High dickkopf-1 levels in sera and leukocytes from children with 21-hydroxylasedeficiency on chronic glucocorticoid treatment

Giacomina Brunetti,1* Maria Felicia Faienza,2* Laura Piacente,2 Annamaria Ventura,2 Angela Oranger,1

Claudia Carbone,1 Adriana Di Benedetto,1 Graziana Colaianni,1 Margherita Gigante,3 Giorgio Mori,3

Loreto Gesualdo,4 Silvia Colucci,1 Luciano Cavallo,2 and Maria Grano1

1Department of Basic Medical Sciences, Neuroscience, and Sense Organs, Section of Human Anatomy and Histology,University of Bari, Bari, Italy; 2Department of Biomedical Sciences and Human Oncology, University of Bari “A. Moro”,Bari, Italy; 3Department of Biomedical Science, University of Foggia, Foggia, Italy; and 4Nephrology, Dialysis, andTransplantation Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy

Submitted 31 October 2012; accepted in final form 3 January 2013

Brunetti G, Faienza MF, Piacente L, Ventura A, Oranger A,Carbone C, Di Benedetto A, Colaianni G, Gigante M, Mori G,Gesualdo L, Colucci S, Cavallo L, Grano M. High dickkopf-1levels in sera and leukocytes from children with 21-hydroxylasedeficiency on chronic glucocorticoid treatment. Am J Physiol Endo-crinol Metab 304: E546–E554, 2013. First published January 8, 2013;doi:10.1152/ajpendo.00535.2012.—Children with 21-hydroxylase de-ficiency (21-OHD) need chronic glucocorticoid (cGC) therapy toreplace congenital deficit of cortisol synthesis, and this therapy is themost frequent and severe form of drug-induced osteoporosis. In thisstudy, we enrolled 18 patients (9 females) and 18 sex- and age-matched controls. We found in 21-OHD patients high serum andleukocyte levels of dickkopf-1 (DKK1), a secreted antagonist of theWnt/�-catenin signaling pathway known to be a key regulator of bonemass. In particular, we demonstrated by flow cytometry, confocalmicroscopy, and real-time PCR that monocytes, T lymphocytes, andneutrophils from patients expressed high levels of DKK1, which maybe related to the cGC therapy. In fact, we showed that dexamethasonetreatment markedly induced the expression of DKK1 in a dose- andtime-dependent manner in leukocytes. The serum from patients con-taining elevated levels of DKK1 can directly inhibit in vitro osteoblastdifferentiation and receptor activator of NF-�B ligand (RANKL)expression. We also found a correlation between both DKK1 andRANKL or COOH-terminal telopeptides of type I collagen (CTX)serum levels in 21-OHD patients on cGC treatment. Our data indi-cated that DKK1, produced by leukocytes, may contribute to thealteration of bone remodeling in 21-OHD patients on cGC treatment.

dickkopf-1; 21-hydroxylase deficiency patients; glucocorticoid-inducedosteoporosis; leukocytes; glucocorticoids

CHILDREN WITH 21-HYDROXYLASE DEFICIENCY (21-OHD), the mostcommon cause of congenital adrenal hyperplasia (CAH) due todeletions or mutations of the P450 21-hydroxylase gene(CYP21), need chronic glucocorticoid (cGC) therapy as soonas the diagnosis has been made to replace congenital deficit incortisol synthesis and to reduce androgen secretion by adrenalcortex (18). One of the most serious problems of cGC therapyis glucocorticoid-induced osteoporosis (GIO), although thepatients are treated with glucocorticoids (GCs) at replacementdoses. However, the risk of GC overreplacement or neuroen-docrine modifications during treatment (i.e., abnormal growth

hormone secretion and pulsatility) should be taken in consid-eration as potential factors determining bone loss even insubjects treated with apparent “physiological doses” of GCs(29, 54). GIO results in an early, transient increase in boneresorption accompanied by a decrease in bone formation,which is maintained for the duration of GC therapy (3).Therefore, 21-OHD patients are at risk of a great incidence oflow bone mass, although studies on bone mineral density(BMD) have reported discordant results. In fact, an increased(46), decreased (2, 8, 12, 15, 16, 20, 36, 44), or normal BMD(5, 13, 14, 30, 47), as well as contrasting results about theevaluation of serum biochemical markers of bone turnover, hasbeen reported in these patients (12, 13, 36, 44). In our previouswork (10), we demonstrated a high osteoclastogenic potentialof peripheral blood mononuclear cells in children with 21-OHD on cGC treatment, which seems to be supported by thepresence of both circulating osteoclast precursors and T cells,expressing high levels of the proosteoclastogenic cytokinereceptor activator of NF-�B ligand (RANKL) as well as lowamounts of the antiosteoclastogenic molecule osteoprotegerin(OPG). Numerous scientists have highlighted the interactionsbetween bone and immune cells, and in particular, the role ofthe T cell in osteoclast formation and activity in many diseases,including 21-OHD, has been highlighted (6, 10, 22). However,little is known about the effect of the immune cell system onthe activity of the osteoblasts (OBs), the bone-forming cells, inbone loss-associated diseases and in 21-OHD patients under-going cGC treatment.

