the present study suggests that the production of big STC is not confined to ovarian steroidogenic cells. Rather, the evidence has revealed that big STC is also synthesized by cells within all three zones of the adrenal cortex, noted for their production of corticosteroids, mineralocorticoids, and androgens (31). Northern blotting and in situ hybridization analysis both showed that adrenocortical cells contained substantial amounts of STC mRNA. Moreover, Western blot analysis of adrenocortical cell CLD proteins revealed that the only form of STC present was a higher-molecular-mass big STC variant. Interestingly, a similar analysis of adipose cells revealed that they also contained big STC variants, which supports the view that big STC is emerging as a new modulator of lipid metabolism. However, in both the present study and a recently published report, STC mRNA levels have proved to be extremely low in adipose tissue (21). Therefore, it is possible that the STC transcript is derived instead from the vasculature, especially in view of the mounting evidence that endothelial cells express the STC gene at very high levels (5, 8, 35). Collectively therefore, it appears that the higher-molecular-mass forms of STC are preferentially synthesized by and/or targeted to cells with a major role in lipid storage and release.
In situ hybridization revealed that the majority of adrenal STC was synthesized in cortical zone cells and that these cells also contained most of the receptor-binding activity, suggesting that the hormone functions in a paracrine manner. Moreover, in both adipocytes and adrenocortical cells, receptors were found on plasma membranes and lipid droplets, and lipid droplets contained high levels of the ligand. These findings are similar to those in ovarian luteal cells (19), where the inhibitory effects of big STC on progesterone release entail its targeting to and sequestration by the CLD fraction, together with the receptor. CLDs are found in all steroidogenic cells and constitute the pool of stored cholesterol that is used for steroid hormone synthesis (31). The droplets are each surrounded by a hemimembrane (11), in which there are embedded proteins such as ADRP (22), TIP47 (33), caveolin (28), perilipins (23), and big STC, along with its receptor (19). Perilipins play a critical role in cholesterol availability, because in the nonphosphorylated state they prevent cytosolic hormone-sensitive lipase (HSL) from accessing the lipid droplets. Hormones that signal via the PKA pathway induce the phosphorylation and inactivation of perilipins and somehow allow HSL to translocate to the CLD and initiate lipolysis (1, 10, 27). Precisely how big STC reduces luteal cell progesterone release is not understood, but in view of the distribution patterns of ligand and receptor within the cell, the effect is likely manifested on or near the surface of the lipid droplet. However, it may not involve the perilipins for the simple reason that big STC is capable of inhibiting both basal and PKA-stimulated progesterone release (19), conditions under which perilipins are active and inactive, respectively (6, 11).
Because of its pronounced inhibitory effects on luteal cell progesterone release, we anticipated that HT-1080-derived big STC would have similar effects on cortisol output by adrenocortical cells. However, the same preparation that had potent inhibitory effects on luteal cell steroid output had only minimal effects on adrenocortical cells. There are several possible explanations for these findings. As the present and previous results (30) have shown, adrenocortical cells have relatively high levels of STC gene expression in vivo. This is not true in the case of luteal cells, which exhibit no endogenous STC gene expression in vivo (3, 30) and only gradually acquire this ability after plating (18). Therefore, the relatively weak effects of HT-1080 big STC on cortisol release could be due to the fact that the cells were already responding to endogenous hormone. Another factor that has to be considered is the nature of the big STC that signals on the two cell types. Luteal cells are targeted by and sequester theca cell-derived big STC, which consists of the same three high-molecular-mass variants (84, 112, and 135 kDa) as those in HT-1080 cells (18, 19). Adrenocortical cells, on the other hand, were shown to possess only a 135-kDa variant. More importantly, the adrenal-derived hormone had substantially greater displacement properties in receptor binding studies, suggestive of marked differences in affinity. Therefore, the HT-1080-derived hormone appeared to be a weaker ligand and may not have contained adequate amounts of the 135-kDa species to elicit a full response in adrenal cells. These differences between the ligands could also have physiological significance by precluding ovarian forms of the hormone from affecting adrenocortical cell function, when the former are circulating during pregnancy and lactation (3).
In view of the fact that adrenocortical cells synthesize big STC and a receptor, it is reasonable to assume that the hormone operates locally within the adrenals via paracrine and autocrine signaling pathways. If so, then adrenal cells would be exposed to much higher ligand concentrations than luteal cells, and this might explain why adrenal receptors were of similar density (6.3 vs. 5 pmol/mg protein) but of lower affinity (6.3 vs. 1.2 nM) than those in luteal cells (19). Furthermore, because adrenal CLDs contain the same complement of perilipins (23), big STC, and STC receptors, it is likely that the hormone has a role in steroid synthesis. However, determining the nature of that role may require that any future studies employ the unique adrenal form of the hormone. Moreover, in view of that fact that different steroids are elaborated by the three cortical zones (mineralocorticoids, glucocortioids, and sex steroids) under widely diverse control systems (31), the zonal regulation of adrenal gland big STC production is likely to be complex.
