Dissecting phenotypic variation among AIS patients

BBRC Biochemical and Biophysical Research Communications 335 (2005) 335–342 www.elsevier.com/locate/ybbrc

Dissecting phenotypic variation among AIS patients q Minghua Wang a, Jiucun Wang a, Zhen Zhang a, Zhimin Zhao b, Rongmei Zhang a, Xiaohua Hu a, Tao Tan a, Shijing Luo a, Zewei Luo a,c,* a

Laboratory of Population and Quantitative Genetics, ChinaÕs State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Morgan-Tan International Center for Life Sciences (MTIC), Fudan University, Shanghai, PR China b Department of Surgery, The First Affiliated Hospital of Guiyang Traditional Chinese Medicine College, Guiyang, PR China c School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Received 28 June 2005 Available online 26 July 2005

Abstract We have created genital skin fibroblast cell lines directly from three patients in a Chinese family affected by androgen insensitivity syndrome (AIS). All patients in the family share an identical AR Arg840Cys mutant but show different disease phenotypes. By using the cell lines, we find that the mutation has not influenced a normal androgen-binding capacity at 37
C but has reduced the affinity for androgens and may cause thermolability of the androgen–receptor complex. The impaired nuclear trafficking of the androgen receptor in the cell lines is highly correlated with the severity of donorsÕ disease phenotype. The transactivity of the mutant is substantially weakened and the extent of the reduced transactivity reflects severity of the donorsÕ disease symptom. Our data reveal that although etiology of AIS is monogenic and the mutant may alter the major biological functions of its wild allele, the function of the mutant AR can also be influenced by the different genetic backgrounds and thus explains the divergent disease phenotypes.  2005 Elsevier Inc. All rights reserved. Keywords: Androgen; Androgen insensitivity syndrome; Androgen receptor; Genital skin fibroblast cells; AR Arg840Cys mutant; Disease phenotypic variation; DHT; Transactivity; Binding; Androgen–receptor complex; Genetic backgrounds

Androgen receptor, an X-linked protein, is a prototypic member of steroid superfamily of ligand-dependent transcription factors [1]. The 110-kDa AR protein, encoded by a single copy gene with eight exons at Xq11-12 [2,3], consists of four major domains: the amino-terminal transcription activation domain, the DNA-binding domain that binds a specific DNA sequence, the hinge region that contains the nuclear local-

q The research was supported in part by grants from ChinaÕs Key Basic Research Program (‘‘973’’) and ChinaÕs National Natural Science Foundation. Z.W.L. is also supported by research grants from the Medical Research Committee of the University of Birmingham, BBSRC, and NERC. * Corresponding author. Fax: +86 21 65643966. E-mail addresses: [email protected], [email protected] (Z. Luo).

0006-291X/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.077

ization signal, and the carboxy-terminal ligand-binding domain [4]. It binds two main androgens, testosterone and dihydrotestosterone (DHT), and mediates their functions in development, maintenance, and regulation of maleness [4]. AR mainly distributes in plasma when it is free of binding with androgens. When present in ligand–receptor complex form, conformation of AR facilitates nuclear transportation, receptor dimerization, and interaction with target sequences, thus facilitating the realization of its normal biological functions [5,6]. With the help of various co-activators and co-repressors, the androgen–AR complex can either induce or suppress the expression of many other genes through its binding to the androgen-responsive elements (ARE) located within the 5 0 flanking regions of these genes [7]. Genetic defects of AR gene may cause a wide spectrum of phenotypes from maleness disorders to mental

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M. Wang et al. / Biochemical and Biophysical Research Communications 335 (2005) 335–342

impairment [8–10], even though a majority of these defects identified to date are associated with androgen insensitivity syndrome. These defects may disrupt partially or completely normal function of the wild type by changing its binding ability to the androgen, disrupting DNA binding, altering its transcriptional activity, or even affecting the rate of its nucleus transfer and the maximal nucleus importation [10,11]. It is well known that AIS patients may display a wide variety of phenotypic abnormalities, ranging in severity from a complete form (CAIS) where affected males have female external genitalia to a partial form (PAIS) where patients show variable degrees of undermasculinization and/or fertility [10]. We have reported an Arg840Cys from a large Chinese family affected with AIS. Genetic analysis showed that the disease trait was completely linked to an Arg840Cys mutant of AR [12] and that all patients shared an identical Arg840Cys mutant but displayed large variation in disease phenotypes. Some of the affected males were infertile and had gynecomastia and/or hypospadias but some had fathered children normally. With an attempt to unveil the molecular mechanisms of the pleiotropic phenomenon, this report investigated the mutantÕs expression, binding properties, trans-activation, and nucleus trafficking in comparison with those of the corresponding wild type. These analyses were carried out by making use of the cells directly isolated from the genital skin tissues of the AIS patients who represented the typical disease phenotypes of the family.