It is recognized that GCs suppress the bone formation byimpairing the replication, differentiation, and function of OBsand inducing the apoptosis of OBs and osteocytes (4, 39). Theinvolved mechanisms include the regulation of the canonicalWnt-�-catenin signaling pathway and its inhibitors (27). Thecanonical Wnt signaling pathway is important for the growthand differentiation of OBs (23) and for the inhibition ofadipocyte differentiation (42). Its activation is followed by�-catenin translocation into the nucleus and, subsequently,transactivation of transcription factor T cell (TCF)-responsivegene (23). Wnt pathway activation is tightly regulated bymultiple families of secreted antagonists, including dickkopf-1(DKK1). The inhibition of Wnt signaling by DKK1 results inthe decrease of bone formation and the increase of boneresorption. In particular, DKK1 blocks the maturation of OBsand decreases OPG levels and increases RANKL expression inOBs, thereby shifting the OPG/RANKL ratio in favor of boneresorption (38).

*These authors contributed equally to this work.Address for reprint requests and other correspondence: B. Giacomina, Dept.

of Basic Medical Sciences, Neuroscience, and Sense Organs, Section ofHuman Anatomy and Histology, Univ. of Bari, Piazza Giulio Cesare, 11 70124Bari, Italy (e-mail: giacomina.brunetti@uniba.it).

Am J Physiol Endocrinol Metab 304: E546–E554, 2013.First published January 8, 2013; doi:10.1152/ajpendo.00535.2012.

0193-1849/13 Copyright © 2013 the American Physiological Society http://www.ajpendo.orgE546

Recently, DKK1 has been described as a central molecularplayer in bone loss-associated diseases such as multiple my-eloma (50), rheumatoid (52), and psoriatic arthritis (7) as wellas Paget’s disease (28).

Thus, in the present study, we evaluated the DKK1 serum levelsand the intracellular expression on leucocytes obtained frompatients affected with 21-OHD, as well as the effects of dexa-methasone on the expression of DKK1 in human leukocytes fromhealthy donors in vitro. Moreover, we examined the effects of theconditioned media by the serum of the patients on OB differen-tiation and RANKL expression. In the same patients, we alsoevaluated the BMD and the biochemical markers of bone turn-over.

MATERIALS AND METHODS

Subjects

The samples included peripheral blood (PB) from 18 Caucasianpatients (9 females) affected by 21-OHD patients aged 3–16 yr.Diagnosis was made on the basis of clinical evidence, basal serumconcentrations, and peaks of 17�-hydroxyprogesterone after adreno-corticotropin test. Molecular analysis of the P450 21-hydroxylasegene (CYP21) gene was performed in all patients and in their parents.Subjects with risk factors for reduced bone mass (familial osteoporo-sis, prematurity, delayed puberty) were excluded from the study. Themain characteristics of the patients and the dose of hydrocortisone atthe time of recruitment are summarized in Table 1. Of all the patients,10 (4 females) had the nonclassical (NC) form of 21-OHD and hadbeen treated at the age of 7.6 � 4.3 yr because of advanced bone ageor hirsutism. Four patients (1 female) had the classical salt-wasting(SW) form, and four subjects (4 females) had the simple virilizing(SV) form. Patients with the SW form received GCs and mineralcor-ticoid (9�-fludrocortisone: 0.1–0.2 mg/day) therapy beginning atearly infancy, whereas the patients with the SV form were treated withGCs alone. Treatment at the time of the study consisted of hydrocor-tisone, expressed as dose per body surface per day (mg·m2·day�1),given twice or three times daily. The four patients with SW formreceived a total hydrocortisone dose of 25 mg·m2·day�1. Thepatients with the SV and NC forms were treated with 10 –15mg·m2·day�1 hydrocortisone. The mean dose of GCs calculated over

the 5 yr preceding the investigation was 17.53 � 4.49 mg·m2·day�1. Thehormonal control was established, with the serum concentrations of17�-hydroxyprogesterone, �4-androstenedione, and testosterone evalu-ated from the patients’ records in the preceding 5 yr (43).

For control group, we studied 18 controls aged 3–16 yr (11 � 5.9yr) recruited from the same geographic area. All subjects werehealthy, and none were involved in competitive sport activities.Candidates were excluded if they had a history of chronic illness, oneor more fractures, or taken any medication, hormone, vitamin prepa-ration, or calcium supplements regularly.

The study was approved by the Ethics Committee of the Universityof Bari Medical School, Bari, Italy, and the informed consent wasobtained from all subjects or from their parents. The study was madein accordance with the principles of the Declaration of Helsinki.

