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Keywords:

  • androgens;
  • osteoblasts;
  • molecular pathway;
  • androgen receptor;
  • nongenomic actions

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Androgens have important effects on the bone metabolism. However, the effect and mechanism of androgen action on the osteoblasts remains unknown. Here we showed that androgens increase phosphorylation and nuclear translocation of Akt. siRNA-AR prevented androgen-induced Akt activation in MC3T3-E1 cells. This suggests that nongenomic androgen activation of Akt is mediated by androgen receptor in osteoblasts.

Introduction: Androgens have important effects on the human skeleton in both males and females. However, the mechanism of androgen action on bone metabolism remains unknown. The aims of this study were to determine the effect and mechanism of androgen action on the osteoblast cells.

Materials and Methods: Here we showed that 5α-dihydrotestosterone (DHT) accelerates cell growth of the MC3T3-E1 cell line in a time- and dose-dependent manner. The specific phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 and kinase-deficient Akt mutant can repress the androgen effect on MC3T3-E1 cells. Western blot analysis showed that DHT, 17β-estradiol, and testosterone (T) induce a rapid and transient phosphorylation of Akt in MC3T3-E1 cells. This activation reached to a plateau after 15 minutes and gradually diminished after 60 minutes of DHT treatment.

Results: Fluorescence microscopy showed a distinct increase in immunostaining intensity in the nuclear interior after androgen treatment but no change in the subcellular distribution of Akt when the cells were pretreated with hydroxyflutamide (HF) or LY294002. In addition, small interfering RNA against androgen receptor (siRNA-AR) prevented DHT-induced Akt phosphorylation and cell growth.

Conclusion: These findings represents the first physiological finding to indicate how steroid hormones such as androgens can mediate the nuclear localization of Akt/PKB in osteoblasts that has previously mainly been linked to growth factor-induced events occurring at the plasma membrane level.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

ANDROGENS CONSTITUTE A CLASS of steroid hormones that are essential for skeletal development as well as the maintenance of bone metabolism throughout adult life. The discovery of the central role of estrogen and androgen deficiency on bone loss by Fuller Albright in 1948 provided a major stimulus to elucidate the mechanism of the sex hormone actions on the skeleton and significant improvements in the clinical management of patients with osteoporosis over the past two decades. Clinical studies indicate that combined therapy of estrogens plus androgens may enhance BMD and bone mass to a more significant degree than estrogen therapy alone in postmenopausal women.(1,2) Administration of flutamide, an androgen receptor antagonist, to female rats results in osteopenia, indicating a role for androgens in the female skeleton.(3) Androgen receptor knockout (ARKO) male mice develop osteopenia, resulting in definitive bone loss in conjunction with changes in histological analysis of bone sections. Cancellous bone volumes are lower in ARKO mice than in both female and male wildtype littermates.(4,5)

The principle steroidal androgens, testosterone (T) and its metabolite 5α-dihydrotestosterone (DHT), are thought to predominantly mediate their biological effects through binding to the androgen receptor (AR). AR, in common with other members of the nuclear receptor superfamily, functions as a ligand-inducible transcription factor. The binding of T or DHT to AR induces receptor dimerization, facilitating the ability of AR to bind to its cognate response element and recruit coregulators to promote the expression of target genes.(6–8) In addition to this transcriptional or genomic mode of action by steroids, an increasing body of evidence suggests that androgens, such as progesterone and estrogen, can induce rapid increases in the levels of conventional second messenger signal transduction cascades, including free intracellular calcium and activation of protein kinase C (PKC).(9) Recent data also suggest a direct link between the AR and the fast and transient activation of the MAPK-signaling cascade.(10) The time course of this rapid activation parallels that induced by peptide hormones, suggesting that these events do not involve the “ classical” genomic actions of androgens.

The nongenomic actions of androgens have been implicated in a number of cellular effects, including gap junc-tion communication, aortic relaxation, and neuronal plasticity.(11–13) In response to DHT or the synthetic androgen R1881, AR interacts with the SH3 domain of Src,(10,14) resulting in stimulation of Src kinase activity within minutes in the AR+ LNCaP prostate cancer cell line in response to 10 nM R1881.(14) R1881 treatment also resulted in stimulation of two members of the MAP kinase signaling cascade, Raf-1 and ERK-2. In addition, androgen treatment reduces etoposide-induced apoptosis in calvarial osteoblasts and MLO-Y4 cells. In MLO-Y4 cells, this effect is abrogated by inhibition or mutations in Src and members of the MAP kinase family.(10)

