Bone morphogenetic protein-4 (BMP4)- A paracrine regulator of human adrenal C19 steroid synthesis

Bone morphogenetic protein-4 (BMP4): A paracrineregulator of human adrenal C19 steroid synthesis

Juilee Rege1, Hiromi Koso Nishimoto1, Koshiro Nishimoto1, Raymond J Rodgers2,Richard J Auchus3, and William E Rainey1

1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA;2School of Pediatrics and Reproductive Health, Robinson Research Institute, University of Adelaide,Adelaide, Australia; 3Division of Metabolism, Endocrinology, and Diabetes, Department of InternalMedicine, University of Michigan, Ann Arbor, MI, USA

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the transforminggrowth factor-� (TGF-�) ligand superfamily. We have identified a functional BMP system equippedwith the ligand (BMP4), receptors (BMP type-II receptor, BMP type IA receptor also called ALK3) andthe signaling proteins (SMADs 1, 4 and 5) in the human adrenal gland and the human adreno-cortical cell line H295R. Microarray, quantitative RT-PCR (qPCR) and immunohistochemistry con-firmed that BMP4 expression was highest in the adrenal zona glomerulosa (ZG) followed by thezona fasciculata (ZF) and zona reticularis (ZR). Treatment of H295R cells with BMP4 caused phos-phorylation of the SMADs and a profound decrease in synthesis of the C19 steroids dehydroepi-androsterone (DHEA), DHEA sulfate (DHEA-S) and androstenedione (A4). Administration of BMP4to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of17�-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on themRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3�-hydrox-ysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse thenegative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in theH295R cell line. Collectively, the present data suggest that BMP4 is an autocrine/ paracrine negativeregulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

The human adrenal cortex is anatomically and func-tionally classified into three zones- zona glomerulosa

(ZG), fasciculata (ZF) and reticularis (ZR), each produc-ing a different class of steroids. ZG and ZF have beenshown to synthesize mineralocorticoids and glucocortico-ids respectively (1, 2). The human ZR is considered theprimary source for the C19 steroids like DHEA and DHEAsulfate (DHEA-S) (3–5). Although some steroidogenic en-zymes and cofactor proteins are common to all zones ofthe cortex, the zone-specific production of steroids is aresult of the differential expression of key steroidogenicenzymes (6–9). The pathway leading to the synthesis of

DHEA requires only two steroidogenic enzymes, namelycholesterol side-chain cleavage cytochrome P450(P450scc) and cytochrome P450 17�-hydroxylase/17,20-lyase (P450c17). While P450scc has been shown to bepresent in all three zones of the adrenal cortex, P450c17 isexpressed in ZF and ZR (10). It is known that the 17�-hydroxylase activity of P450c17 is required for the pro-duction of the glucocorticoid cortisol in the ZF, and boththe 17�-hydroxylase and 17,20-lyase activities of theP450c17 enzyme are mandatory for the synthesis of C19

steroids in the ZR (11–13). Other vital factors that con-tribute towards the C19 steroid production by the ZR are:

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2015 by the Endocrine SocietyReceived November 24, 2014. Accepted April 7, 2015.

Abbreviations: ZF, zona fasciculata; ZR, zona reticularis; ZG, zona glomerulosa; BMP, Bonemorphogenetic protein; CYP17A1, 17�-hydroxylase/17,20-lyase mRNA; P450c17, 17�-hydroxylase/17,20-lyase protein; CYP11A1, cytochrome P450 cholesterol side-chain cleav-age; HSD3B2, type 2 3�-hydroxysteroid dehydrogenase; ALK3 /BMPR1A, BMP type IAreceptor; ALK6 /BMPR1B, BMP type IB receptor; BMPRII, BMP type II receptor; Preg, Preg-nenolone; Prog, Progesterone; 17OHPreg, 17�-hydroxypregnenolone; 17OHProg, 17�-hydroxyprogesterone; DHEA, Dehydroepiandrosterone; 11-DOC, 11-deoxycorticoste-rone; Preg-S, Pregnenolone sulfate; 17OHPreg-S, 17�-hydroxypregnenolone sulfate;DHEA-S, DHEA sulfate; Adiol-S, 5-androstenediol sulfate.

G R O W T H F A C T O R S - C Y T O K I N E S

doi: 10.1210/en.2014-1942 Endocrinology endo.endojournals.org 1

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the ZR dominance of cytochrome b5, an allosteric regu-lator of P450c17, enhancing the 17,20-lyase activity of theenzyme, and the distinctly reduced expression/ activity ofHSD3B2 in the ZR (8, 14, 15).