Bone Mineral Measurements

BMD of 21-OHD patients and controls was measured at theproximal femur and lumbar spine (L2–L4), using dual-energy X-rayabsorptiometry (ACN Unigamma X-Ray Plus; L’ACN ScientificLaboratories), and converted to SD scores (z-scores) in relation to age-and sex-matched normal population. Calcium and phosphate werealso measured in the patients and controls.

Cells and Culture Condition

CD14� and CD2� cell and neutrophil isolation. PB samples ob-tained from controls or 21-OHD patients were subjected to His-topaque 1077 density gradient (Sigma-Aldrich, St. Louis, MO) cen-trifugation. The obtained buffy coat cell fraction was submitted toCD14� monocyte and CD2� T lymphocyte isolation using anti-CD14and anti-CD2 antibody (Ab)-coated immunomagnetic Dynabeads(Dynal, Lake Success, NY), respectively. Only samples with a purityof �97%, checked by flow cytometry, were used. After Ficoll-Paquecentrifugation, neutrophils were separated from erythrocytes by 3%dextran (GE Healthcare) density gradient sedimentation. Purity, de-termined by flow cytometry analysis on forward scatter/side scatterparameters, was routinely �98%. Freshly isolated CD2� and CD14�

cells and neutrophils were plated for confocal immunostaining orsubjected to RNA extraction.

Leukocyte stimulation. To stimulate DKK1 synthesis, 200 l ofheparinized PB from healthy donors was either nonstimulated or

Table 1. Main clinical and hormonal characteristics of patients with 21-OHD

PatientNo. Sex Age, yr Weight, kg Height, cm

BMI,kg/m2

ClinicalForm

17�-OHP,ng/ml*

�4-A,ng/ml*

Testosterone,ng/ml*

Dose ofHydrocortisone,

mg/m2

1 M 16 61.5 157 25 SW 30 4 4.2 202 M 16 68 168 24 SW 40 3 4.5 203 M 16 70 170 24.2 NC 15 1.3 3.0 19.94 M 16 75.5 175.7 24.5 NC 25.5 2.7 4.0 15.45 M 15 65 165 24 SW 30 2.4 4.0 206 M 11 38.2 149.5 17 NC 40 6 3.4 17.47 M 16 70.5 169.3 24.6 NC 10 0.7 3.2 158 M 13 50 153.2 21.3 NC 5.6 0.6 3.3 109 M 5 22 98 22 NC 10 2.5 3.2 10

10 F 14 60.5 155 25.2 SW 30 2.3 0.3 2011 F 16 53.4 156.4 21.8 NC 11 2.4 0.3 1512 F 16 48.2 148.2 22 SV 40 3.7 0.5 2513 F 15 56 151.2 24.5 NC 10 1.5 0.3 17.514 F 14 58.6 158.7 23.4 NC 21 2.4 0.3 1515 F 16 62.9 147.9 28.8 NC 5 2.3 0.4 10.316 F 16 52 155.1 21.6 SV 20 2.4 0.3 2017 F 3 11.7 82 17.5 SV 30 0.1 0.1 2518 F 14 60 157.2 24.3 SV 30 2.5 0.3 20

21-OHD, 21-hydroxylase deficiency; M, male; F, female; SW, salt wasting; NC, nonclassical; SV, simple virilizing; *Mean value of the preceding 5 yr (exceptfor patient no. 17).

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stimulated with different concentrations of dexamethasone (rangingfrom 10�7 to 10�9 M) for 3 and 6 h. At the indicated times, thesamples were processed for the DKK1 intracellular staining by flowcytometry analysis.

Human osteoblasts. Clonetics Normal Human Osteoblasts (LonzaWalkersville, Walkersville, MD) were plated in six-well plates at adensity of 10 � 104/well, using a medium composed of �-minimalessential medium supplemented with 20 ng/ml bone morphogeneticprotein 2 (BMP2; R & D Systems, Minneapolis, MN) and 10% fetalcalf serum (FCS; Gibco Life Technologies, Milan, Italy) or humanserum obtained from controls or 21-OHD patients in the presence orabsence of 5 g/ml anti-DKK1 monoclonal Ab (mAb) (R & DSystems) or an anti-IgG Ab. After 48 h of culture, OBs were subjectedto alkaline phosphatase activity evaluation or to protein extraction.

Flow Cytometry Analysis

For the DKK1 intracellular staining, 50 l of heparinized PB fromcontrols and 21-OHD patients and unconjugated anti-DKK1 mAbswere used. In particular, we utilized two different anti-DKK1 mAbs:clone 141119 (R & D Systems) and clone 2B12 (Abnova). Intracel-lular staining for unconjugated DKK1 was preceded by fixation andpermeabilization with the Intraprep TM kit (Instrumentation Labora-tory) and then incubated for 25 min at 4°C. Cells were then washedand labeled with F(ab=) fragment secondary antibody Alexa Fluor 488(Life Technologies, Milan, Italy) for an additional 25 min. Then, cellswere washed twice and data acquired using a FC500 (BeckmannCoulter) flow cytometer and analyzed using Kaluza software. The areaof positivity was determined using an isotype-matched mAb, and atotal of 104 events for each sample were acquired.