Akt, also call protein kinase B (PKB), is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses, including inhibiting apoptosis and stimulating cellular growth. In response to a variety of stimuli, Akt, which contains a pleckstrin homology domain, is recruited to the plasma membrane by the lipid products of PI 3-kinase.(15,16) The double phosphorylation of Akt at Thr-308 and Ser-473 results in full activation of Akt kinase activity.(17,18) PI-3K/Akt pathway provides the survival signal in diverse cell types. Many growth factors such as platelet-derived growth factor (PDGF), nerve growth factor (NGF), insulin, insulin-like growth factor (IGF)-1, and cytokines such as interleukin (IL)-2, IL-3, and IL-6 can activate the PI-3K/Akt pathway through their cognate receptors. Deprivation of these growth factors blocks the PI-3K/Akt activation, leading to cell apoptosis, and reactivation of PI-3K/Akt can rescue the cell apoptosis in response to the growth factor deprivation. A large variety of Akt substrates have been identified and these include, among others, BAD, CREB, members of the forkhead family of transcriptional factors, IkB kinase, procaspase-9, GSK-3-a/β, mTOR/FRAP, and p21WAF1.(15) Previously, we reported that Akt phosphorylates AR at serine 210 and serine 790 and suppresses AR activity.(19) In addition, we further showed that activation of the PI 3-kinase/Akt pathway promotes AR ubiquitination and leads to AR degradation through proteasome-dependent pathways.(20)

In these studies, we showed that androgens stimulate a rapid and transient phosphorylation of Akt in osteoblasts. This activation may lead to increase the endogenous Akt immunostaining intensity in the nucleus. Moreover, antiandrogen such as hydroxyflutamide or small interfering RNA against androgen receptor (siRNA-AR) prevented androgen-induced Akt phosphorylation, intranuclear translocation, and cell growth. In addition, G-proteins, phospholipase C (PLC), Src kinase, and intracellular calcium mobilization are essential for androgen-mediated Akt activation. These findings strongly suggest that this androgen nongenomic action mediated by AR and androgen-induced Akt activation may play important roles in osteoblasts.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Chemicals and plasmids

LY294002, PP2, PP3, pertussis toxin, U-73122, U73343, and BAPTA/AM were purchased from Calbiochem. DHT, EGTA, testosterone, testosterone 3-(o-carboxymethyl)oxime bovine serum albumin, Nifedipine, and Verapamil were purchased from Sigma. Hydroxyflutamide (HF) was obtained from Schering. pCDNA3 cAkt (a constitutively active Akt with a deletion at amino acids 4-129 replaced with a consensus myristoylation domain) and pCDNA3 dAkt (a kinase-deficient mutant, K179A) were from Dr R Freeman (University of Rochester, Rochester, MN, USA). Antibodies to total Akt and phospho-Akt (S473) were from New England Biolabs, Upstate Biotechnology (Lake Placid, NY, USA). The anti-AR polyclonal antibody, NH27, was produced as described,(4,5) and N20 was purchased from Santa Cruz.

Cell lines

U2OS and SaOS2 cells were purchased from ATCC. MC3T3-E1 cells were kindly provided by Dr Renny T Franceschi (University of Michigan, Ann Arbor, MI, USA). For routine cellular maintenance, MC3T3-E1 mouse calvaria osteoblast cells were plated as monolayer cultures and maintained in α-MEM medium (Life Science) containing 10% FBS.

Cell proliferation assay

Cells were plated on 96-well plates in α-MEM medium containing 10% FBS. Once attached, they were grown in α-MEM medium containing 5% charcoal-dextran-treated FBS. The cells were then treated with DHT or various agents for 15 minutes and incubated for 6, 12, or 24 h, and absorbance was measured using the XTT assay kit (Roche). This method used a tetrazolium salts XTT {3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)} benzene sulfonic acid hydrate labeling reagent that is reduced by living cells to yield a soluble formazan product that can be assayed in a colorimetric reaction (absorbance measured between 492 and 690 nm). To determine the relationship between absorbance and cell numbers, increasing concentrations of MC3T3-E1 cells (103-5 × 103) were added to microtiter wells and incubated for 24, 48, or 72 h, and absorbance was measured using the XTT assay kit. The absorbance in wells containing medium with the XTT reagents (but without cells) was subtracted as background. A linear relationship between cell number and absorbance was observed over the range of cell concentrations examined (data not shown). For all experiments, these standard curves of cell numbers were plated for each experiment to verify cell viability, and a 1-h incubation with XTT was used to calculated cell numbers. All results were expressed as a percentage of the time-matched medium control value. For each experiment, n = 6-10 (where each n represents a single well) unless otherwise noted.