Bone morphogenetic proteins (BMPs) comprise one ofthe largest subgroups in the transforming growth factor-�(TGF-�) ligand superfamily. The name BMP was initiallygiven to three proteins purified from a bovine bone prep-aration that were independently capable of inducing theformation of cartilage and bone in vivo (16). Currently,fifteen BMPs have been described and recognized as mul-tifunctional regulators involved in a myriad of processes,in numerous tissues, including cell growth, apoptosis, dif-ferentiation and migration (17). BMPs are known to actthrough a combination of type II and type I BMP-recep-tors. Binding of BMP to its type II receptor in concert witha type I receptor leads to formation of a receptor complex.The constitutively active kinase domains of the type IIreceptor phosphorylate type I receptor rendering it active.The active type I receptor subsequently phosphorylates areceptor-regulated SMAD (R-Smad; eg, SMAD1/5/8) al-lowing it to associate with the co-SMAD (SMAD 4) toform a heteromeric complex that translocates to the nu-cleus and stimulates the expression of target genes (18).

The expression levels of individual members of theBMP superfamily not only vary among different tissues,but each BMP member also differs in its functional effect,depending on the cell type. Some BMPs (BMP2, 4, 6, 7, 15)have also been shown to regulate steroidogenesis in thehuman ovarian tissue and cell lines (19–21). However,currently there is limited knowledge regarding the role ofthe BMP system in human adrenal steroidogenesis. BMP6has been shown to augment angiotensin II (AngII)- in-duced aldosterone secretion in the human adrenocorticalH295R cell line (22, 23). In contrast, BMP2 and BMP5were able to suppress forskolin-stimulated steroidogenesis[aldosterone, cortisol and DHEA(S)] in H295R cells (24).

In this study, we identified a functional BMP systemequipped with the ligand (BMP4), receptors (BMP type-IIreceptor, activin receptor-like kinase receptors 3; alsocalled ALK3 or BMP type-IA receptor) and the signalingproteins (SMADs1, 4 and 5) in the human adrenal gland.Using the H295R human adrenocortical cell line, we dem-onstrate that BMP4 can act as an autocrine/paracrine neg-ative regulator of adrenal C19 steroid synthesis.

Materials and Methods

Human tissue preparationFollowing informed consent from the family, human adult

adrenal glands were obtained from cadaveric kidney donors that

were transplanted at the Georgia Regents University. Use of thesetissues was approved by the Institutional Review Board of Geor-gia Regents University.

Isolation of ZG, ZF and ZR tissues by laser-capturemicrodissection (LCM) from human adult adrenals

Adrenal pieces of 3 mm were cut from donor adrenal glandsand frozen in optimum cutting temperature compound (O.C.T.block; Sakura Finetek, Torrance, CA). Frozen adrenal glands inO.C.T. compound were cut into 7 �m sections and mountedonto Superfrost Plus Microscope Slides (Fisher Scientific,Waltham, MA). To recognize enriched populations of aldoste-rone-producing ZG, the sections were immunostained forCYP11B2 (aldosterone synthase; monoclonal mouse antibodygenerously provided by Dr. Celso Gomez-Sanchez, University ofMississippi Medical Center, Jackson, Mississippi). For the im-munohistochemistry, sections were fixed with 100% acetoneand incubated with primary/secondary antibodies without an-tigen retrieval. The remaining tissue sections were stained withcresyl violet and used for laser-capture microdissection as pre-viously reported (25, 26). ZG cells were captured fromCYP11B2-positive cells based on CYP11B2-stained sections. ZFand ZR were captured for transcriptome comparison from lipid-rich cells in the middle layer (ZF) or compact cells outside of themedulla (ZR).

Transcriptome analysis of laser-captured ZG, ZFand ZR

RNA from ZG, ZF, and ZR cells was isolated using a Pico-Pure RNA isolation kit (Life Technologies, Grand Island, NY).Total RNA (1–10 ng) from ZG, ZF and ZR samples was sub-mitted to the University of Michigan DNA Sequencing and Mi-croarray Cores for RNA amplification to reverse-transcriptionwhich were performed using the Ovation Pico WTA System V2(NuGEN Technologies, San Carlos, CA). The cDNA was puri-fied using QIAquick PCR Purification Kit (Qiagen, Hilden, Ger-many) and biotin-labeled using Encore Biotin Module (NuGENTechnologies), followed by hybridization to GeneChip HumanGenome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA).This array was designed to investigate 21 702 genes including18 802 distinct, unambiguous genes on the human genome with54 675 probe sets comprised of 11 perfect match (PM) probesper probe set. Expression values for each probe set were calcu-lated using a robust multiarray average method (27). We fit two-way ANOVA models with terms for 3 tissues (ZG, ZF, ZR) and4 people (respective patients) to log-transformed data for eachprobe set, and used the resulting F-tests to compare pairs oftissues. To identify the differences among the 3 zones, a list ofZG, ZF and ZR markers was created by using transcripts satis-fying the conditions including fold expression differences andstatistical differences between the respective zones.