From a structural standpoint, what distinguishes big STC from STC50 has not yet been determined. The mammalian STC cDNA sequence predicts a protein of 247 residues with a single glycosylation consensus site (2, 17, 30). An unpaired cysteine residue in the molecule allows it to form homodimers with a combined molecular mass of 50 kDa (30, 34). The big STC monomer is at least 10 kDa larger. However, the increased mass does not appear to be due to an enhanced degree of glycosylation because, unlike STC50, big STC does not bind to plant lectins such as concanavalin A and does not undergo a downward size shift following PNGase F treatment (18). Hence, big STC could be differentially glycosylated or not glycosylated at all. Other possibilities could include posttranslational modifications to the STC core sequence, such as phosphorylation (7), or additional yet uncharacterized exons. In the context of the present study, it was noteworthy that the 135-kDa variant in adrenocortical cells was resistant to chemical reduction. In this respect, adrenocortical STC resembles STC50 from the mitochondrial matrix (12), which is also entirely resistant to chemical reduction (unpublished observations). The significance of this remains to be established, but it suggests that both forms of the hormone are subject to covalent modifications at some point in their targeting pathways, thereby rendering their quaternary structures more or less permanent.
The adrenals are one of the few mammalian organs in which STC gene expression is readily detectable by conventional Northern blotting (30). In situ hybridization analysis has now revealed that, in bovine and rodent adrenals, the majority of this gene activity is confined to the cortex. The medullary zone also showed evidence of gene activity, but the levels of expression here were substantially lower than in cortical zone cells. These findings support, in large part, the pattern of expression in human adrenals, where the STC transcript is reported to be most prevalent in the outer glomerulosa layer and in the medulla (15). The identification of big STC in adrenal steroidogenic cells also makes it highly likely that a related variant will be present in testicular Leydig cells and perhaps the placenta, both of which are known to contain high levels of STC immunoreactivity (25, 26). If this proves to be the case, it may then be possible to formulate a unifying hypothesis of big STC action, perhaps as a general regulator of steroid production.
The discovery of big STC ligand and receptor on adipocytes and their lipid droplet membranes is implicit of a role in fat storage and/or release independent of its regulatory effects on luteal cell steroid hormone synthesis. A metabolic role for STC has already been attested to in two separate models of mouse transgenesis, where the hallmarks of STC overexpression include increased food and oxygen consumption in combination with reduced body weight (4, 29). This energy-wasting phenotype is likely due to the stimulatory effects of STC on mitochondrial electron transport (14), which in excess can result in overt mitochondrial hypertrophy (4). In addition, recent studies have catalogued the colocalization patterns of STC and perilipin in human white and brown fat cells (21) and of STC and its receptor in human mammary gland stromal adipocytes (13). The results of all of these studies are collectively supportive of a central role for STC in intermediary metabolism. However, perhaps the most convincing evidence of STC's importance to metabolism can be found in the magnitude of STC receptor levels on fat cells, particularly on the lipid droplets. The best possible comparator in this case is the ovarian luteal cell (19), in which quantifiable estimates of binding capacity have been obtained for both membranes (8 pmol/mg protein) and the CLD fraction (25 pmol/mg protein). Comparatively, adipocyte membranes had a 10-fold greater capacity for ligand binding (64 pmol/mg protein), whereas the capacity of adipocyte lipid droplets was 50-fold higher (1,234 pmol/mg protein). Because the number of membrane and mitochondrial receptors in liver and kidney (12) are even lower than those in either luteal cells or adipocytes, this suggests that fat contains the greatest number of cellular STC receptors among the tissues that have been analyzed to date. At this point in time, it is premature to speculate on the central role of STC in adipocytes. Nonetheless, it is clear that an appreciation of how big STC regulates adipocyte function will be crucial to a better understanding of its overall role in metabolism.
In summary, we have shown that the big STC variants are not unique to ovarian steroidogenic cells but are also found in adrenocortical cells and adipocytes. Moreover, in both cell types both the ligand and receptor were concentrated in the lipid droplet fraction. The findings collectively support the notion that big STC has a role in lipid metabolism in different types of cells.
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GRANTS
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This study was made possible with grant support from the Canadian Institutes of Health Research and the Kidney Foundation of Canada (G. F. Wagner), and a Natural Sciences and Engineering Council of Canada Summer Studentship awarded to E. R. Hirvi
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FOOTNOTES
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Address for reprint requests and other correspondence: G. F. Wagner, Dept. of Physiology and Pharmacology, Faculty of Medicine and Dentistry, Univ. of Western Ontario, London, ON, Canada N6A 5C1 (email: graham.wagner{at}fmd.uwo.ca)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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