Akt immunoprecipitation kinase assay kit

MC3T3-E1 cells were maintained in 5% charcoal-dextran-treated FBS overnight, stimulated with DHT in different time intervals, and lysed in 1 ml of lysis buffer (50 mM Tris-HCL, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% [vol/vol] 2-mercaptoethanol, 1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 μg/ml of aprotinin, pepstatin, and leupeptin). After centrifugation as above, the supernatants were incubated for 2 h at 4°C with protein A-Sepharose beads coated with 2.5 μl of anti-Akt1, PH domain antibodies. Immunoprecipitates were washed three times with the lysis buffer and twice with the kinase assay buffer (50 mM Tris-HCl, pH 7.5, 0.03% [wt/vol] Brij-35, 0.1 mM EGTA, and 0.1% [vol/vol] 2-mercaptoethanol) and assayed using GSK-3 peptide (RPRAATF) as substrate. After the kinase reaction, the phosphorylated peptide was separated from unincorporated [γ-32P] ATP on a 40% polyacrylamide gel containing 6 M urea. The phosphopeptide spots were excised and counted. Control assays using protein A-Sepharose beads preabsorbed with normal rabbit serum were run concurrently, and the values from these were subtracted from the experimental.

Production of stably transfected clone cell line clones

MC3T3-E1 and SaOS2 cells were plated 1 day before transfection. Transfection was carried out by using Lipofection performed according to manufacturer's instructions. The wildtype AR, kinase-deficient Akt, and control expression constructs were transfected into the individual cell lines. Two days after transfection, cells were split into 100-mm dishes with medium containing G418 at 250 μg/ml.

Immunoblot of phosphorylated Akt kinase

This assay uses a polyclonal antibody specific against the phospho-Akt kinase (Cell Signaling). MC3T3-E1 cells were seeded and allowed to attach overnight, and the media were replaced with 10% CD-FBS in α-MEM media. The cells were pretreated with PI 3-kinase inhibitor, LY294002, and followed by DHT or vehicle treatment in the indicated time. Cells were washed and harvested at indicated times. Twenty micrograms of each cell lysate was resolved by 8% SDS-PAGE, immunoblotted with anti-phospho-Akt antibody, and incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody. Proteins were visualized by the enhanced chemiluminescence system (Amersham, Pharmacia Biotech), and images were obtained with a model FluorMax2 (Bio-Rad). Bands were quantified by Quantity One software (Bio-Rad). Akt kinase (total Akt) was blotted as a control.

Immunocytofluorescence

MC3T3-E1 cells were seeded with α-MEM-10% FBS on 4-well Lab Tek chamber slides (Nalge) overnight and treated with LY294002 (Calbiochem, San Diego, CA, USA), a PI 3-kinase inhibitor, or 1 μM HF (Zeneca), followed by treatment with 10 nM DHT. Cells were fixed, and immunostaining was performed by incubating anti-Akt kinase antibody, followed by incubation with fluorescein-conjugated goat anti-rabbit antibody. Slides were mounted with mounting medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), sealed, and observed under the fluorescent microscopy.

Construction and transfection of DNA vector-based siRNA plasmids

The selection of coding sequences was determined empirically and was analyzed by blast search to avoid any significant sequence homology with other genes. The AR siRNA oligonucleotides is 5′-GGGCCCTATCCCAGTCCCACTTGCTCGAGCAAGTGGGACTGGGATAGGGCTTTTTGAATTC-3′, and the nonspecific negative control oligonucleotides (siRNA-renilla) are from Ambion. After annealing the oligonucleotides, the fragments were cloned into the ApaI/ EcoRI site of pMSV/U6 vector from Dr Eric Devroe. Transfection with siRNA plasmids using Lipofectamine 2000 (Invitrogen) was performed according to manufacturer's instructions. For each single cell type experiment, the cells were seeded at an initial density of 2 × 106 cells in 60-mm dishes and cultured for at least 24 h before transfection. pMSV/U6-siRNA AR or negative control plasmids were incubated with Lipofectiamine 2000 in α-MEM for 20 minutes at room temperature. Plates of subconfluent cells were growth in medium containing 5% charcoal-dextran-treated FBS without antibiotics before addition of siRNA-Lipofectamine 2000 mixtures, the DNA/medium mixture was added, and the plates were placed into an incubator for a further 36 h. The cells were then treated with DHT or various agents for 15 minutes and harvested for Akt phosphorylation assay.