tility was confirmed by repeated semen test data and child-given status after a more than 3-year marriage history. Clinical descriptions of these three patients are given in Table 1. The research was conducted with an official approval from the Academic Advisory Board of the Institute of Genetics, Fudan University. Cell culture. After obtaining written consent from each of the three patients described above and a normal control subject, a surgeon performed the punch biopsy operation under local anesthesia for collecting their genital skin tissues. Fibroblasts were grown from these skin samples and the cell strains were marked as MJ for the proband object (IV54), ZGJ and YS for the AIS patients IV28 and IV39, respectively, and N for the normal control subject. The cells were cultured in DulbeccoÕs modified high-glucose essential medium (DMEM-H, Gibco-BRL, MD) at 37
C in an incubator maintained at 5% CO2. The medium was prepared with 10% fetal calf serum (FCS, Hyclone, UT), 2.0 mM L-glutamine, 20 mM N 0 -2-hydroxyethylpiperazine-N 0 -ethanesulfonic acid (Hepes), 100 U/ml penicillin, and 100 lg/ ml streptomycin. The fibroblasts cultured at the stage of passage 5 were used for the experiment. These cell lines had been sequenced for the AR gene before they were used for the analyses described below and no new mutation was detected during their culturing. Two repeat sequences, CAG (Gln)21 and GGC (Gly)23, in exon I were checked in all cell lines under study and no variation was observed. Northern analysis. Probes of AR and b-actin genes were first amplified from total RNA of the cell strain N by RT-PCR. The forward and the reverse primer pairs were 5 0 -gactactacaactttccactggctc-3 0 and 5 0 -tcccagagtcatccctgcttcataac-3 0 for the AR gene, and 5 0 -acc ctgaagtaccccatc-3 0 and 5 0 -ctagaagcatttgcggtg-3 0 for b-actin. Amplified fragments were purified by use of QIAquick PCR Purification kit (Qiagen, Germany) and labeled with digoxigenin by random-primed DNA synthesis using DIG high-prime DNA labeling and detection starter kit II (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturerÕs protocol. Total RNA extracted from approximately 1 · 106 cells was denatured at 65
C for 10 min in RNA loading buffer and then separated on 1.0% (w/v) formaldehyde agarose electrophoresis gels. The bands were blotted in 7.5 mM NaOH onto nylon membranes [13]. The membranes were pre-hybridized in DIG easy hybridization buffer for 30 min at 42
C and then hybridized with digoxigenin-labeled AR and b-actin DNA probes at 42
C for 16 h. The membranes were washed twice in 2· SSC, 0.1% SDS for 5 min at room temperature and twice in 0.5· SSC, 0.1% SDS for 15 min at 68
C. Chemiluminescent test was performed as described in the DIG Northern Starter kit II (Roche). The light density was recorded on an X-ray film. Total RNA was extracted from COS-7 cells and from the genital skin fibroblast of the normal subject N and tested in the same way. Western analysis. The cultured cells were washed three times with 20 nM Tris–HCl (pH 7.4) containing 0.15 M NaCl [14]. After microfuging, the cells were re-suspended in 100 ll of 0.25 M Tris–HCl (pH

Materials and methods Family and patients. A large Chinese family, which consisted of a total of 132 members across five generations, 14 males of them affected with AIS, was described in our previous genetic analysis. It showed that the patients in the pedigree shared an identical Arg840Cys substitution in the androgen receptor but displayed divergent disease phenotypes [12]. The patients who donated their genital skin tissues for the present study included the proband IV54 (cell strain MJ), who was fertile but was born with penile hypospadia, and two infertile individuals IV28 (cell strain ZGJ) and IV39 (cell strain YS) whose infer-

