Neuroendocrinologia
Endocrinologia
Oncologica
Selezione dei lavori piu' recenti
Selection of most recent papers
Poletti A., Weigel N.L., McDonnell D.P., Schrader W.T. and O'Malley
B.W. A novel, highly regulated, rapidly inducible system for the expression
of chicken progesterone receptor, cPRa, in Saccharomyces cerevisia.
Gene
(1992) 114:51-58.
Poletti A., Weigel N.L. Identification of a Hormone-dependent
phosphorylation site adjacent to the DNA binding domain of the chicken
progesterone receptor.Mol. Endocrinol. (1993) 7:241.
Poletti A., Conneely O.M., McDonnell D.P., Schrader W.T., O'Malley
B.W., Weigel N.L. Chicken progesterone receptor expressed in Saccharomyces
cerevisiae is correctly phosphorylated at all four Ser-Pro phosphorylation
sites. Biochemistry (1993) 32:9563-9569.
Poletti A., Melcangi R.C., Negri-Cesi P., Maggi R., Martini L.
Steroid binding and metabolism in the LHRH-producing neuronal cell line
GT1-1. Endocrinology (1994) 135:2623-2628.
Zhang Y., Beck C.A., Poletti A., Edwards D.P., Weigel N.L. Identification
of phosphorylation sites unique to the B form of the human progesterone
receptor: in vitro phosphorylation by Casein Kinase II. J. Biol. Chem.
(1994) 269:31034-31040.
Zhang Y., Beck C.A., Poletti A., Edwards D.P., Weigel N.L. Identification
of a group of Ser-Pro motif hormone inducible phosphorylation sites in
human progesterone receptor. Mol.Endocrinol. (1995) 9:1029-1040.
Poletti A., Celotti F., Motta M., Martini L. Characterisation
of rat 5alpha-reductases type 1 and type 2 expressed in yeast Saccharomyces
cerevisiae Biochem. J. (1996) 314:1047-1052.
Guarna A., Poletti A., Catrambone F., Danza G., Marrucci A.,
Serio M., Celotti F., Martini L.Synthesis of a chemiluminescent probe useful
for the purification of steroid 5alpha-reductase. Bioorg. Med. Chem.
Lett. (1996) 6:1997-2002.
Moretti R.M., Montagnani-Marelli M., Dondi D., Poletti A., Martini
L., Motta M., Limonta P. Luteinizing hormone-releasing hormone (LHRH) agonists
interfere with the stimulatory actions of the epidermal growth factor in
human prostatic cancer cell lines, LNCaP and DU145. J. Clin. Endocr.
Metab. (1996) 81:3930-3936.
Poletti A., Rabuffetti M., Martini L. Effect of suramin on the
biological activity of the two isoforms of the rat 5alpha-reductase. Steroids
(1996)
61: 504-505.
Negri-Cesi P., Poletti A., Celotti F. Metabolism of Steroids
in the Brain: a New Insight into the Role of 5alpha-Reductase and Aromatase
in Brain Differentiation and Functions. J. Steroid Biochem. Mol.
Biol. (1996) 58:455-466
abstract
Poletti A., Negri-Cesi P., Melcangi R.C., Colciago A., Martini
L., Celotti F. Expression of androgen-activating enzymes in cultured cells
of developing rat brain. J. Neurochem. (1997) 68:1298-1303.
abstract
Zhang Y., Beck C.A., Poletti A., Clement IV J.P., Prendergast
P., Yip T.-T., Hutchens T.W., Edwards D.P., Weigel N.L. Phosphorylation
of human progesterone receptor by Cyclin-dependent kinase 2 on three sites
that are authentic basal phosphorylation sites in vivo. Mol. Endocrinol.
(1997)
11:823-832
Poletti A., Celotti F., Rumio C., Rabuffetti M., Martini L. Identification
of type 1 5alpha-reductase in myelin membranes of male and female rat brain.
Mol.
Cell. Endocr. (1997) 129:181-190.
Celotti F., Negri-Cesi P., Poletti A. Steroid metabolism in the
mammalian brain: 5alpha-reduction and aromatization. Brain Res.Bull.