Statistical analysis

Statistical significance of the data were evaluated using Student's t-test, and p < 0.05 was considered significant. Results are expressed as means ± SD from the indicated set of experiments.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Androgen stimulates the phosphorylation of Akt in osteoblast cells

Both AR mRNA and proteins have been shown to be present in osteoblastic cell lines as well as primary cultures of bone marrow and of stromal/osteoblastic cells.(21,22) To characterize the effect of androgen exposure on osteoblast and examine the biological consequences of bone cells in terms of androgen responsiveness, MC3T3-E1 calvaria osteoblastic cells were chosen as an osteoblastic model because they have been shown to express high levels of alkaline phosphatase activity, AR mRNA, and proteins, and to be androgen responsive.(23,24) To investigate the effect of DHT on Akt activation in MC3T3-E1 cells, DHT-treated MC3T3-E1 cell lysates were analyzed by Western blotting using an anti-phospho-Akt-specific antibody, which recognizes activated Akt kinase. Immunoblotting showed that Akt was activated in response to DHT in a time- and dose-dependent manner (Figs. 1A and 1C). We measured the kinase activity of the Akt from DHT-treated MC3T3-E1 cell lysates at different time points. Striking activation of Akt activities in DHT treatment occurred somewhere between 15 and 60 minutes, with activation reaching a plateau after 15 minutes and gradually diminishing after 60 minutes of DHT treatment (Fig. 1B). Because this activation reaches the optimal condition at 10−8 M DHT (Fig. 1C), we also tested the effect of different steroid hormones at 10−8 M on Akt phosphorylation. In MC3T3-E1, we were able to observe the activation of Akt by T, 17-β-estradiol, and DHT (Fig. 1D). The kinetics of DHT-induced Akt activation was rapid and transient, suggesting that activation of Akt by DHT may be nongenomic and transcriptional independent. To test this possibility, we treated MC3T3-E1 with actinomycin D (RNA polymerase I inhibitor) followed by DHT treatment. As expected, inhibition of transcription did not affect DHT-induced Akt phosphorylation (Fig. 1E). To test whether DHT-induced Akt activation is PI 3 dependent, we treated MC3T3-Z, cells with LY294002 (a specific PI 3-kinase inhibitor), followed by DHT treatment. As shown in Fig. 1F, when we treated MC3T3-E1 cells with LY294002, a specific PI 3-kinase inhibitor, followed by DHT treatment, inhibition of PI 3-kinase by LY294002 abolished DHT-induced Akt phosphorylation.

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Figure FIG. 1.. Activation of Akt kinase phosphorylation by androgen in osteoblast. (A) MC3T3-E1 cells were treated with 10−8 M DHT (lanes 2-8) for different times as indicated and lysed. Equal amounts of cell lysates were analyzed by 8% SDS-PAGE and subsequent immunoblotting with anti-phospho-Akt and anti-Akt antibodies. (B) Equal amounts of cell lysates from A were immunoprecipitated with anti-Akt antibodies, and the immuno-complexes were assayed for Akt enzymatic activity as described in the Materials and Methods section. Each bar represents means ± SD of three independent sets for each experiment. Samples significantly different to controls: *p < 0.05. (C) MC3T3-E1 cells were incubated for 15 minutes with various concentration of DHT as indicated. (D) Cells were treated with 10−8 M 17-β-estradiol, 10−8 M progesterone, 10−8 M dexamethasone, 10−8 M T, and 10−8 M DHT. (E) Cells were incubated with Actinomycin D (10 μg/ml) before the addition of 10−8 M DHT. (F) Cells were treated with 20 μM LY294002, followed by incubation with 10−8 M DHT. Representative blots are shown, and the results were verified in at least three independent experiments.

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Androgen receptor is required for activation of Akt

To examine whether AR is required for activation of Akt, we used the siRNA-AR to block AR expression in MC3T3-E1 cells. Interestingly, inhibition of AR expression significantly decreased the activation of Akt phosphorylation (Fig. 2A). On the other hand, expression of constitutive active AR increased the basal levels of Akt phosphorylation, and DHT was able to further enhance Akt activity in the AR stably transfected MC3T3-E1 cells (Fig. 2B). To investigate whether the effect of androgen-dependent Akt activation is through classic androgenic action within 30 minutes, we first transfected the (ARE)4-luc reporter plasmids into MC3T3-E1, DU145, SaOS2-AR3, and LNCaP cells and assayed for the androgen-induced reporter gene activity. On 18 h treatment of DHT, AR-mediated transactivation was observed in AR+ cells, such as MC3T3-E1, SaOS2-AR3, and LNCaP, but not in AR DU145 cells. However, we were unable to detect the AR-mediated transactivation within 30 minutes (Fig. 2C). Testosterone covalently bound to high molecular weight bovine serum albumin (BSA), which did not enter the cell,(25,26) was also used to test the Akt activation in different cell lines. As shown in Fig. 2D, activation of Akt phosphorylation was observed in MC3T3-E1 and SaOS2 cells but not in LNCaP cells, which Akt is constitutive activation because of PTEN mutation. In addition, MC3T3-E1 cells pretreated with an AR antagonist (HF) also remained at control levels (Fig. 2E), and the dose-dependent activation of Akt by DHT stimulation was not detected in ARDU145 cells (Fig. 2F).

