69 research outputs found
Hormonal control of spermatogenesis: expression of FSJH receptor and androgen receptor genes
FSH and testosterone are the main hormonal regulators of spermatogenesis. The actions of
androgens and FSH are mediated by their respective receptors. Receptor gene expression
(mRNA and protein). is an important determinant of hormone action. Biochemical aspects of
the regulation of androgen and FSH receptor gene expression in the testis were chosen as
the subject of the studies described in this thesis. Regulation of the expression of the receptor
genes was studied at the level of gene transcription, and at the level of mRNA and protein
expression.
In Chapters 2-4, a detailed characterization is given of the effects of FSH on androgen
and FSH receptor mRNA and protein expression in cultured immature Sertoli cells. For the
androgen receptor, these findings were extended by measurements of androgen receptor
gene transcription initiation rate in cultured immature Sertoli cells and LNCaP Oymph node
carcinoma of the prostate) cells (Chapter 5).
Preliminary results concerning a putative paracrine factor, produced by Sertoli cells and
affecting androgen receptor mRNA expression in peritubular myoid cells, are presented in Chapter 6.
The effects of testosterone deprivation iD. vivo on androgen receptor mRNA and protein
expression in the adult rat testis were examined as described in Chapter 7.ffi vitro effects of
testosterone on androgen receptor gene expression in cultured testicular cells and LNCaP
cells are described in the Chapters 2, 3 and 5.
In the General Discussion (Chapter 8) we have considered some aspects of regulation
of spermatogenesis by FSH and testosterone and have discussed them in relation to our
experimental data as well as in a broader perspective. This way, we hope that the results
which we have presented, and discussions which we have tried to initiate, may contribute to
research concerning hormonal control of spermatogenesis, now and in the futur
Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP
Elevation of intracellular calcium levels in the presence of normal
androgen levels has been implicated in apoptotic prostate cell death.
Since the androgen receptor (AR) plays a critical role in the regulation
of growth and differentiation of the prostate, it was of interest to
determine whether Ca2+ would affect the expression of androgen receptor
messenger RNA (mRNA) and protein, thus affecting the ability of androgens
to control prostate function. AR-positive human prostate cancer cells,
LNCaP, were incubated with either the calcium ionophore A23187 or the
intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin.
Subsequently, AR mRNA and protein levels were assessed by Northern and
Western blot analysis. Both A23187 and thapsigargin were found to
down-regulate steady state AR mRNA levels in a time- and dose-dependent
manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M
A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of
incubation, respectively. In contrast, control mRNA (glyceraldehyde
3-phosphate dehydrogenase) did not change significantly during the
treatments with either A23187 or thapsigargin. AR protein levels were
found to be decreased after 12 h of incubation with either 10(-6) M A23187
or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to
precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was
found to alter cell morphology within the treatment time. Cycloheximide
and actinomycin D were unable to change the calcium-mediated decrease in
AR mRNA, ruling out the necessity for de novo protein synthesis or a
change in mRNA stability. Moreover, the decrease in AR mRNA induced by
calcium does not seem to involve protein kinase C- or calmodulin-dependent
pathways, since inhibitors of these cellular components had no effect.
Nuclear run-on assays demonstrated little or no effects of either A23187
or thapsigargin treatment on AR gene transcription (8 h and 10 h). In
conclusion, these studies show that intracellular calcium seems to be a
potent regulator of AR gene expression in LNCaP cells
Regulation of inhibin βB-subunit mRNA expression in rat Sertoli cells: Consequences for the production of bioactive and immunoreactive inhibin
Abstract
In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the α-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of βB-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the βB-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of βB-subunit mRNA on the production of bioactive and immunoreactive inhibin.
During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the βB-subunit of inhibin changed markedly. The meaning of this changing ratio between βB-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of βB-subunit mRNA correlated with the in vitro production of bioactive inhibin as published earlier.
Prolonged stimulation of cultured Sertoli cells from 14-day-old rats with 4β-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the βB-subunit mRNAs, presumably by down-regulation of protein kinase C. A similar effect was obtained after addition of the calcium ionophore A23187. Concomitantly, a decreased production of bioactive inhibin was observed. Furthermore, Western blotting revealed that secretion of the 32 kDa inhibin αβ-dimer was decreased, whereas secretion of the combination of the C-terminal part with the pro-region of the α-subunit was increased.