Table 1 Phenotypes of the three patients from a Chinese AIS family who donated genital skin fibroblasts to the present study Patients

Cell line

Fertilitya

Age (year)

Laryngeal protuberance

Pubic hair

Breast (tanner stage)

Penis size (length/circumference, cm)

Hypospadias status

Scrotum

Testis volume left/right (ml)

IV 54

MJ

Yes

29

Impalpable

I

3.2/6.0

Penile

Normal

24.2/19.0

IV 39

YS

No

31

Small

III

4.0/10

Penile

Normal

20.3/22.5

IV 28

ZGJ

No

26

Small

Sparse, inverted triangular-shaped Sparse, inverted triangular-shaped No

II

3.0/6.5

Scrotal/penile

Bifid

a

7.3/19.0

Fertility of the AIS patients was determined by their status of fathering child, and their semen and hormone tests as given below: IV 28—semen level: absence of primary spermatocyte; hormone level: not tested. IV 39—semen level: azoospermia; hormone level: 34.70 nM (T), 16 IU/L (LH), and 10 IU/L (FSH). IV 54—semen level: 0.6 ml (volume), 35.7 million/ml (density), 40% (motility), and 15% (abnormal sperm); hormone level: 58.48 nM (T), 6.3 IU/L (LH), and 2.3 IU/L (FSH).

M. Wang et al. / Biochemical and Biophysical Research Communications 335 (2005) 335–342 7.4) containing 10 lg/ml Aprotinin (Amresco, USA), 100 lg/ml PMSF (Amresco), and 2 lg/ml Leupeptin (Amresco), lysed by sonication, and microfuged (13,000 rpm, 30 min, 4
C). Total protein was measured by use of the Bradford protein assay [15] and 50 lg proteins from each sample were subjected to 10% SDS–PAGE and transferred to PVDF membranes according to the standard method [16]. The PVDF filters were blocked by immersion in TBS (20 mM Tris–HCl, pH 7.4, and 500 mM NaCl) with 0.5% Tween 20 and 5% BSA for 1 h at room temperature and incubated overnight at 4
C with mouse monoclonal IgG1 antibody diluted to 1:500 in TBS with 0.05% Tween 20 (Santa Cruz Biotechnology). The antibody targets amino acids 299–315 of the human androgen receptor. After washing four times in TBS with 0.5% Tween 20, the filters were incubated with 1:5000 horseradish peroxidase rabbit anti-mouse IgG (CNI) for 1 h at room temperature. After washing in TBS with 0.5% Tween 20 seven times, the blot was developed using the ECL chemiluminescence detection system (PFBIO, China) and the light density was recorded on an X-ray film. The same treatments were carried out on COS-7 and the N cells. Monolayer-binding experiment. Fibroblasts grown to 80% confluence in flasks were digested with 0.05 M trypsin–0.02% EDTA at 37
C and then seeded into six-well microplates. Each of the wells had a volume of 2 ml and contained approximately 2 · 105 cells in the DMEM with 10% FCS, glutamine, Hepes, and antibiotics. After being incubated for 48 h, the medium in each well was replaced by DMEM without serum and the incubation was continued for another 24 h. After the media were removed, the monolayers were rinsed with the same DMEM and were then incubated with the DMEM that contained varying concentrations of [3H]DHT with or without adding unlabeled DHT. In the presence of unlabeled DHT, its concentration was as much as 200-fold of [3H]DHT. After being incubated for 1 h at 37
C, the monolayers were rinsed gently with 4
C pre-cold PBS for five times and lysed with lysis buffer (Roche) for 30 min at room temperature under constant gentle shaking. Aliquots of the cell extracts were collected to measure the total protein concentration and the radioactive density by making use of a liquid scintillation counter (Beckman Coulter, CA, USA). The binding data were collected from a two-way factorial experimental design. The specific binding for each of the four cell lines was calculated from an average of the difference between total binding and nonspecific-binding records over three repeated observations at each of seven concentrations: 0.2, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 nM [3H]DHT. The experiments were repeated twice and, thus, yielded two sets of data. For each of the two data sets, the specific-binding measure was regressed on the ratio of the binding measure over the concentration of unbound [3H]DHT across the above [3H]DHT gradients as suggested in the Scatchard analysis [17]. The regression analysis produced estimates of two regression parameters (regression intercept, aˆ, and regression coefficient, ^ bÞ for each of the four cell lines and their corresponding variances s2a and s2b . The two most important parameters are the maximum binding capacity (Bmax) and the apparent dissociation constant (Kd), which can be calculated from the regression
1 parameter estimates as
^a=^b and
^b , respectively. By following the standard statistical formulae [18], we calculated sampling variances of 2 4 4 the Bmax and Kd estimates as s2a =^b þ ^a2 s2b =^b and s2b =^b , respectively. Means and standard errors of the parameter estimates were calculated by averaging the estimates from the two experimental replicates and used to compare the binding parameter estimates among the different cell lines. To investigate the effect of temperature on binding capacity, we adjusted the incubation temperature from 37 to 42
C. The specific binding at 42
C was determined at the [3H]DHT concentration of 2 nM. Thermolability of binding was declared if the binding capacity at 42
C decreased by more than 40% of that at 37
C as suggested in Griffin and Durrant [19]. Dissociation of the androgen–receptor complex in monolayers. To estimate the rate of dissociation of the [3H]DHT–receptor complex in monolayer cells, we incubated the monolayer cells at 37
C in the