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Figure FIG. 2.. AR is required for activation of Akt. (A) MC3T3-E1 cells were transiently transfected with pMSCV/U6 siRNA-AR, nonspecific siRNA control (siRNA-renilla), or empty control vector (pMSCV/U6). After transfection, cells were treated with vehicle or DHT for indicated periods of time, and Akt phosphorylation and AR protein expression were determined. (B) Akt phosphorylation was detected in AR stable-transfected MC3T3-E1 cell line and wildtype cells in the presence or absence of androgen. (C) MC3T3-E1, DU145, SaOs2-AR, and LNCaP cells were transfected with synthetic (ARE)4-luc reporter plasmid, followed by incubation with or without 10−8 M DHT for 30 minutes or 18 h. Cell lysates were harvested for reporter gene assay. (D) Akt phosphorylation was detected in MC3T3-E1, LNCaP, and SaOs2-AR3 cells in the presence of 1.5 × 10−7 M testosterone-conjugated BSA or BSA control. (E) MC3T3-E1 cells were pretreated with 10−6 M HF, followed by incubation with 10−8 M DHT. (F) DU145 was treated with DHT for different concentrations of DHT in indicated time points. Quantification of Western blot bands is shown in each graph: each bar represents means ± SD of independent triplicate blots for each treatment. There were no significant differences in total Akt band intensity between treatments. Samples (n = 3) significantly different to controls: *p < 0.05 with respect to the corresponding control.

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Androgens induces osteoblast growth activation in part through AR and the PI 3-kinase/Akt pathway

To establish the time course and dose-response characteristics of androgen action on osteoblast growth, we first determined the MC3T3-E1 cell growth curve after treatment with vehicle or increasing concentrations of DHT from 10−12 to 10−8 M for 15 minutes. Cells were then incubated in charcoal-treated FBS medium for 1, 2, or 3 days. As shown in Fig. 3A, DHT concentrations were associated with an increase in MC3T3-E1 cell growth. The apparent maximal effect was seen with 10−8 M DHT, which is in a range associated with physiological relevance. To determine whether functional AR regulated the increase in androgen-mediated cell proliferation, MC3T3-E1 cells were treated with DHT along with an AR antagonist (HF). As shown in Fig. 3B, the increase in cell growth after 10−8 M DHT treatment was blocked by the addition of 10−6 M HF. Because the increase of cell growth examined by XTT assay can result from decreased cell apoptosis as well as increased in cell proliferation, we further investigated the effects of DHT on MC3T3-E1 cell proliferation and survival. Our results show that DHT was able to increase cell numbers by direct cell counting as well as decreased Dex-induced cell apoptosis by trypan blue in a dose-dependent manner (Figs. 3C and 3D). To examine whether AR is required for androgen action on osteoblast growth, we used the specific siRNA-AR to block AR expression in MC3T3-E1 cells. Interestingly, inhibition of AR expression decreased androgen-mediated cell growth in MC3T3-E1 cells (Fig. 3E). To study the potential roles of Akt kinase in androgen action on osteoblast growth, we transfected MC3T3-E1 with a kinase-deficient Akt that has been shown to act as a dominant-negative mutant of Akt. The cells were treated with DHT, and cell growth was measured. Expression of kinase-deficient Akt blocked DHT-mediated MC3T3-E1 cell growth (Fig. 3F). We also treated MC3T3-E1 cells with LY294002 or transfected MC3T3-E1 cells with a dominant-negative p85 regulatory subunit that blocks the enzymatic activity of the catalytic p110 subunit of PI 3-kinase. As expected, inhibition of PI 3-kinase by LY294002 or a dominant-negative p85 suppressed DHT-mediated cell growth (data not shown).

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Figure FIG. 3.. Androgen-stimulated osteoblast growth in part through PI 3-kinase/Akt pathway. (A) MC3T3-E1 cells were incubated with various concentrations of DHT for indicated periods of time and applied to XTT assay for measuring cell growth. (B) Cells were pretreated with AR antagonist, HF (10−6 M), followed by incubation with 10−8 M DHT stimulation. Subsequently, the cells were quantified by XTT assay. (C) Cells were incubated with various concentrations of DHT for indicated periods of time, and the total cell number was counted for measuring cell growth. (D) Cells were pretreated with various concentrations of DHT for 15 minutes and followed by incubation with 10−6 M Dex to induce apoptosis for 3 h. Subsequently, the cells were quantified by trypan blue exclusion assay. (E) Cells were transiently transfected with siRNA-AR expression vector or empty control vector, followed by treatment with vehicle or DHT for indicated periods of time and applied to XTT reagents. Bars indicate means ± SD of triplicate determinations. (F) MC3T3-E1 cells were stably transfected with kinase deficient Akt expression vector or empty control vector and incubated in the presence or absence of DHT for indicated periods of time to assay cell growth. Each data point indicates means ± SD of at least six independent sets for each experiment. *p < 0.05 with respect to the corresponding control.