Residual RNA, which was prepared in the transcriptomeanalysis, was used for confirmation qPCR of BMP4. These RNAwere again amplified and reverse-transcribed to cDNA as de-scribed above. For qPCR, 1 ng of prepared cDNA was mixedwith Fast Universal PCR Master Mix (Applied Biosystems, Fos-ter City, CA), following the manufacturer’s recommendations,and stored at –80°C until further use.

2 BMP4 inhibits adrenal C19 steroid production Endocrinology


Cell culture and treatmentsThe H295R human adrenocortical cell line was cultured in

DME/F12 medium (Life Technologies) supplemented with 10%cosmic calf serum (GE Healthcare Life Sciences, HyClone Lab-oratories, Logan, UT) and antibiotics including penicillin/strep-tomycin and gentamycin (Sigma-Aldrich, St. Louis, MO). Thecells were incubated under a humid atmosphere of 5% CO2, at37°C, and medium was changed every two days. H295R cellswere plated at a density of 100 000 cells / well (48-well dish) ingrowth medium and grown to 60% confluence after which theywere starved in low serum medium (1% cosmic calf serum andantibiotics) 18 hours prior to treatment with BMP4 (ProSpec,East Brunswick, NJ, USA).

Primary adrenal cells were isolated as previously described(28). Briefly, adrenal tissue was minced and dissociated into asingle cell suspension by repeated exposure of the tissue frag-ments to DMEM/F12 medium containing 1 mg/mL collagenasedispase and 0.25 mg/ml DNase-1 (Hoffmann-La Roche Ltd, Ba-sel, Switzerland). Four 1 hour cycles of digestion at 37°C andmechanical dispersion were performed. Cells were collected be-tween each digestion and combined prior to storage at –150°C.Primary adrenal cells were plated at a density of 20 000 cells /well (48-well dish) in growth medium and grown to 60% con-fluence after which they were starved in low serum medium 18hours prior to treatment with BMP4.

To study the effect of BMP4 on dibutyryl cyclic AMP (db-cAMP)-stimulated steroidogenesis, H295R cells were initiallytreated with 30 ng/mL BMP4 for 24 hours. The medium was thenreplaced with that containing 30 ng/mL BMP4, with and without1 mM dbcAMP (Sigma-Aldrich). After 24 hours, media and cellswere collected. For inhibitor studies, cells were preincubatedwith the BMP inhibitor, Noggin (ProSpec), for 30 minutes beforeBMP4.

For the LC-MS/MS studies, H295R cells were plated at adensity of 100 000 cells / well (48-well dish) in growth mediumand grown to 60% confluence after which they were starved inlow serum medium (1% cosmic calf serum and antibiotics) 18hours prior to treatment with 30 ng/mL BMP4 (ProSpec, EastBrunswick, NJ, USA). After 24 hours, the medium was replacedwith that containing fresh 30ng/mL BMP4. Medium was thencollected after 24 hours for steroid quantitation. At the end ofeach treatment, medium was collected from each well and storedat –20°C for steroid immunoassays or LC-MS/MS, while cellswere frozen at –80°C for protein assay, western blot or RNAisolation, with subsequent qPCR analysis, as described below.

In order to quantify the basal BMP4 secretion in the adrenalcell types, the H295R and primary adrenal cells were incubatedin low serum medium for 48 hours after reaching 60% conflu-ence. BMP4 content in the experimental media was quantified byEIA.

qPCR analysisqPCR was performed using the ABI 7500 Fast Real-Time

PCR System (Applied Biosystems, Foster City, CA, USA). Theprimer and probe sets for BMP4, CYP11A1, HSD3B2 andCYP17A1 were designed using Primer Express 3.0 (Applied Bio-systems) or purchased from Integrated DNA Technologies (Cor-alville, IA, USA) as published previously (29). Quantitative nor-malization of cDNA in each tissue-derived sample and cellsample was performed using expression of peptidylprolyl

isomerase A (PPIA, Cyclophilin A), purchased from AppliedBiosystems, as an internal control.

Protein extraction and protein assayCells were lysed in 100 �L Mammalian Protein Extraction

Reagent (Pierce Chemical Co., Rockford, IL, USA), and the pro-tein content was estimated by the bicinchoninic acid (BCA) pro-tein assay using the micro BCA protocol (Thermo Scientific,Rockford, IL, USA).

Steroid immunoassays for cell culture experimentsThe aldosterone content of the cell culture medium was an-

alyzed using Coat-a-Count radioimmunoassay (RIA) kits fromSiemens (Los Angeles, CA, USA) and radioactivity was quanti-fied using a Wallac Wizard 1470 multicrystal gamma counter(Perkin Elmer, Waltham, MA, USA). Cortisol and DHEA werequantified by enzyme immunoassay (EIA) (Alpco, Salem, NH,USA). Assays were conducted following the manufacturer’s rec-ommendations except that the standard curves were prepared inthe experimental cell culture medium. The results were normal-ized to protein content and shown as fold change or % changeover basal condition.