Confocal Microscopy

For each experiment, 1 � 105/cm2 CD14� cells, CD2� cells, orneutrophils were plated on poly-L-lysine-coated coverslips (Sigma)and fixed in 3.7% paraformaldehyde.

Fixed cells were washed three times with PBS, permeabilized with0.3% Triton X-100-PBS for 30 min, and blocked in 1% BSA and 1%fetal bovine serum in PBS for 1 h. CD14� cells and neutrophils wereincubated with mouse anti-DKK1 mAb. CD2� cells were incubatedwith mouse anti-DKK1 mAb and rabbit anti-CD4 and anti-CD8polyclonal Ab (10 g/ml in blocking buffer; Santa Cruz Biotechnol-ogy). After washing, bound antibodies were detected using 10 g/mlfluorescent-labeled goat anti-mouse or anti-rabbit F(ab’) fragmentsecondary antibody, Alexa Fluor 488, or Alexa Fluor 555 (LifeTechnologies). Nuclei were counterstained with TO-PRO-3 Iodide(Life Technologies). The cells were then visualized and photographedby laser confocal microscopy TCS SP5 (Leica Mycrosystems, Mann-heim, Germany). Images (2,048 � 2,048 pixels) were acquired withan oil immersion objective (�63 1.4 HCX PL APO; Leica Mycro-systems). Overlay images were assembled.

RNA Isolation and Real-Time PCR

Total RNA was extracted from CD2� cells, CD14� cells, andneutrophils isolated from controls and 21-OHD patients using spincolumns (RNeasy; Qiagen, Hilden, Germany) according to the man-ufacturer’s instructions. The extracted RNA was reverse-transcribedusing the Super Script First-Stand Synthesis System kit for RT-PCR(Life Technologies, Carlsbard, CA); an RT mixture containing 1 gof total RNA, deoxyribonucleoside triphosphates, oligo(dT), RT buf-fer, MgCl2, dithiothreitol, RNaseOUT, SuperScript II RT, and diethylpyrocarbonate-treated water to final volume of 100 l was preparedaccording to the manufacturer’s instructions. cDNA was amplifiedwith the iTaq SYBR Green supermix with ROX kit (Bio-Rad Labo-ratories, Hercules, CA), and the PCR amplification was performed usingthe Chromo4 Real-Time PCR Detection System (Bio-Rad Laboratories).The following primer pairs were used for the real-time PCR amplifica-

tion: sense DKK1, 5=-TTCAACGCTATCAAGAACCTG-3=; antisense5=-DKK1, CGCACTCCTCGTCCTCTG-3= (NM_012242.2); senseciclophylin, 5=-CAGGTCCTGGCATCTTGTCC-3=; antisense ciclophy-lin, 5=-TTGCTGGTCTTGCCATTCCT-3= (NM_021130.3). The runningconditions were as follows: incubation at 95°C for 3 min and 40 cyclesof incubation at 95°C for 15 s and 60°C for 30 s. After the last cycle, themelting curve analysis was performed into a 55°C to 95°C interval byincrementing a temperature of 0.5°C. The fold change values werecalculated by Pfaffl’s method (37).

Alkaline Phosphatase Activity

Alkaline phosphatase activity was determined in cell lysates usingthe colorimetric Alkaline Phosphatase Assay Kit (Abcam, CambridgeScience Park). The kit uses p-nitrophenyl phosphate as a phosphatasesubstrate, which turns yellow when dephosphorylated by alkalinephosphatase. The absorbance at 405 nm was measured using amultiwell plate reader (550 Microplate Reader; Bio-Rad Laborato-ries). Cell lysates were analyzed for protein content using the Bio-RadDC Protein Assay (Bio-Rad Laboratories), and alkaline phosphataseactivity was normalized for total protein concentration.

Western Blot Analysis

Cells were lysed by incubation on ice for 30 min in lysis buffercontaining 50 mmol/l Tris·HCl (pH 8.0), 150 mmol/l NaCl, 5 mmol/lethylenediaminetetraacetic acid, 1% NP-40, and a commercial pro-tease inhibitor mixture (Sigma-Aldrich). Western blot analysis wasperformed as described previously (10). The following primary Abswere used: anti-collagen 1 (Santa Cruz Biotechnology), anti-RANKL(Abcam), anti-OPG (Abcam), and anti-�-actin (Santa Cruz Biotech-nology). After incubation with the appropriate fluorescent dye-conju-gated secondary Ab (LI-COR Biosciences, Bad Homburg, Germany),specific reactions were revealed with the LI-COR’s Odyssey InfraredImaging System (LI-COR Biotechnology, Lincoln, NE).