(1997) 44:365-375.
Negri-Cesi P., Poletti A., Colciago A., Magni P., Martini P.,
Motta M. Presence of androgen-activating enzyme in the human prostatic
cancer cell line LNCaP and in Begnin prostatic hyperplasia. The Prostate.
(1998)
34:283-291.
Poletti A., Negri-Cesi P., Rabuffetti M., Colciago A., Celotti
F., Martini L. Transient expression of the type 2 5alpha-reductase isozyme
in the brain of the late fetal and early post-natal life. Endocrinology
(1998)
139: 2171-2178.
abstract
Poletti A., Coscarella A., Negri-Cesi P., Colciago A., Celotti
F., Martini L. The 5alpha-reductase isozymes in the Central Nervous System.
Steroids
(1998) 63:246-251.
Poletti A., Martini L. Androgen-activating enzymes in the Central
Nervous System. J. Steroids Biochem. Mol. Biol. (1998) 65:295-299
abstract
Negri-Cesi P., Colciago A., Poletti A., Motta M.5alpha-reductase
isozymes and aromatase are differentially expressed and active in androgen-independent
human prostate cancer cells. The Prostate (1999) 41:224-232.
Simeoni S., Mancini M.A., Stenoien D., Marcelli M., Weigel N.L., Zanisi
M., Martini L., Poletti A. Motoneuronal cell death is not correlated
with aggregate formation of androgen receptors containing an elongated
polyglutamine tract. Hum. Mol. Genet. (2000) 9:133-144.
abstract
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Selection of most recent abstracts
5th International Conference on Hormones, Brain, and Behavior
Torino, Italy. August 25-30, 1996.
Gene expression of the 5alpha-reductase in the rat brain.
Poletti A., Paola Negri-Cesi, Monica Rabuffetti, Fabio Celotti,
Luciano Martini.
abstract
16th Joint Meeting of the "British Endocrine Societies"
Harrogate, North Yorkshire, U.K. April 7-10, 1997.
Allopregnegnolone must be converted into 5alpha-dihydrotestosterone
to regulate the transcriptional activity of progesterone receptor expressed
in yeast Saccharomyces cerevisiae.
Rabuffetti M., Motta M., Celotti F., Martini L., Poletti A.
abstract
XVIII Meeting of the "International Study Group for Steroid Hormones"
Rome, Italy. November 29-December 2, 1997.
The 5alpha-reductase isozymes in the central nervous system.
Poletti A.
abstract
37th Annual Meeting of "The American Society for Cell Biology"
Washington, D.C. USA. December 13-17, 1997.
Adverse effects of the elongated polyglutamine tract of the androgen
receptor in transfected motoneuronal cells.
Poletti A., Marcelli M., Celotti F., Martini L.
abstract
IV European Congress of the "European Federation of Endocrine Societies"
Sevilla, Spain. May 9-13, 1998.
Androgens may directly act on LH-RH-synthesising neurons.
Poletti A., Coscarella A., Martini L., Zanisi M.
abstract
Xth International Congress on Hormonal Steroids
Quebec City, CA. June 17-21, 1998.
Androgen-activating enzymes in the central nervous system.
Poletti A.
abstract
80th Annual Meeting of the"Endocrine Society"
New Orleans, LA, USA. June 24-27, 1998.
Androgen receptor containing polyglutamine tract elongation induces
apoptosis in motoneuronal cells
Poletti A., Coscarella A., Marcelli M., Celotti F., Martini
L.
abstract
GENE EXPRESSION OF THE 5a-REDUCTASE IN THE RAT BRAIN.
A. Poletti*, P. Negri-Cesi, M. Rabuffetti, F. Celotti, L. Martini.
Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano, Italy.
In rodens, testosterone (T) controls the sexual differentiation of the
peripheral androgen-dependent structures, is responsible of the male sexual
differentiation of the brain and of the maintainance of male sexual behaviour.