BMP4 immunoassay for cell culture experimentsThe BMP4 content of the cell culture medium was analyzed

using the RayBio EIA kit (Raybiotech, Norcross, GA). Assayswere conducted following the manufacturer’s recommendationsexcept that the standard curves were prepared in the experimen-tal cell culture medium. The results were normalized total pro-tein content.

Steroid measurement by liquid chromatography-mass spectrometry (LC-MS/MS)

21- and 19-carbon steroids were also measured by LC-MS/MS to determine the effects of BMP4 on H295R steroido-genesis, where specified. The LC-MS/MS method is described indetail in the manuscript’s Supplemental Methods section.

Western blot analysisH295R cells were plated at a density of 200 000 cells / well

(24-well dish), in growth medium and grown to 60% confluenceafter which they were starved in low serum medium 18 hoursprior to treatment with BMP4 for the indicated times. Immuno-blotting was performed using XCell SureLock system (Life Tech-nologies) following the manufacturer’s recommendation.Briefly, samples were lysed with lysis buffer (2% sodium dodecylsulfate, 62.5 �M Tris, 0.04% bromophenol blue, 0.5 M dithio-threitol) and heated at 95°C for 5 minutes. Proteins were thenloaded in equal amounts on 10% Bis-Tris gel and allowed toseparate for 1.5 hours before transferring to polyvinylidene di-fluoride membranes. The membranes were then blocked with5% BSA for 1 hour and incubated with either pSMAD1/5/8antibody (polyclonal rabbit antihuman, 1:1000 in BSA; Cell Sig-naling, Beverly MA) or P450c17 antibody (polyclonal rabbitantihuman, 1:1000; generously provided by Dr. Michael R Wa-terman, Vanderbilt University School of Medicine, Nashville,TN), and secondary antibody (goat antirabbit, 1:2000; LifeTechnologies). The Pierce ECL Western Blotting Substrate kit(Life Technologies) was used for signal development.

doi: 10.1210/en.2014-1942 endo.endojournals.org 3


ImmunohistochemistryRabbit polyclonal antibody against human BMP4 was di-

luted 1:100 with the antibody diluent solution (DAKO, Carpin-teria, CA, USA). The slides with tissue sections were first heatedat 225°F for 15 minutes. After deparaffinization slides were in-cubated in target retrieval solution (diluted 1:100 with distilledwater, pH 9) (DAKO) for antigen retrieval. Immunohistochem-ical analysis was performed employing Envision� kit (DAKO) asper manufacturer’s instructions. Nonimmunized rabbit antise-rum was used as a negative control for immunohistochemistry.

Data analysis and statistical methodsResults are presented as mean � SEM where appropriate. For

the microarray analysis, we fit two-way ANOVA models withterms for 3 tissues (ZG, ZF, ZR) and 4 people (respective pa-tients) to log-transformed data for each probe set, and used theresulting F-tests to compare pairs of tissues. Significance wasaccepted at the P � .05 level of probability. Statistical analysis forBMP4 mRNA comparison between ZG, ZF and ZR by qPCRwas determined using One-Way ANOVA followed by a Holm-Sidak correction. Significance was accepted at the P � .05 levelof probability.

Comparison between two treatment groups with H295R cellsfor enzyme expression and steroid analysis was performed usingunpaired t test and significance was accepted at the P � .05 levelof probability. For comparison between more than two treat-ment groups with H295R or primary cells, One-Way ANOVAwas employed followed by a Holm-Sidak correction. Signifi-cance was accepted at the P � .05 level of probability.

Results

A functional BMP system in the human adrenalLaser-captured, microdissected, ZG, ZF and ZR from

human adrenals were analyzed by microarray for the pres-ence of different BMPs as well as the entities involved inBMP signaling (Supplemental Figure 1 and Figure 1A re-spectively) in order to define potential candidates for func-tional studies. We demonstrated that the ligand BMP4was up-regulated in the ZG and ZF compared to ZR,whereas there was abundant expression of the receptorsALK3 (BMPR1A) and BMPR2, and the signaling proteinsSMAD 1, 4 and 5 in all the three zones (Figure 1A). SMAD8 expression was mainly restricted to the ZG. We vali-dated the microarray results obtained for BMP4 usingqPCR on the ZG, ZF and ZR (Figure 1B) and showed thatthe expression of BMP4 in the ZR is lower than the outertwo zones of the adrenal cortex. In accordance with theexpression analysis, BMP4 immunoreactivity was highestin the ZG (Figure 1C). Cultured primary adult adrenalcells and H295R cells were also found to secrete abundantBMP4 under basal conditions (Table 1).