ELISA

DKK1, RANKL, OPG (Biomedica, Vienna, Austria), bone-specificalkaline phosphatase (Dade Behring, Newark, DE), and COOH-terminal telopeptides of type I collagen (CTX) (Serum CrossLaps;Immunodiagnostics Systems, Fountain Hills, AZ) were measured inthe sera obtained from the 21-OHD patients as well as in the sera ofage-matched healthy donors, using a commercially available ELISAkit according to the manufacturer’s instructions. The absorption wasdetermined with an ELISA reader (550 Microplate Reader; Bio-Rad),and the results were expressed as means � SE.

Statistical Analyses

Statistical analyses were performed by Mann-Whitney and Wil-coxon tests with the Statistical Package for the Social Sciences(spssx/pc) software (SPSS, Chicago, IL). Correlations were analyzedwith the Spearman test. The effect of the type of 21-OHD and sexDKK1 serum levels was evaluated by the Kruskal-Wallis test. Theresults were considered statistically significant for P 0.05.

RESULTS

DKK1 Serum Levels in 21-OHD Patients

DKK1 serum concentrations were significantly elevated(P 0.008) in patients with 21-OHD (2,413 � 218 pg/ml; range:1,255–3,776 pg/ml) compared with control subjects (1,660 � 88pg/ml; range: 1,019–2,318 pg/ml) (Fig. 1). In particular, in themajority of 21-OHD patients, serum levels of DKK1 were abovethe mean serum amounts of controls. No significant correlationwas found between DKK1 serum levels and GC dose (Rho �0.02, P � 0.86), duration of treatment (Rho � 0.23, P � 0.72),

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androgen levels (Rho � 0.14, P � 0.61), and age (Rho � �0.02,P � 0.94). Type of 21-OHD and sex of patients had no effect onserum DKK1 levels (P � 0.73).

DKK1 Expression in Circulating Leukocytes From 21-OHDPatients

To investigate which cells are responsible for DKK1 pro-duction, we analyzed the intracellular expression of DKK1 on

leukocytes from 21-OHD patients on cGC therapy and fromcontrols through flow cytometry and confocal microscopy.

Flow cytometry analysis revealed a significant increase inDKK1 expression in all the three blood subpopulations (lympho-cytes, monocytes, and neutrophils) in patients (Fig. 2B) comparedwith controls (Fig. 2A), as assessed by intracellular labeling ofDKK1 vs the side scatter of cells (P 0.025). Furthermore, inpatients we observed that DKK1 was highly expressed in aportion of lymphomonocyte (5.4%) compared with controls,where it was barely detectable (0.2%) (P 0.001).

Coimmunostaining revealed DKK1 protein expression inCD8� and CD4� T lymphocytes (Fig. 2, C and D) frompatients. Interestingly, we also observed strong DKK1 stainingin monocytes and neutrophils (Fig. 2, E and F). A weakstaining was found when cells from controls were evaluated(data not shown). To confirm DKK1 expression in whole bloodleukocytes in 21-OHD patients, we next amplified RNA frompurified monocytes, T lymphocytes, and neutrophils. As can beseen in Fig. 2, G–I, the lowest mRNA levels of DKK1messenger RNA were detected in samples from controls. Instriking contrast, DKK1 expression was higher in all investi-gated cell types (monocytes, T cells, neutrophils) isolated frompatients. In detail, we found that, compared with controls, in21-OHD patients DKK1 mRNA levels were 2.3 � 0.4- (P 0.001), 3.7 � 0.4- (P 0.001), and 4.8 � 0.7-fold (P

Fig. 1. Dickkopf-1 DKK1 serum levels (Œ) in patients with 21-hydroxylase-deficient (21-OHD). DKK1 serum concentrations were significantly elevated inpatients with 21-OHD (2,413 � 218 pg/ml; range: 1,255–3,776 pg/ml) comparedwith control subjects (1,660 � 88 pg/ml; range: 1,019–2,318 pg/ml), P 0.004.

Fig. 2. DKK1 expression in circulating leukocytes from 21-OHD patients. Flow cytometry analysis of DKK1 expression in permeabilized leukocytes revealedthat DKK1 was significantly higher in all cell blood populations from patients (B) compared with the age-matched normal controls (A), especially inlymphomonocytes. Data shown are gated on all blood cell populations, and quadrants were established on the basis of DKK1 intracellular staining vs. cell sidescatter (SS). C and D: confocal micrographic images showing coimmunostaining of DKK1 (green) protein and CD8� (red) or CD4� (red) T lymphocytes frompatients. E and F: strong DKK1 staining in monocytes and neutrophils was also observed. Nuclei were counterstained with TO-PRO-3 iodide (blue). G–I: theexpression of DKK1 was detected in T lymphocytes, monocytes, and neutrophils by real-time PCR. Results are depicted for 1 patient and 1 control but arerepresentative of 8 different expriments.

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0.0001) increased in T lymphocytes, monocytes, and neutro-phils, respectively (Fig. 2, G–I). Taken together, these datastrongly suggest that a variety of different cell types contributeto the total pool of secreted DKK1 in the peripheral blood.