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Activation of Akt by androgen is involved in specific G-proteins, PLC, and Src kinase

In rat osteoblasts, T induces inositol trisphosphate (InsP3), and diacylglycerol formation is linked to intracellular effector coupled to PLC through a pertussis toxin-sensitive G-protein.(25) To evaluate whether PLC is part of androgen nongenomic signaling mechanism involving Akt phosphorylation in osteoblast cells, we investigated the effect of the U73122 (specific PLC inhibitor), and U73343, an inactive form of U73122 analog, on Akt phosphorylation induced by DHT. MC3T3-E1 cells were treated with 2 μM U73122 or U73343 for 2 minutes, followed by exposure to 10−8 M DHT. Under these conditions, the effects of the hormone on Akt phosphorylation were abolished by U73122 but not by U73343 (Fig. 4A). Previous studies have shown that preincubation of the osteoblasts with pertussis toxin totally abolishes T-induced InsP3 and diacylglycerol formation, and the toxin seems to uncouple the androgen nongenomic receptor from its G-protein by blocking the signal transduction that activates PLC.(25) To examine the role of pertussis toxin-sensitive G-proteins in DHT-induced activation of Akt, we treated the osteoblasts with pertussis toxin before DHT stimulation. Recently, AR, progesterone receptor, and estrogen receptor have been found to interact with the intracellular tyrosine kinase Src, triggering Src activation.(10,14) In response to DHT or the synthetic androgen R1881, AR interacts with the SH3 domain of Src.(10,14) Because the possible role of Src tyrosine kinase in DHT-induced activation of Akt remains unknown, the requirement of Src for phosphorylation of Akt was examined. As shown in Fig. 4C, DHT induced Akt phosphorylation, and this effect was effectively suppressed by soluble Src family tyrosine kinase-selective inhibitor (PP2) but not by PP3, an inactive form of PP2.

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Figure FIG. 4.. DHT-induced Akt activation is dependent on PLC, G-protein, Src kinase, and calcium mobilization. (A) Cells were incubated with 2 μM U73122 or U73343 before the addition of 10−8 M DHT, and Akt phosphorylation was determined. (B) Cells were pretreated with pertussis toxin before the addition of 10−8 M DHT (C) Cells were incubated with 10 μM PP2 or PP3 before the addition of 10−8 M DHT, and Akt phosphorylation was determined. (D) Cells were incubated with 3 mM EGTA for 1 or 15 minutes before 10−8 M DHT stimulation. (E) Cells were incubated with 10 μM nifedipine and verapamil followed by incubation with 10−8 M DHT. (F) Cells were incubated with 10 μM BAPTA/AM followed by incubation with 10−8 M DHT.

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Calcium mobilization mediates DHT-induced Akt activation

Recently, Ca2+ has been proven to promote cell survival through activation of the Akt pathway.(27,28) The basal level of intracellular calcium concentration in confluent male osteoblasts was 140 ± 3 nM, and 1 nM testosterone as well as DHT were able to induce the transient increase(29) up to 240 nM within 5-30 s through the intracellular calcium influx. The ability of physiological levels of T to induce a rapid increase in intracellular free Ca2+ concentrations has also been reported in primary cultures of rat osteoblasts.(25) When extracellular Ca2+ in MC3T3-E1 cells was removed by pretreating with EGTA for 1 minute, DHT was still able to induce Akt phosphorylation (Fig. 4D). However, when both extracellular and intracellular Ca2+ was removed by pretreating with EGTA for 15 minutes, DHT-induced Akt phosphorylation was significantly suppressed (Fig. 4D). Interestingly, both Nifedipine and Verapamil, the L-type voltage-gated Ca2+ channels blockers, caused an increase in DHT-stimulated Akt phosphorylation (Fig. 4E). A similar result was obtained by thapsigargin, the endoplasmic reticulum ATP-dependent Ca2+ pump blocker, by releasing Ca2+ from intracellular stores, which also enhanced the phosphorylation of Akt induced by DHT (data not shown). Blockade of the intracellular calcium by BAPTA/AM prevented DHT-induced Akt phosphorylation (Fig. 4F).