BMP-pathways are inducible in H295R cellsWe examined and confirmed the expression of tran-

scripts associated with BMP signaling in the H295R hu-man adrenocortical cell line, primary adrenal cells, andnormal adrenal tissue, by microarray (Supplemental Fig-ure 2). To ensure that the BMP-dependent pathways areintact in an adrenocortical tumor cell model, the H295Rcells were incubated with 30 ng/mL of recombinant hu-man (rh) BMP4, for increasing time intervals (5–30 min-utes).A time-dependentBMP-inducedphosphorylationofdownstream SMAD1/5/8 proteins was observed (Figure2), indicating that the BMP-pathways are active in H295Rcells.

Concentration-dependent effects of BMP4 onsteroidogenesis in H295R cells

To examine the concentration-dependent effect ofBMP4 on steroidogenesis, H295R cells were treated for 48hours with control medium plus BMP4 in increasing doses(0.3, 1, 3, 10, 30 and 100 ng/mL). Accumulation of aldo-sterone, cortisol and DHEA in the culture medium wasthen evaluated using immunoassays. No significantchanges were found in aldosterone and cortisol levels fol-lowing treatment with increasing concentrations of BMP4(Figure 3A). However, BMP4 was shown to significantlyreduce the level of DHEA (Figure 3A) in a dose-dependentmanner for all concentrations equal to or greater than 3ng/mL (P � .001).

We further examined the effect of BMP4 on DHEAlevels following treatment with dibutyryl cAMP. Db-cAMP is an analog of cAMP that stimulates cAMP-de-pendent protein kinase)-stimulated steroid production.Treatment of H295R cells with BMP4 inhibited both basaland dbcAMP-increased DHEA production (Figure 3B)whereas, levels of aldosterone and cortisol remained un-changed under both basal and dbcAMP-stimulated con-ditions (Figure 3B) (P � .001).

Effects of BMP4 on the expression of keysteroidogenic enzymes in H295R cells

We sought to elucidate the effect of BMP4 on H295Rcell expression of key determinants of steroidogenesis,namely CYP11A1, CYP17A1 and HSD3B2. In accor-dance with the decreasing levels of DHEA (Figure 3A),qPCR revealed BMP4-dependent down-regulation ofCYP17A1 in a dose-dependent manner in H295R cells(Figure 4A). We observed a similar dose-dependent de-crease in the mRNA levels of CYP17A1 in primary adrenalcells (Figure 4B). Western analysis indicated that althoughBMP4 was able to decrease the levels of P450c17 inH295R cells, it was not able to completely abolish its cel-



Figure 1. Panel A. Heatmap representation of microarray analysis for transcripts encoding genes involved in the BMPsignaling pathway in adrenal ZG, ZF and ZR. Normalized log2 signal intensity acquired from microarray analysis is represented by color(see color bar). Average ratio was calculated as average of normalized ZG or ZF signal intensities divided by that of normalized ZR signal intensities(n � 4) as indicated. A two-fit ANOVA (P � .05) confirmed that BMP4 was higher in the ZG as compared to both ZF and ZR. BMPR1A, BMP typeIA receptor; BMPR1B, BMP type IB receptor; BMPR2, BMP type II receptor. Panel B. Quantitative RT-PCR analysis for BMP4 in theadrenal ZG, ZF and ZR obtained by laser capture microdissection. Validation of microarray analysis by real-time qPCR for BMP4 in ZGvs. ZF vs. ZR. BMP4 showed higher expression in ZG and ZF as compared to ZR. Three ZG, ZF and ZR samples were used for qPCR analysis. Data arerepresented as Mean � SEM. Different letters above the bars indicate statistically significant differences (P � .05) between the zones (n � 4).Statistical significance was determined using one-way ANOVA followed by a Holm-Sidak test. Panel C. Immunohistochemical localizationfor BMP4 in the adrenal capsule, ZG and ZF. Immunohistochemical analysis showed that BMP4 levels (left panel) were highest in ZG.



lular expression after 48 hours of incubation (Figure 4C,top panel).

To further detail the BMP4-dependent effects on ste-roidogenesis, we also evaluated the regulation of otherenzymes located at key branch points in the steroidogenicpathway – CYP11A1 and HSD3B2. Contrary toCYP17A1, we found that there was no significant changein the expression of these enzymes (Figure 4C).

Noggin prevents the negative regulatory effectsof BMP4 on adrenal CYP17A1 mRNA levels andDHEA synthesis

In order to further confirm the role of BMP4 in de-creasing C19 steroid levels in H295R cells, a BMP antag-onist Noggin (which acts by limiting BMP signaling byligand sequestration) was used in combination with BMP4followed by analysis of CYP17A1 mRNA and DHEA ste-roid levels. We observed that noggin alone (20–80 ng/mL)did not affect the CYP17A1 mRNA and DHEA levels (Fig-ure 5). However, 40–80 ng/mL of Noggin was able toreverse the negative effects of BMP4 (30 ng/mL) with re-spect to both CYP17A1 transcription and DHEA secre-tion (Figure 5) (a vs b; P � .01).