Effect of Dexamethasone on DKK1 Expression in HumanLeukocytes

The enhanced expression of DKK1 by leukocytes from21-OHD patients prompted us to investigate the effect ofdexamethasone on human leukocytes from healthy donors. Tothis end, heparinized blood from controls was incubated withdexamethasone (10�9 to 10�7 M) for different times (0, 3, and6 h). By flow cytometry, we found that dexamethasone inducedthe expression of DKK1 significantly compared with that in theunstimulated condition in a time- and dose-dependent manner.In particular, after 3 h of dexamethasone treatment, no differ-ences in DKK1 expression were detected between the unstimu-lated and stimulated condition (data not shown). DKK1 ex-pression increase effect was observed at 6 h of treatment,reaching the maximum amounts at the highest dose of dexa-methasone used (10�7 M, P 0.001), maintaining elevatedlevels at 10�8 M dexamethasone (P 0.001) and thus return-ing to control levels at 10�9 M (Fig. 3). These results suggestthat GCs induce the expression of DKK1 specifically in humanleukocytes.

Effect of Serum From 21-OHD Patients on OsteoblastDifferentiation In Vitro

To assess whether the high serum levels of DKK1 in the21-OHD patients would be able to inhibit OB differentiation,we cultured human OBs in the basal condition (medium �10% FCS), in conditioned media with sera from patients orcontrols, and in the presence or absence of anti-DKK1-neutral-izing antibody or an anti-IgG. To promote OB differentiation,these cells were cultured in the presence of BMP2, which canstimulate OB differentiation through a mechanism that in-volves Wnt/�-catenin signaling (17).

In the cultures treated with BMP2 and FCS or serum fromcontrols, the presence or absence of the anti-DKK1-neutraliz-ing mAb did not exert effects on the activity of alkalinephosphatase, a specific marker of OB differentiation. On thecontrary, OBs cultured with BMP2 and conditioned medium

containing the serum of patients showed a significant reduc-tion in alkaline phosphatase activity (21 � 2%, P 0.003;Fig. 4A) with respect to the OBs cultured with the serum fromcontrols (or FCS). Moreover, we found that anti-DKK1-neu-tralizing mAb increased the alkaline phosphatase activity in the

Fig. 3. Effect of dexamethasone on DKK1 expression in human leukocytes. Flow cytometry analysis of DKK1 expression in 6-h dexamethasone-stimulatedheparinized blood showed that in permeabilized leukocytes DKK1 expression increase reached the maximum amounts at 10�7 M dexamethasone, maintainedelevated levels at 10�8 M, and thus returned to control levels at 10�9 M. The gray histograms signify staining with isotype control, and the white histogramsrepresent the staining with anti-DKK1 monoclonal antibody. Results are depicted for 1 experiment that was representative of 5 independent assays. OD, opticaldensity.

Fig. 4. Effect of serum from 21-OHD patients on osteoblast (OB) differenti-ation in vitro. Human OBs were cultured with bone morphogenetic protein 2(BMP2) in the basal condition (medium � 10% FCS), in conditioned mediawith sera from patients or controls, or in the presence or absence of anti-DKK1-neutralizing antibody. In all of the described conditions, the activity ofalkaline phosphatase (A) as well as collagen I expression by Western blotanalysis (B) was evaluated. The intensity of the bands obtained by Western blotwas quantified by densitometry (histogram) and normalized to �-actin. Thehistograms represent means � SE of 5 independent experiments, whereas theblots correspond to 1 representative experiment. *P 0.007.

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OBs significantly after 48 h of culture (20% � 3%, P 0.006)compared with conditioned medium containing the patientserum alone. No effect was found using anti-IgG antibody (notshown).

In the same culture system, we also evaluated the expressionof collagen I as an additional marker of OB differentiation(Fig. 4B). In OBs cultured with FCS or serum from controls,the anti-DKK1-neutralizing mAb did not exert effects oncollagen I expression. On the contrary, we found that condi-tioned medium containing the serum from 21-OHD patientsand anti-DKK1-neutralizing mAb increased the collagen Iexpression in the OBs significantly after 48 h of culture (P 0.007). No effect was found using anti-IgG antibody (notshown).

Effect of Serum From 21-OHD Patients on OsteoblastExpression of RANKL and OPG

On the basis of the knowledge that RANKL/OPG ratio canbe unbalanced by Wnt inhibitors (38), we investigated whetherin our culture system the serum from 21-OHD patients couldaffect, through DKK1 production, the expression of RANKLand OPG with pro- and antiosteoclastogenic activity, respec-tively. In OBs cultured with FCS or serum from controls, theanti-DKK1-neutralizing mAb did not exert effects on RANKLor OPG expression (Fig. 5, A and B). On the contrary, theanti-DKK1 mAb strongly downregulated the RANKL expres-sion (P 0.003) and did not affect the OPG expression byOBs (Fig. 5, A and B). No effect was exerted by anti-IgGantibody (not shown). These data are supported further by thedirect correlation between DKK1 and RANKL serum levels(Rho � 0.62, P 0.003) in sera from 21-OHD patients (Fig.5C). No significant correlation was found between DKK1 andOPG serum levels (Rho � 0.31, P 0.12).