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presence of 2 nM [3H]DHT for 1 h before DHT in 200-fold excess was added. In parallel, incubation was carried on in the presence of serumfree DMEM in order to monitor the stability of the androgen–receptor complexes. The cells were lysed at 0, 30, 60, and 90 min and the specific binding was measured at these time points. Confocal immunofluorescence microscopy. The GSFs were cultured directly on microscope glass slides and treated in the presence of four concentrations of DHT (0, 1, 20, and 50 nM) overnight. After washing three times with PBS (pH 7.4), the GSFs were fixed in 3% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X-100 in PBS for 5 min [20]. After rinsing with PBS, the GSFs were incubated with 5% BSA in PBS for 30 min and then with mouse monoclonal IgG1 antibody (1:100), which targets amino acids 299–315 of the androgen receptor of human origin (Santa Cruz Biotechnology), at 4
C overnight. Washed with PBS, the GSFs were stained with FITC-conjugated goat anti-mouse antibody (1:300) at room temperature for 1 h. Images of the AR trafficking were viewed on a Zeiss LSM510 confocal microscope (Germany). Plasmid construction. Expression vectors for wild type and mutant androgen receptor genes were constructed using RT-PCR. The cDNAs extracted from MJ and N cells (corresponding to IV54 subject and normal subject) were used as templates for amplifying mutant and wild types of the AR gene, respectively, in PCR experiments, which used TaKaRa LA taq DNA polymerase (TaKaRa, Japan), primer 1 (5 0 -ca tcacctcgagttgaactcttctg-3 0 ), and primer 2 (5 0 -aacaggcagtcgacatctga aaggg-3 0 ). After an initial 5 min denaturation at 95
C, a touchdown thermocycling profile was performed: 15 cycles of 94
C for 45 s; 63– 49
C for 60 s (
1
C per cycle); 72
C for 5 min, then 25 cycles at 94
C for 45 s; 51
C for 60 s; 72
C for 5 min, and a final extension for 10 min at 72
C. The PCR products were purified using QIAquick PCR Purification kit (Qiagen) and inserted into the mammalian expression vector pTarget (Promega, USA) directly, yielding the ARArg840Cys expression vector (pT-AR-Arg840Cys) and the wild type AR vector (pT-wt-AR). Sequences of the constructs were confirmed by direct sequencing on an ABI PRISM377 DNA Sequencer with BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystem). The plasmid carrying a firefly-luciferase-reporter gene (pGL3MMTV) was kindly provided by Dr. Nawata (Kyushu University) and its co-transfection with pRL-SV40 vector (Promega) has been reported [21]. DNA transfection and luciferase reporter assay. GSFs were seeded into six-well plates at a density of approximately 3 · 105 cells per well and incubated for 24 h. The incubation was continued for another 3 h in the presence of the Superfect Reagent (Qiagen, Germany), 1.5 lg pGL3-MMTV, 3 ng pRL-SV40 vector, and 0.2 lg of each of the two AR expression vectors: pT-AR-Arg840Cys or pT-wt-AR. The medium was replaced by 10% charcoal-treated fetal calf serum (DCC, Hyclone) in the presence or absence of 100 nM DHT. The cells were incubated for another 48 h and then lysed with 150 ll lysis buffer. Luciferase reporter gene activity was determined from the Dual-luciferase Reporter Assay System (Promega) on a Lumat LB 9507 model (EG&G Berthold, Germany).