BMP4 effects on the H295R steroid metabolomeWe also defined the global effects of BMP4 on steroid-

ogenesis in the H295R adrenocortical cell line using LC-MS/MS analysis of secreted steroids (Figure 6). We mea-sured nine 21-carbon and two 19-carbon unconjugated

steroids along with three steroid sulfates. Our LC-MS/MSstudies confirmed that incubation of H295R cells withBMP4 (30 ng/mL) for 48 hours decreased the levels of bothC19 steroids - DHEA and androstenedione (Figure 6C)(P � .05). DHEA sulfate (DHEA-S) also decreased fol-lowing BMP4 treatment (Figure 6D) (P � .05). On theother hand, we detected a discernible increase in the con-centrations of pregnenolone, progesterone, 17�-hydroxy-progesterone and 11-deoxycorticosterone (Figure 6B)(P � .05). Levels of other unconjugated and sulfated C21

steroids, including aldosterone and cortisol, did notchange with BMP4 treatment (Figure 6B and D).

Discussion

The purpose of this study was to demonstrate the existenceof an endogenous BMP system in the human adrenal glandand to investigate its autocrine/paracrine role in the reg-ulation of adrenal C19 steroid biosynthesis via modulationof key enzymes involved in steroidogenesis.

The fetal and adult human adrenals have been shown toproduce a variety of factors that affect growth, and pro-liferation as well as steroidogenic capacity (30–37). Mem-bers of the TGF-� family, including TGF�1, activin andBMPs, have been shown to play a significant role in theregulation of key steroidogenic enzymes and thereby hu-man adrenal steroidogenesis (22–24, 30, 31, 35, 36, 38–41). The present study has demonstrated the existence of

Legend to Figure 1 Continued. . .Middle and right panel represent corresponding negative control (rabbit antiserum used instead of primary antibody) and Hematoxylin-Eosin (HE)stained sections respectively.

Table 1. Basal BMP4 secretion in primary adult adrenal cell cultures and the H295R adrenocortical cell line cultures

Cell Type BMP4 (pg/mg total protein�48 h)

Primary adrenal cells 893 � 134H295R 507 � 82

EIA was used to quantify the BMP4 in the medium from the primary adrenal cells and the H295R adrenocortical cell line after 48 h of incubation.Results are normalized to total cellular protein content. Data are presented as mean � SEM (n � 3 experiments).

Figure 2. Effects of BMP4 on SMAD1/5/8 phosphorylation in H295R adrenocortical cells. Cells were incubated with BMP4 (30 ng/ml) for the indicated time points. The cells were then lysed and subjected to SDS-PAGE and immunoblotting analysis using an anti-pSMAD1/5/8antibody. Treatment of adrenocortical tumor cells with BMP4 shows integrity of BMP-dependent pathways by phosphorylation of SMAD 1/5/8.Results shown are representative of those obtained from three independent experiments.



a complete BMP system composed of ligand (BMP4), typeI receptor (ALK3), type II receptor (BMPRII) and signalingproteins (SMAD 1, 4 and 5) in the human adrenal cortex.

In addition, we observed the existence of the same en-tities in the H295R cell line by microarray. Our results arepartially in agreement with studies by Suzuki et al thatsuggest that the H295R cell line expresses entities likeALK2, ALK3, ALK4 and BMPRII (22). However, theyalso demonstrate that the H295R cells express the ligandBMP6, rather than BMP4. The expression of other BMPligands like BMP2 and BMP5 has also been established inthe adrenal cortex (24). We have also showed that theH295R cells and the primary adult adrenal cells secretesignificant quantities of the BMP4 protein.

In the present study, the treatment of H295R cells withBMP4 caused a concentration-dependent inhibition of thetwo adrenal C19 steroids, DHEA and A4. The effect of

BMPs on adrenal C19 steroids has been evaluated by onlyone other study (24). Johnsen et al showed that BMP2 andBMP5 were able to abate the production of aldosterone,cortisol and DHEA sulfate (DHEA-S) in a dose-dependentmanner in the H295R cell line (24). In contrast, our studiesshow that aldosterone and cortisol levels remained un-changed after BMP4 treatment. BMP2 and BMP5 havebeen shown to suppress the overall forskolin-induced ste-roidogenic capacity of H295R cells (24). In other studiesBMP6 has been shown to augment angiotensin II (AngII)-induced aldosterone secretion through SMAD1/5/8 (22,23). However, potassium-induced aldosterone secretionremained unchanged. An interesting follow-up study byOtani et al demonstrated that the endogenous BMP6 in theH295R cells was involved in ‘aldosterone breakthrough’caused by chronic blockage of AngII type 1 receptors (AT-1R) (38). It would be interesting to determine if BMP4 is