Bone Mineral Measurements and Bone Biochemical Markersin 21-OHD Patients

Proximal femur and lumbar spine BMD z-scores of 21-OHDpatients were in the normal range (z-scores greater than �0.22)according to World Health Organization criteria for osteopeniaand osteoporosis (19). However, a slight but significant reduc-tion in bone mass was detected in 21-OHD patients (P 0.05)with respect to the controls. The quantification of degradationproducts of CTX, an indicator of bone resorption, showed thatpatients had significantly higher serum levels with respect tocontrols (0.824 � 0.17 ng/ml, range 0.34–1.70 ng/ml; 0.41 �0.09 ng/ml, range 0.15–0.87 ng/ml, P 0.006). Interestingly,the higher levels of CTX were found in patients with lowerBMD (P 0.01). Moreover, we found that in sera frompatients, DKK1 levels correlate with CTX concentrations(Rho � 0.65, P 0.02). Serum levels of bone-specific alkalinephosphatase, total calcium, and phosphate of 21-OHD patientswere within the normal range.

DISCUSSION

The current study results in three important observations.First, elevated serum levels of DKK1 have been found in21-OHD patients on cGC treatment with respect to controls.The study included only 18 patients, with subjects with boththe classical (SW or SV) and NC forms of 21-OHD, with theconsideration that the incidence of the most severe forms (SW

or SV) is about 1 in 10,000–15,000 people, whereas theincidence of milder forms (NC) is probably 10 times higher,with a prevalence ranges from 1 in 30 to 1 in 1,000 (33).Furthermore, whereas in the subjects with classical forms theGC treatment is started at the time of the diagnosis, generallyin the newborn period, in those affected by the NC form theGC treatment needs to be directed toward the symptoms andshould not be initiated merely to decrease abnormally elevated

Fig. 5. Effect of serum from 21-OHD patients on OB expression of receptoractivator of NF-�B ligand (RANKL) and osteoprotegerin (OPG). Human OBswere cultured with BMP2 in basal conditions (medium � 10% FCS), inconditioned media with sera from patients or controls, or in the presence orabsence of anti-DKK1-neutralizing antibody. In all of the described conditions,RANKL (A) as well as OPG expression (B) was evaluated by Western blotanalysis. The intensity of the bands obtained by Western blot was quantified bydensitometry (histogram) and normalized to �-actin. The histograms representmeans � SE of 5 independent experiments, whereas the blots correspond to 1representative experiment. C: graph shows the direct correlation betweenDKK1 and RANKL serum levels (Rho � 0.62, P 0.003) in sera from21-OHD patients. *P 0.003. Œ, DKK1 and RANKL serum levels.

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hormone concentrations but in children and adolescents withsignificantly advanced skeletal maturation. Interestingly, in our21-OHD patients on cGC therapy, we found no significantdifference between DKK1 serum level duration of treatmentand type of 21-OHD. DKK1 is a critical regulator of systemicbone mass (26, 31), and its role as bone-remodeling regulatorin pathological states is emerging. In fact, according to ourdata, DKK1 circulating levels are higher in bone loss-associ-ated diseases such as multiple myeloma (50), rheumatoid (52),and psoriatic arthritis (7) as well as Paget’s disease (28). On thecontrary, its serum levels are reduced in bone diseases associ-ated with the increase of bone mass, such as ankylosing spondy-litis (9), and diffuse idiopathic skeletal hyperostosis (45).

Second, monocytes, T lymphocytes, and neutrophils from21-OHD patients on cGC treatment expressed high levels ofDKK1. Previous reports have demonstrated that in the physi-ological condition the OBs are the bone main source of DKK1,but in pathological states other cells secreted it. In particular, inthe bone marrow from patients with multiple myeloma bonedisease, the malignant plasma cells showed high DKK1 levels(50). In the rheumatoid arthritis subjects, DKK1 was widelyexpressed in the inflamed synovium, especially in fibroblast-like synoviocytes embedded in the inflamed tissue, in synovialmicrovessels, and in cartilage adjacent to inflammatory tissue(9). In Paget’s disease, DKK1 expression and protein havebeen reported to be elevated in osteoblastic cell and stromalcell cultures from Paget’s lesions compared with control cul-tures from unaffected bone from the same patients or fromother control subjects (32). During acute intestinal inflamma-tion, DKK1 expression is strongly induced in intestinal tissuein T lymphocytes, macrophages, neutrophils, and platelets(21). Here, we reported for the first time that circulating Tlymphocytes, monocytes, and neutrophils from 21-OHD pa-tients express higher levels of DKK1 than controls. In the lastdecade, numerous scientists have highlighted the interactionsbetween bone and immune cells, specifically in pathologicalconditions in which activation of both systems occurs (48).However, the biology regulating osteoimmune cross-talk isincompletely understood, and much research focuses on theidentification of common molecules that can modulate theactivation of immune cells as well as bone cells, and DKK1could be another link between the two tissues. Moreover, in ourprevious work we demonstrated in 21-OHD subjects on cGCtreatment a significant percentage of monocytes strongly com-mitted toward osteoclasts as well as the high RANKL expres-sion by T cells (10). Thus, the fact that monocytes, lympho-cytes, and neutrophils also express DKK1 further supports therole of cell immune systems in regulating bone remodeling in21-OHD patients on cGC treatment.