Results Expression of AR in GSF cell lines Fig. 1 shows Northern (A) and Western (B) blotting assays of AR in the GSF cell lines from the three AIS patients in the same family and from one independent normal donor. It does not reveal any viewable change in both transcriptional and translational levels of the AR gene among the four cell lines (N, MJ, YS, and

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M. Wang et al. / Biochemical and Biophysical Research Communications 335 (2005) 335–342

Fig. 1. Northern (A) and Western (B) blot assays of androgen receptor gene in the five cell lines: COS-7, N, MJ, YS, and ZGJ. COS-7 cell here plays a role of negative control in the assays.

Binding capacities and disassociation rates of the AR mutant in the four cell lines Table 2 shows the estimates and sampling variances of the maximum binding capacity (Bmax) and the apparent dissociation constant (Kd) for each of the four cell lines from two independent experiments. Means and standard errors of the parameter estimates were calculated from averaging over the repeated experimental estimates and listed as the last panel in the table. Based on the mean estimates of the binding parameters and their standard errors, we carried out a t test for significance of difference in each of these two parameter estimates between the cell lines. Since each of the means was calculated from averaging two independent estimates, the t test had 2 degrees of freedom. It can be seen from the t test analyses that there is no significant change in the maximum binding capacity between normal and patientsÕ donated cells. However, the difference in the Kd estimate was significant between the normal cell line and the patient cell lines (0.0025 < P < 0.05). In addition, the maximum binding capacities measured at 42
C were compared with those at 37
C for these four cell lines under a given [3H]DHT concentration of 2 nM. The Bmax estimates at 42
C for the cells donated by the two infertile pa-

tients were decreased by more than 40% when compared to those estimates at 37
C, and thus the ligand–receptor binding in these two cell lines was inferred to be thermolabile according to the criterion set in [19], whilst the binding in the fertile patient cell line was thermostable (Table 2). Fig. 2 shows the amount of androgen receptor remaining in the four cell lines cultivated below 37
C over a time span from 0 to 90 min. It shows a clear differentiation in the dissociation rates of the androgen receptor among the four cell lines and demonstrates a good correlation between the affinity and the cell donorsÕ severity of the AIS symptom. 100

% Androgen-receptor complexes remaining

ZGJ). The data first exclude the possibility of ascribing the divergent disease phenotypes of the three patients to any variation in the mRNA and protein levels of the mutant that these patients carry, and suggest the absence of any mutation in the promoter region, which may alter the expression pattern.

N MJ YS ZGJ

10 0

30

60

90

Time (min) at 37 C

Fig. 2. The androgen–receptor compound remained over a time span in the four cell lines (N, MJ, YS, and ZGJ) that were cultivated at 37
C.

Table 2 The binding properties of genital skin fibroblasts Cell lines

N (normal) MJ (fertile) YS (infertile) ZGJ (infertile)

Experiment I Bmax

s2b

102.12 87.46 83.23 107.62

74.38 80.79 71.53 82.12

Experiment II Kd

s2K

0.6374 1.1529 1.1339 1.4490

0.0064 0.0582 0.0673 0.0631

Bmax

s2b

103.03 98.60 79.21 105.39

63.73 90.87 72.61 89.48

Means (SE) Kd

s2K


max B


d K

0.6941 1.1477 1.3806 1.2555

0.0110 0.0170 0.0112 0.0553

102.56(5.87) 93.03(6.55) 81.22(6.14) 106.51(6.56)

0.6658(0.0663) 1.1503(0.1370) 1.2623(0.1400) 1.3523(0.1720)

Bmax 42
C/ 37
C (%)

Thermolability

74.9 67.5 51.9 47.8

No No Yes Yes

Bmax ðs2b Þ, the maximum binding capacity (sample variance); K d ðs2K Þ, the apparent dissociation constant (sample variance).