Figure 3. Panel A. Dose-dependent effects of BMP4 on aldosterone, cortisol and DHEA production in H295R adrenal cells.Cells were treated with the indicated concentrations of BMP4 for 48 hours. Steroid content of media was measured by immunoassays. Data areshown as the percent fold change compared with no treatment. Results represent the Mean � SEM from four independent experiments. Statisticalsignificances with the no treatment control were determined using one-way ANOVA followed by a Holm-Sidak test. *,P � .001. Panel B. Effectof BMP4 on dbcAMP-induced steroidogenic hormone production in H295R cells. Cells were initially treated with the 30 ng/mLBMP4 for 24 hours. The medium was then replaced with that containing 30 ng/mL BMP4, with and without 1 mM dbcAMP. After 24 hours,media and cells were collected. Data are shown as the fold change compared with no treatment. Results represent the Mean � SEM from fourindependent experiments. Statistical significance with the no treatment control was determined using one-way ANOVA followed by a Holm-Sidaktest. *,P � .001.

Table 2.

Peptide/protein target Antigen sequence (if known) Name of AntibodyManufacturer, catalog #, and/or

name of individual providing the antibodySpecies raised in;

monoclonal or polyclonal Dilution used

BMP4 Thermo Scientific, Rockford, IL, Cat # PA5–32 279 polyclonal; rabbit 1:100 (IHC)

P450c17 Dr. Michael Waterman, Vanderbilt University polyclonal; rabbit 1:1000 (WB)

CYP11B2 Dr. Celso Gomez-Sanchez, University of Mississippi Medical Center monoclonal; mouse 1:100 (IHC)

pSMAD 1/5/8 Cell Signaling, Beverly, MA, Cat# 9511 polyclonal; rabbit 1:1000 (WB)



able to cause similar effects in H295R cells under AngIIinduction.

Despite the substantial reduction in the mRNA levels ofCYP17A1 with BMP4 treatment, LC-MS/MS profiles

Figure 4. Effects of BMP4 on expression of keysteroidogenic enzymes. Panels A and B represent dose-dependent effects of BMP4 on CYP17A1 mRNA levels in humanH295R adrenal cells and primary human adrenal cells, respectively.Cells were treated with the indicated concentrations of BMP4 for 48hours. CYP17A1 mRNA levels were quantified by real-time qPCRanalysis. Data are shown as the percent fold change compared with notreatment. Results represent the Mean � SEM from at least three

Legend to Figure 3 Continued. . .independent experiments. Statistical significance was determined usingone-way ANOVA followed by a Holm-Sidak test. *,P � .001. Panel Cshows the effects of BMP4 on mRNA levels of key steroidogenicenzymes CYP11A1, CYP17A1 and HSD3B2. The top panel representseffect of BMP4 on P450c17 protein levels in H295R adrenal cells. Cellswere treated with or without BMP4 (30 ng/mL) for 48 hours. The cellswere then lysed and subjected to SDS-PAGE and immunoblot analysisusing an anti- P450c17 antibody. Results shown are representative ofthose obtained from three independent experiments.

Figure 5. Action of Noggin on BMP4- induced effects onCYP17A1 (Panel A) mRNA levels and DHEA production(Panel B) in H295R cells. Cells were treated with 30 ng/mL ofBMP4 for 48 hours. qPCR was used to quantify cellular CYP17A1mRNA and media content of DHEA was measured by immunoassay.Data are shown as the percent fold change compared with notreatment (basal). Results represent the Mean � SEM from threeindependent experiments. Statistical significance was determined usingone-way ANOVA followed by Holm-Sidak test. (a vs b, P � .01).



showed that BMP4 prominently decreased synthesis ofonly the C19 steroids DHEA, DHEA-S, and A4 but notcortisol. Interestingly, BMP4 did not have any effect on themRNA levels of CYB5A, an allosteric regulator of the17,20-lyase activity of P450c17 (data not shown). Ourresults are predictable for changes in P450c17, an enzymethat catalyzes two sequential reactions. Reduced P450c17

activity with sustained CYP11A1 and HSD3B2 expres-sion allowed precursors pregnenolone and progesteroneto rise. Steroids that require only the 17�-hydroxylaseactivity of P450c17 for their production either remainedunchanged (cortisol) or showed a slight increase(17OHProg) upon BMP4 treatment. The dominant C21

steroid product of H295R cells, 11-deoxycortisol, fell

Figure 6. Effects of BMP4 on global steroidgenesis in H295R cells. The adrenal steroidogenic pathway including C21 and C19 steroids,and steroid sulfates is described in Panel A. Cells were incubated with BMP4 (30 ng/ml) for 48 hours with medium/BMP4 changed after 24hours. Medium steroid content from the final 24 hours was determined using LC-MS/MS (Panels B,C and D) and normalized with cellularprotein content. Results represent the Mean � SEM from three independent experiments. Statistical significance with the untreated control wasdetermined using unpaired t test *,P � .05.



slightly but not significantly. Thus, precursor-to-productratios rose for both the 17�-hydroxylase and 17,20-lyasereactions. Our results mirror the consequences of treat-ment with the P450c17 inhibitor abiraterone acetate inmen with prostate cancer (42).