Androgen induces nuclear translocation of active Akt in MC3T3-E1 cells

It is commonly thought that Akt is activated after its recruitment to the plasma membrane. In this regard, the direct binding of the PI 3-kinase-generated phospholipids to the pleckstrin homology domain of Akt stimulates Akt translocation to the membrane. However, several growth factors recently have been shown to be capable of inducing intranuclear migration of Akt, such as IGF-1 and PDGF, in osteoblast cells.(30) Therefore, we investigated whether Akt translocates into the nucleus of MC3T3-E1 cells in response to androgen and whether this event is dependent on PI 3-kinase activity. Using immunofluorescence studies using anti-Akt antibody, it was shown that the majority of Akt has a diffuse cytosolic distribution, with some labeling of the plasma membrane in unstimulated cells (Fig. 5A). After 30 minutes of treatment with 10−8 M DHT, a portion of Akt localized into the cell nucleus, as indicated by reinforcement of the fluorescence signal (Fig. 5C). An increase of intranuclear translocation in DHT-exposed cells was observed with a maximal nuclear translocation after 40 minutes of DHT treatment (Fig. 5D). After 60 minutes of stimulation with DHT, Akt immunoreactivity was again dispersed throughout the cytoplasm (Fig. 5E). Results obtained by fluorescence microscopy were also confirmed by cell fractionation experiments (data not shown). We next examined the possible role of PI 3-kinase and AR in DHT-dependent localization of Akt. Treatment of cells with the specific PI 3-kinase inhibitor, LY294002, and AR antagonist, HF, for 60 minutes did not influence the subcellular distribution of Akt (Figs. 6C and 6E versus 6A). In the case of DHT stimulation, the nuclear interior was immunostained with a brilliant fluorescence after 40 minutes of exposure (Fig. 6B). Cells pretreated with LY294002 and exposed to DHT up to 40 minutes did not show any intranuclear migration or subcellular modification of Akt (Fig. 6D). Similar results were obtained when cells were treated with HF and exposed to DHT stimulation (Fig. 6F).

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Figure FIG. 5.. Androgen-dependent nuclear translocation of Akt kinase in MC3T3-E1 cells. MC3T3-E1 cells in separate culture dishes were treated with 10−8 M DHT and fixed at different time intervals (0, 10, 20, 30, 40, 50, and 60 minutes) as indicated on the left panel of the figure. Cells stained with anti-Akt antibody. Cells stained with DAPI are as indicated on the middle panel of the figure. Merge images are as indicated on the right panel of the figure.

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Figure FIG. 6.. Conversion of Akt nuclear distribution in LY294002 and HF-treated cells subsequently exposed to DHT. (A) The fluorescence pattern of Akt kinase localization was detected in unstimulated MC3T3-E1 cells. (B) Cells stimulated with 10−8 M DHT for 40 minutes. (C) Cells treated with LY294002 (20 μM) for 60 minutes. (D) Cells treated with LY294002 and exposed to DHT for 40 minutes. (E) Cells treated with 1 μM HF for 60 minutes. (F) Cells treated with HF and exposed to DHT for 40 minutes. Cells stained with anti-Akt antibody are as indicated on the left panel of the figure. Cells stained with DAPI are as indicated on the middle panel of the figure. Merge images are as indicated on the right panel of the figure.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

These studies represent the first demonstration of activation of the Akt signal pathway in response to sex steroid hormone androgens in osteoblast cells. Figure 7 shows a possible model for early nongenomic signaling of androgen-mediated Akt activation in osteoblast proliferation. Rapid action of androgens may occur through multiple pathways. DHT may stimulate second messenger cascades in a nongenomic manner through more than one mechanism. DHT may induce the Akt pathway through stimulation of Src, PI 3-kinase, and PLC. In addition to these effectors, G-protein-coupled receptors may also directly bind androgens or indirectly influence androgen mediated-Akt activation. One of the effects mediated by G-protein-coupled receptors is to increase intracellular calcium levels. The elevation of intracellular calcium may also be involved in androgen-mediated Akt signal transduction cascades. However, the relationship between DHT/AR and these different effectors in Akt activation remains unclear; future studies will be needed to characterize the mutual interaction between these critical upstream regulators of the androgen-mediated Akt activation pathway.

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Figure FIG. 7.. The schema of androgen/AR/Akt signaling pathways in osteoblast cells. A simplified model for rapid androgen action in osteoblasts occurs through multiple pathways.