These actions are essentially due to the formation of active metabolites
of T, formed respectively by the enzymes aromatase (e.g., estradiol) and
5alpha-reductase (e.g., dihydrotestosterone, DHT). Many experimental data
suggests that estradiol is an important mediator of T actions on sexual
functions of the rat brain; however, a specific role for DHT has also been
postulated. Two 5alpha-R isoforms, with different biochemical properties
and functional roles, have been identified and cloned. The 5alpha-R type
1 is present in a wide variety of tissues, while the 5alpha-R type 2 appears
to be present only in androgen-dependent structures. No data are available
on the expression of the two 5alpha-R isoforms in the rat brain, which
possesses a considerably high level of 5alpha-R activity. In this study,
the gene expression of the two isozymes has been analysed in the rat brain
by the reverse transcription-polymerase chain reaction (RT-PCR) technique,
followed by Southern analysis. Particular attention has been given to the
control of the 5alpha-R expression in the perinatal life, a period in which
the endocrine-priming of the male rat brain occurs. RT-PCR analysis has
been performed using specific sets of oligoprimers on the total RNAs obtained
from brains of fetal, postnatal and adult male and female rats. The results
have shown that the mRNA for 5alpha-R type 1 is detectable, in the rat
brain, at all the ages examined, i.e. at gestational days (GD) 14, 16,
18, at postnatal (PN) days 2,7,10,14,18,22 and 28, and in adulthood. On
the contrary, the expression of the 5alpha-R type 2, analysed in the same
brain samples, is almost undetectable at GD14 and 16, increases progressively
after GD18, and shows a peak of maximal expression at PN2. After this,
the gene expression of this isozyme decreases gradually, is still detectable
at PN 28, but becomes almost undetectable in adulthood. The data demonstrate
that the 5alpha-R type 1 is constantly expressed in the rat central nervous
system (CNS), while the type 2 isoform is expressed only transiently in
the late fetal and in the early post-natal life. The CNS expression of
5alpha-R type 2 appears to correlate with the secretion patterns of T from
the rat fetal testis, suggesting a possible involvement of androgens in
its control. Also, in other studies performed in cultured rat hypothalamic
neurons the expression of 5alpha-R2 was found to be testosterone-dependent.
However, treatment of pregnant rats with an antagonist of the androgen
receptor (flutamide) was unable to counteract the peak of 5alpha-R2 in
the brain of newborn male or female rats studied independently (sex assignement
performed by determining the presence of the SRY gene located of the Y-chromosome
using PCR). The data suggest that other factors are involved in the regulation
of this isozyme. A possible involvement of 5alpha-R2 in the process of
sexual differentiation of the brain is also postulated. Grants funding
ACRO95.00395.PF39, Aging95.00470PF40, FATMA95.00868PF41 is gratefully acknowledge.
ALLOPREGNENOLONE MUST BE CONVERTED INTO 5a-DIHYDROPROGESTERONE
TO REGULATE THE TRANSCRIPTIONAL ACTIVITY OF PROGESTERONE RECEPTOR EXPRESSED
IN YEASTS SACCHAROMYCES CEREVISIAE.
M Rabuffetti, M Motta, F Celotti, L Martini, A Poletti.
Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano, Italy.
Allopregnenolone (THP) locally derived from progesterone in the brain,
is a potent positive modulator of the GABAA receptor, which exerts anxiolytic
and hypnotic activities. It is not known whether THP is also active in
inducing the transcriptional activity of the progesterone receptor. To
verify this hypotesis we have co-transformed yeasts Saccharomyces cerevisiae
with two different plasmids containing: a) the cDNA encoding the chicken
progesterone receptor (cPRB) linked to the copper-responsive yeast metallothionein
promoter (CUP1); b) a progesterone responsive element (PRE) inserted into
the CYC1 promoter fused to the lacZ gene. The resulting b-galactosidase
activity, measured after addition of P, DHP or THP, is proportional to
the levels of transcriptional activation of cPRB. The results show that
P induces the lacZ gene in a dose dependent manner; DHP is also very efficient
with a bell shape profile and a peak of activity at doses of 10-8 M. Surprisingly,
THP is also active, but only at higher concentrations. At the highest dose
(10-6 M), DHP and THP possess the same transcriptional activity. Hormone-binding
experiments have shown that DHP is a high affinity ligand for cPRB, while
THP does not bind to the receptor. Incubation of [3H]THP with yeast cell
lysates produces [3H]DHP, demonstrating the presence of an active 3a-hydroxysteroidoxidoreductase
(3a-HSOR). These results indicate that THP exerts its progestational action
after oxidation into DHP by the 3a-HSOR. Moreover, alfaxalone, an anaesthetic
steroid which activates the GABAA receptor, but cannot bind the cPRB and
it cannot be converted into DHP, is unable to activate PR-mediated transcription
of the lacZ reporter gene. Taken together these results indicate that THP
either interacts with transmitter-gated ion channels (GABAA receptor) or
intracellular steroid receptors following retroconversion into DHP and
in this way regulates neuronal function. Grants funding MURST, ACRO95.00395.PF39,
Aging95.00470PF40, FATMA95.00868PF41 are gratefully acknowledge.