P450c17 executes the switch between the production ofmineralocorticoids, glucocorticoids and C19 steroids. Theproduction of mineralocorticoids in the ZG relies on lowlevels of P450c17 expression, while C19 steroid produc-tion in the ZR is controlled through the differential reg-ulation of its 17�-hydroxylase and 17,20-lyase activities.One interpretation correlating our immunohistochemicalanalysis and the decreased CYP17A1 mRNA could be thatBMP4 acts as a paracrine/ autocrine factor involved insuppression of P450c17 in the ZG and outer ZF. It canthereby contribute to functional adrenocortical zonation.

The intracellular mechanism of BMP4 action on ste-roidogenic enzymes is still unknown. The BMP2/5 studyby Johnsen et al (24) showed that along with decreasingthe expression of the steroidogenic enzymes (P450c17 andHSD3B2) and adrenocorticotropic hormone receptors(MC2R), that BMP2/5 increased H295R cell ID1 tran-script levels under both basal and forskolin-stimulatedconditions. ID1 (member of the Inhibitor of DNA bindingproteins family) can inhibit proteins of the basic helix-loop-helix (bHLH) family. The same study (24) also ex-amined the MC2R promoter and showed that it possessestwo consensus sites for E-box elements which could betargeted by the transcription factors of the bHLH family.Hence, they speculated that the inhibition of MC2R andthe genes encoding the steroidogenic enzymes could be adirect consequence of the BMP-dependent up-regulationof the ID1 levels, since ID1 could in turn inhibit the stim-ulating transcription factors of the bHLH family (24). In-terestingly, the bHLH factor Bmal1 is required for circa-dian glucocorticoid secretion through E-box elements onthe StAR promoter (43, 44). Hence it would be interestingto determine if BMP4 down-regulation of P450c17 occursby a similar mechanism.

Studies by Glister et al using ovarian cells indicate analternate mechanism by which BMP4 could regulateCYP17A1 mRNA levels in the human adrenal (45). Thismicroarray study involving bovine theca cells (TC) culti-vated in the presence and absence of BMP6, suggested alink between INSL3 signaling and thecal cell steroidogen-esis, since the treated cells showed a reduction in INSL3 aswell as CYP17A1 and the mRNA levels of other steroid-ogenic enzymes, along with a concomitant decrease in A4secretion (45). The authors not only proved that INSL3-RXFP2 (INSL3 receptor) signaling plays a pivotal role inthecal androgen secretion but also confirmed that auto-crine/paracrine INSL3-RXFP2 signaling increases TC an-

drogen synthesis under both basal and LH-stimulatedconditions (45).However,microarrayanalysis of the threezones of the human adrenal showed that the endogenouslevels of both INSL3 and RXFP2 are very low (data notshown) suggesting that the INSL3-RXFP2 signaling path-way does not operate in the adrenal. Thus, it appears thatCYP17A1 is likely regulated differently between theca andadrenal cells.

Collectively, we have demonstrated the presence of aBMP system in the human adrenal gland. BMP4 was ableto decrease adrenal C19 steroid production without sig-nificantly affecting aldosterone and cortisol synthesis atbasal conditions. Such an endogenous BMP system mightplay a crucial role in regulation of adrenal steroidogenesisin an autocrine/ paracrine manner and could thereby mod-ulate functional adrenocortical zonation.

Acknowledgments

We thank Dr. Mary Bassett for her editorial assistance. Thiswork was supported by the National Institutes of Health (GrantsDK43140 and DK069950 to W.E.R.). Mass spectrometry usedcore services supported by Grant DK089503 from the NationalInstitutes of Health to the University of Michigan under theMichigan Nutrition Obesity Center (NORC).

Address all correspondence and requests for reprints to: Cor-responding author and person to who reprint requests should beaddressed: William E Rainey, Ph.D., Director of Endocrine Neo-plasia Basic Research, Departments of Molecular and IntegrativePhysiology and Internal Medicine, University of Michigan, AnnArbor, MI 48 109–5622. Email: [email protected], Tel(734).-764–7514.

Disclosure Summary: The authors have nothing to declare.This work was supported by Funding: This work was sup-

ported by grants from the National Institutes of HealthDK43140 and DK069950 to W.E.R.

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Bone morphogenetic protein-4 (BMP4)- A paracrine regulator of human adrenal C19 steroid synthesis

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