Our results also showed that the expression of DKK1 byleukocytes in 21-OHD patients may be related to the cGCtherapy. In fact, we found that in vitro dexamethasone treat-ment markedly induced the expression of DKK1 in a dose- andtime-dependent manner in leukocytes from controls. Dexa-methasone can be used as glucocorticoid in the therapy ofchildren with CAH (40), and it is recommended in the prenataltreatment of 21-OHD patients (24). Moreover, several in vitroand in vivo models have been used to explore the role of DKK1in GIO, and most of them have used dexamethasone as GC, aswe have in our experiments. In particular, our used amountshave been selected from Ohnaka et al. (34), who demonstrated

that in cultured human OBs dexamethasone induces a dose-dependent increase in DKK1 expression that is mediated by theactivation of transcription via the GC-responsive element ofthe DKK1 gene promoter. These authors also reported thatWnt3a upregulated TCF/LEF transcriptional activity (indicat-ing increased Wnt pathway activation); this process is inhibitedby dexamethasone in a dose-dependent manner (35). More-over, the addition of anti-DKK1 mAb partially restored (by40%) the dexamethasone-induced suppression of the TCF/LEFtranscriptional activity. In another study, it was shown thatcorticosterone upregulated DKK1 expression two- to threefoldin cultured mouse OBs (27). These data indicate that GCsinhibit the Wnt pathway and that this effect may be at leastpartially mediated by DKK1. Wang et al. (51) explored the roleof DKK1 further in GIO. These authors treated MC3T3-E1preosteoblasts with dexamethasone and reported the suppres-sion of OB activity. Knockdown of DKK1 expression byDKK1-AS alleviated dexamethasone-induced suppression ofOB activity and decreased OB apoptosis. Investigators alsostudied the changes in gene expression following GC treatmentin a mouse model, using microarrays, and found that GCtreatment led to a significant upregulation of DKK1 (53).Consistent with this finding, in the present study we demon-strated clearly that dexamethasone can induce the expression ofDKK1 in human leukocytes.

Third, the serum from patients can directly inhibit OBdifferentiation in vitro as well as RANKL expression in OBs,and these effects were neutralized by the addition of ananti-DKK1 antibody in the cultures. We also found the corre-lation between DKK1 and RANKL or CTX serum levels in21-OHD patients on cGC treatment. These positive correla-tions also suggest an effect of DKK1 on the bone resorptionactivity of osteoclasts that together with the DKK1 inhibitoryeffect on OB differentiation sustain the bone impairment thatmay be associated with cGC therapy. Thus, in these patients,the effects of DKK1 on bone seem to point to an importantcross-talk between the bone-anabolic Wnt signal and the bone-catabolic RANKL pathway. Other studies have demonstrated acoregulation between DKK1 and RANKL expression in arange of diseases, including osteosarcoma (25) and prostatecancer (41), and in vitro studies using vascular progenitor cells(1) and murine mesenchymal stem cells (11). Additionally, ithas been demonstrated that in sera from multiple myelomapatients, high DKK1 levels correlate with elevated CTXamount, a bone resorption marker (49).

In conclusion, the present study showed for the first time thatdexamethasone treatment induces DKK1 expression in leuko-cytes, and in 21-OHD patients on cGC therapy the same cellsrepresent an important source of this cytokine. Therefore, ourfindings suggest that the involvement of DKK1 secreted byleukocytes could be a general mechanism occurring not only in21-OHD patients but in other forms of GIO. Thus, the period-ical monitoring of DKK1 levels could be useful for controllingthe bone metabolism in these patients, and in those with aserious osteoporotic degree, DKK1 could represent a promis-ing therapeutic target.

DISCLOSURES

The authors state that they have no conflicts of interest, financial orotherwise.

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AUTHOR CONTRIBUTIONS

G.B. drafted the manuscript; M.F.F. and M. Grano contributed to theconception and design of the research; L.P., A.V., A.O., C.C., A.D.B., G.C.,and M. Gigante performed the experiments; G.C. and L.G. interpreted theresults of the experiments; G.M. analyzed the data; G.M. prepared the figures;S.C. edited and revised the manuscript; L.C. and M. Grano approved the finalversion of the manuscript.

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