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Evidence from observations that transcriptional inhibitors such as actinomycin D do not affect androgen-mediated Akt phosphorylation (Fig. 1D) and that androgen initiates Akt phosphorylation that is too rapid (5-10 minutes) to involve transcriptional regulation supports the nongenomic nature of androgen-induce Akt activation. Furthermore, our studies suggest that Akt-mediated osteoblast proliferation may be regulated by AR. First, AR antagonist HF was able to block androgen-mediated cell proliferation, Akt phosphorylation, and nuclear translocation (Figs. 3B, 1E, and 6F). Second, overexpression of AR enhances the basal level of Akt phosphorylation in the absence of DHT (Fig. 2C). Third, siRNA-AR was able to inhibit androgen-mediated Akt activation and cell proliferation (Figs. 2A and 3C). Together, these results suggest that AR is indeed capable of mediating osteoblast proliferation when Akt is activated.

The existence of a novel membrane-bound AR has been postulated by a number of experiments based on the detection of specific androgen binding to plasma membrane.(9,26,31) However, it has not yet been determined whether the nongenomic effects are mediated through a membrane AR. A novel family of high-affinity membrane progestin receptors with structure and signaling similar to G-protein-coupled receptors has recently been cloned.(32,33) The identification of distinct membrane receptors for other steroid hormones suggests a novel membrane receptor for androgens may also exist. At this point, it cannot be excluded that unidentified membrane receptors for androgens may mediate androgen-promoted osteoblast proliferation. Perhaps both the classical AR and novel unidentified androgen membrane receptors are used, depending on the target cells and signal cascade involved. Future studies will be needed to determine the extent to which the nongenomic mechanism of androgen action interacts to result in a specific rapid androgen effect.

Addition of androgens suppresses osteoblast and osteocyte apoptosis induced by a variety of proapoptotic stimuli in vitro, and both effects are caused by activation of an Src/Shc/ERK signaling pathway through a nongenotropic action of the classical sex steroid receptors, which is sex nonspecific and dissociable from the transcriptional activity of the receptor.(10) In agreement with the antiapoptotic effects of androgen on osteoblast cells and osteocytes,(10,34) we have shown that addition of androgens increases the osteoblast proliferation through activation of the PI 3-kinase/Akt pathway. Because androgen stimulation of proliferation of these cells and possibly also of differentiation have been reported with increased expression of TGF-β mRNA and increased responsiveness to fibroblast growth factor (FGF) and IGF-II,(35,36) it is likely that androgens may also potentiate the actions of cytokines or growth factors acting through the similar pathways in osteoblast cells.

In addition to its well-established role at the plasma membrane, it is now agreed that Akt may also be involved in nuclear signal transduction events. Several growth factors have been shown to be capable of inducing intranuclear migration of Akt, such as PDGF and IGF-1.(18,30) Recently, it has been reported that the TCL1 oncogene can bind to Akt and mediate the formation of oligomeric TCL1-Akt high molecular weight protein complexes in vivo.(37) Within these protein complexes, Akt is preferentially phosphorylated and activated. Because we have previously shown that AR can also interact with Akt,(19,20) it is likely that androgens may promote AR to interact with TCL1/Akt complex. The consequence of such multiple complexes between AR and TCL1/Akt may enhance Akt kinase activity and promote its nuclear transport.

It has been shown that Akt regulates cell proliferation by phosphorylating p27(kip1) and causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest.(38,39) Because downregulation of osteoprogenitor proliferation is a critical step for osteoblast differentiation, it has been indicated that p27(kip1) may play a key role in regulating osteoblast differentiation by controlling proliferation-related events in bone cells during differentiation of osteoprogenitor cells derived from the bone marrow of p27−/− mice.(40) Interestingly, DHT was also able to cause an accumulation of the cyclin-dependent kinase inhibitor p27(Kip1).(41) Androgens are able to promote osteoblast proliferation through activating Akt phosphorylation and nuclear translocation. Therefore, it is quite possible that androgens may activate Akt to phosphorylate and accumulate p27(kip1) in the cytoplasm of osteoblast cells. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, could be a possible mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated by androgens and the proliferation of osteoblast cells is sustained.

Our knowledge of the sex hormone signaling pathway has advanced recently because of the recognition that AR is one of the major mediators of nongenomic androgen action, and the results presented here expand the contention that AR, besides the role it plays as a transcription factor, is involved in transducing key signals to the PI 3-kinase/Akt pathway. This mechanism is increasingly appreciated in that it plays important roles in the bone cell formation of androgen actions, and manipulation of these pathways could therapeutically modulate the metabolism of androgens in the bone microenvironment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Drs Renny T Franceschi and R Freeman for valuable plasmids and cells. We also thank Karen Wolf for manuscript preparation. This work was supported by the following grants: NSC Grants NMRPD1073 (NSC91-2320-B-182-040), CGMH Grant CMRP1287 to HYK; NSC Grants NMRPD0143, NMRPD0139, and CMRP845 to KEH; and NIH Grants CA55639, CA68568, and CA75732 to CC.

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  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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