M. Wang et al. / Biochemical and Biophysical Research Communications 335 (2005) 335–342

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Trafficking of the androgen receptors

Assay of transactivities of the ARs

Fig. 3 shows confocal views of immunocytochemical analysis on the four cell lines. As expected, ARs in all cell lines distribute in the cytoplasm when the hormone (DHT) is absent. When treated with the three concentrations of DHT, wild type AR in the N cells was seen as a clear assembling into the nucleus. A clear trafficking of AR in the MJ cells from cytoplasm to nucleus occurred only when the DHT concentration was equal to or greater than 20 nM. However, it needed even a higher DHT concentration (P50 nM) of AR in the YS cells to accomplish a normal trafficking. In contrast, the trafficking seemed to be remarkably delayed in the ZGJ cells, which were donated from the patient who suffered the most severe AIS symptom among the samples. Thus, data from the immunocytochemical analysis revealed that severity of the mutant caused disease was closely correlated to the degree of abnormality in the mutant trafficking from cytoplasm to nucleus.

The assay was designed to test if any variation exists in the ability of the receptors from the four cell lines in stimulating transcription of the genes whose transcription is regulated by the AR complex. The transcriptional activation was defined in the present study as an average of three repeated observations of relative luciferase unit (RLU). Fig. 4 shows a comparison of the transcriptional activity between the normal and mutant ARs when DHT was present (+) or absent (
). The transcriptional activation measures were trivial in all of the four cell lines when DHT was absent but increased dramatically when DHT was present, suggesting that the reporter geneÕs expression was well controlled by the AR complex. It can be seen from the figure that the transcriptional activities induced by the wild type AR (solid bars) were significantly and consistently higher than those by AR-Arg840Cys (open bars), a strong evidence supporting the fact that the AR complexÕs transcription-

Fig. 3. Confocal views of immunocytochemical analysis of androgen receptor in four cell lines (N, MJ, YS, and ZGJ) under treatment of four concentrations of DHT.

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M. Wang et al. / Biochemical and Biophysical Research Communications 335 (2005) 335–342

Fig. 4. Transcriptional ability of the wild type (solid bars) and mutant (open bars) AR in the four cell lines (N, MJ, YS, and ZGJ), which is represented as the mean of three repeated measures of relative luciferase unit (RLU) in the presence of 100 nM DHT (+) or absence (
) of DHT.

al activity had been impaired by the Arg840Cys mutation. Moreover, a comparison between the three patientsÕ donated cell lines, which were induced either with pT-wt-AR or pT-AR-Arg840Cys, showed that the transcriptional activity decreased as the donorsÕ maleness disorder became even more severe (open bars for cell lines from MJ to ZGJ). An interesting phenomenon observed was that the impaired transactivation of AR in the MJ cells had been remarkably recovered whenever the wild type AR was induced. This observation was interpreted in light of the fact in two aspects: (1) the fertile status of the MJ cellsÕ donor, (2) that the MJ cells showed a significantly higher transactivity than the N cells when both of them were induced with the mutant plasmids. We might anticipate that there existed some cofactors in the MJ cells which played a superior role in compensating its defective transcriptional activation due to the mutation in the AR gene.

Discussion There have been more than 300 mutations in the human androgen receptor gene [22]. Understanding the genetic effects of these mutants on the development of human maleness has been one of the active areas in human molecular genetics and reproductive physiology [20,23,24]. Many studies have focused on correlating the different mutants to different disease phenotypes of androgen insensitivity syndrome, based either on a few sporadic cases and/or on exogenous, such as COS or