It is concluded that the level of the βB-subunit of inhibin is rate-limiting for the production of bioactive inhibin in cultured Sertoli cells, and that its expression can be influenced by modulation of protein kinase C, and/or intracellular calcium levels
Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein
In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding
the alpha-subunit of inhibin, and the production of immunoreactive inhibin
are stimulated by follicle-stimulating hormone (FSH). In contrast, the
amount of beta B-subunit mRNA is not increased after FSH treatment of the
cells, and the ratio between bioactive and immunoactive inhibin decreases
after stimulation with FSH. These data suggest that the beta B-subunit is
the limiting factor in the production of bioactive inhibin. The aim of the
present experiments was to investigate the effect of changes in the amount
of beta B-subunit mRNA on the production of bioactive and immunoreactive
inhibin. During early postnatal testicular development, the relative
amounts of the 4.2 kb and 3.5 kb mRNAs encoding the beta B-subunit of
inhibin changed markedly. The meaning of this changing ratio between beta
B-subunit mRNAs is not clear, since both mRNAs are actively translated, as
demonstrated by polysomal analysis. The total amount of beta B-subunit
mRNA correlated with the in vitro production of bioactive inhibin as
published earlier. Prolonged stimulation of cultured Sertoli cells from
14-day-old rats with 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a
decreased expression of the beta B-subunit mRNAs, presumably by
down-regulation of protein kinase C. A similar effect was obtained after
addition of the calcium ionophore A23187. Concomitantly, a decreased
production of bioactive inhibin was observed. Furthermore, Western
blotting revealed that secr
Activin is produced by rat Sertoli cells in vitro and can act as an autocrine regulator of Sertoli cell function
Regulation of androgen receptor (AR) mRNA expression was studied in
Sertoli cells and peritubular myoid cells isolated from immature rat
testis, and in the lymph node carcinoma cell line derived from a human
prostate (LNCaP). Addition of dibutyryl-cyclic AMP (dbcAMP) to Sertoli
cell cultures resulted in a rapid transient decrease in AR mRNA expression
(5 h), which was followed by a gradual increase in AR mRNA expression
(24-72 h). This effect of dbcAMP mimicked follicle-stimulating hormone
(FSH) action. In peritubular myoid cells, there was only a moderate but
prolonged decrease during incubation in the presence of dbcAMP, and in
LNCaP cells no effect of dbcAMP on AR mRNA expression was observed. When
Sertoli cells or peritubular myoid cells were cultured in the presence of
androgens, AR mRNA expression in these cell types did not change. This is
in contrast to LNCaP cells, that showed a marked reduction of AR mRNA
expression during androgen treatment. In the present experiments,
transcriptional regulation of AR gene expression in Sertoli cells and
LNCaP cells was also examined. Freshly isolated Sertoli cell clusters were
transfected with a series of luciferase reporter gene constructs, driven
by the AR promoter. It was found that addition of dbcAMP to the
transfected Sertoli cells resulted in a small but consistent increase in
reporter gene expression (which was interpreted as resulting from AR
promoter activity); a construct that only contained the AR 5' untranslated
region of the cDNA sequence did not show such a regulation. The same
constructs, transfected into LNCaP cells, did not show any transcriptional
down-regulation when the synthetic androgen R1881 was added to the cell
cultures. A nuclear transcription elongation experiment (run-on), however,
demonstrated that androgen-induced AR mRNA down-regulation in LNCaP cells
resulted from an inhibition of AR gene transcription. The present results
indicate that in Sertoli cells and LNCaP cells, hormonal effects on AR
gene transcription play a role in regulation of AR expression. However, AR
gene transcription in these cells is differentially regulated
Transcriptional regulation of androgen receptor gene expression in Sertoli cells and other cell types
Cooperative actions of FSH and androgens on initiation, maintenance, and
restoration of spermatogenesis have been described. In the present
experiments the regulatory effects of FSH on androgen receptor (AR) gene
expression in Sertoli cells were studied. In immature rats injection of
FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of
testicular AR mRNA expression (4 h), followed by recovery to the control
level (10 h). Using cultured immature Sertoli cells, a similar transient
effect on AR mRNA expression was observed after the addition of FSH (500
ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not
prevent the rapid FSH-induced down-regulation of AR mRNA expression,
indicating that de novo protein synthesis is not required for this effect.
Furthermore, using a transcriptional run-on assay, no marked decrease in
the rate of AR gene transcription was found upon treatment of the cultured
Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term
effect of FSH or AR mRNA expression reflects a change in mRNA stability.
The AR protein level was not markedly affected by the transient decrease
in AR mRNA expression. When immature Sertoli cells were incubated with FSH
for longer time periods (24-72 h), both AR mRNA and protein expression
were increased. In Sertoli cells isolated from 15-day-old rats, this
increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli
cell
Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression
The androgen receptor (AR) is activated upon binding of testosterone or
dihydrotestosterone and exerts regulatory effects on gene expression in
androgen target cells. To study transcriptional regulation of the rat AR
gene itself, the 5' genomic region of this gene was cloned from a genomic
library and the promoter was identified. S1-nuclease protection analysis
showed two major transcription start sites, located between 1010 and 1023
bp upstream from the translation initiation codon. The area surrounding
these start sites was cloned in both orientations in a CAT reporter
plasmid. Upon transfection of the constructs into COS cells, part of the
promoter stimulated transcription in an orientation-independent manner,
but the full promoter showed a higher and unidirectional activity. In the
promoter/reporter gene constructs, transcription initiated from the same
positions as in the native gene
The rat androgen receptor gene promoter
The androgen receptor (AR) is activated upon binding of testosterone or
dihydrotestosterone and exerts regulatory effects on gene expression in
androgen target cells. To study transcriptional regulation of the rat AR
gene itself, the 5' genomic region of this gene was cloned from a genomic
library and the promoter was identified. S1-nuclease protection analysis
showed two major transcription start sites, located between 1010 and 1023
bp upstream from the translation initiation codon. The area surrounding
these start sites was cloned in both orientations in a CAT reporter
plasmid. Upon transfection of the constructs into COS cells, part of the
promoter stimulated transcription in an orientation-independent manner,
but the full promoter showed a higher and unidirectional activity. In the
promoter/reporter gene constructs, transcription initiated from the same
positions as in the native gene. Sequence analysis showed that the
promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but
one SP1 site is located at about 60 bp upstream from the major start site
of transcription. Other possible promoter elements are TGTYCT sequences at
positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505,
resembling half-sites of the glucocorticoid-responsive element (GRE).
Furthermore, a homopurine stretch containing a total of 8 GGGGA elements
and similar to sequences that are present in several other GC-rich
promoters, is located between -89 and -146 bp upstream from the major
start site of transcriptio
Regulation of androgen receptor mRNA and protein in the rat testis by testosterone
__Abstract__
Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration.
In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment.
In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered.
Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens
Phosporylation of androgen receptor isoforms
Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed
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