CV1, cells. Distinguished from those in the literature, the present study created genital skin fibroblasts (GSFs) directly from the three patients in a large Chinese family affected by partial androgen insensitivity syndrome, in which all those affected share an identical mutation Arg840Cys in the AR [12]. These tissue donors represent the divergent pattern of the disease phenotypes, varying from morphological disorders in genital virilization to loss of fertility as shown in Table 1. Use of these materials allows us to investigate the defective effect of the AR mutant on the patientsÕ maleness disorders under their own physiological backgrounds, to compare functional features of the wild type and mutant of the gene, and to interpret the pleiotropic phenomenon of the AR mutant on an endogenous basis. We first compared the mRNA and protein expression between wild type and mutant ARs in their corresponding cell lines and neither detected a viewable change either between the normal and affected groups or among the affected groups. This may exclude the possibility of ascribing the different disease phenotypes to variations in both transcriptional and translational levels of this mutant as well as the possibility of mutations in both 5 0 -untranslated and promoter regions of the AR gene which affect the stability and/or the efficiency of the gene expression. This contrasts with the observation that expression of Arg773Cys was reduced by sevenfold at the mRNA level in GSFs of patients with complete AIS and protein expression of the same mutation was considerably decreased in COS-7 cells [25]. Our ligand-binding experiment did not detect any significant change in the maximum binding capacity of the mutant AR when compared to its wild type. However, the experiment revealed a significant decrease in binding affinity of the ligand–receptor complex in the cell lines that carried the mutant AR when compared to the normal cell line. This agrees with the fact that the Arg840Cys mutant locates within the ligand-binding domain of the AR gene. Moreover, the dissociation data, which were collected over a wide time span, reveal a clear correlation with severity in disease symptom (Fig. 2). In addition, the AR binding in the cells donated by two infertile patients is found to be thermolabile. All these may suggest that the disease causing mutation detected in this affected pedigree alters the AR-binding affinity and thermostability. By artificially introducing two AR mutants Arg840Cys and Glu743Val into the COS-7 cell line, respectively, Georget et al. [11] demonstrated correlation between the nuclear transfer capacities and distinguishable symptom grades of PAIS patients caused by these two different types of AR mutation. Our data illustrate a close correlation between the nuclear trafficking of the same AR Arg840Cys mutant in the different cell lines and the AIS severity of these cell donors who are genetically related members of the AIS family. In com-

M. Wang et al. / Biochemical and Biophysical Research Communications 335 (2005) 335–342

parison, the present experiment would have effectively controlled the background effects on the observed trafficking capacities by making use of the endogenous cell lines and it demonstrates that correlation exits not only between the wild type and mutant of AR but also between different carriers of the same AR mutant when their disease phenotype varies. This thus reveals a more complicated genotype/phenotype relationship of the AR function in patients with AIS on one hand and may suggest a useful complementary tool for grading the AR patients on the other. Like other steroid hormone receptors, the androgen receptor has two major transaction function domains [26]. Adachi et al. [21] detected the disruption of transmission of the activation signal in one of these domains in the GSF cells cultured from a patient who had normal AR gene but developed complete AIS. The mutation under our investigation locates in the androgen-binding domain other than in the activation signal domains but our data demonstrate a clear picture that the transactivation of the mutant carriers is substantially disrupted when compared to the normal subject and that the decrease in transcriptional activation is closely correlated with the disease severity of the patients: the poorer the transactivation, the more severe the disease symptom would be. The experiment was carried out by paralleling the transactivation under inducing the plasmids bearing the wild type AR with that under inducing the different plasmids bearing the AR mutant. Comparison between these experimental treatments reveals an interesting observation that there was a clear and quick recovery in the disrupted transactivation in the GSF cells, whose donor was the mutant carrier but had much milder disease symptom, when the normal AR was induced through the pT-wt-AR plasmid. This enables us to anticipate that mutation occurring in the regions other than the activation signal domains of the AR gene may also play a significant and interactive role in influencing transmission of the activation signal because, otherwise, the defects in transactivation should not have been highly associated with such a mutation in the present investigation and in McPhaul et al. [27]. In addition, there would be an effective mechanism that plays a role in regulating and compensating the defective transmission of the activation signal because, otherwise, we should not expect to see that the transactivation in MJ cells is significantly higher than in both normal and mutant cells under inducement of the pT-AR-Arg840Cys plasmid and that the disrupted transactivation in the mutant cells is recovered when the pT-wt-AR plasmids are introduced into all these cell lines, particularly the MJ cells. The present study provides a primary analysis on the functional mechanisms underlying phenotypic variation among the patients sharing an Arg840Cys mutant in the AIS pedigree. Although the variation can be explained

341

to a great extent by the altered binding property, nuclear trafficking, and transactivation of the AR mutant, the study reveals very likely existence of other interactive factors with the AR gene in contributing to the disease phenotypic variation.

Acknowledgment We are grateful to the patients for their participation and cooperation in the present study.

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