THE 5a-REDUCTASE ISOENZYMES IN THE CENTRAL NERVOUS SYSTEM.
A. Poletti.
Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano, Italy.
The 5a-reductase (5a-R) activates several 3keto-D4 steroids to more
potent derivatives which may also acquire new biological actions. In males,
testosterone (T) is the main substrate and gives rise to the most potent
natural androgen dihydrotestosterone (DHT). In females, progesterone (P)
is reduced to dihydroprogesterone (DHP), a precursor of tetrahydroprogesterone
(THP) a natural anxyolytic/ anesthetic steroid. Other possible substrates
are some gluco- and minero-corticoids. Two isoforms of the 5a-R, with limited
degree of homology, different biochemical properties and tissue distribution,
have been cloned: 5a-R type 1 and type 2. The two recombinant isoforms
expressed in yeast are differentially localized within the cells possibly
reflecting different physiological roles. In androgen-dependent structures
DHT is almost exclusively formed by the 5a-R type 2. The 5a-R type 1 (widely
distributed in the body with the highest levels in liver) might be involved
in steroid catabolism. In the brain, the role of the two isozymes is unknown.
By evaluating the gene expression of the two 5a-Rs in the whole rat brain
we have found that the type 1 isoform is constantly expressed throughout
life, while the 5a-R type 2 is expressed at high levels only in the perinatal
period. The exposure 'in utero" to the antiandrogen flutamide counteracts
the increased expression of the type 2 gene at time of birth only in male
rat brain. In adulthood the type 2 gene is expressed, at low level, in
particular brain structures, like the hypothalamus, the hyppocampus, and
at high levels in the spinal cord. Among the various brain cell populations,
both the glial and the neuronal cells express the type 1 gene; the 5a-R
type 2 gene is expressed normally in neuronal hippocampal cells, while
it becames detectable in neuronal hypothalamic cells only after induction
with T or TPA. Finally, a particular type of hypothalamic neurons (the
GT1-1 cells, which originates from LHRH producing system and which secrete
the hypothalamic hormone, expresses both 5a-R isoforms, but apparently
only the 5a-R type 2 is efficiently translated into a protein. The colocalization
of 5a-R type 2 and of the androgen receptor in GT1-1 cells suggests that
these neurons are potential androgen-target structures. The physiological
significance of the data summarized will be discussed. Grants funding ACRO95.00395.PF39,
CNR CT96.03105.CT04, Aging95.00470PF40, FATMA95.00868PF41 is gratefully
acknowledge.
ADVERSE EFFECTS OF THE ELONGATED POLYGLUTAMINE TRACT
OF THE ANDROGEN RECEPTOR IN TRANSFECTED MOTONEURONAL CELLS.
((A. Poletti, M. Marcelli¡, F. Celotti, L. Martini))
Istituto di Endocrinologia, Università di Milano, Italy; ¡Dpt.
of Medicine, Baylor College of Medicine, Houston, TX, USA.
Spinal and bulbar muscular atrophy (SBMA) is an X-linked recessive disease
in which an abnormal increase of the number of (CAG)n repeats has been
detected in the first coding exon of the androgen receptor (AR) gene. This
results in an elongation of the N-terminal polyglutaminic (polyGln) tract
normally present in AR, which becomes at least twice as long as usual.
The physiological function(s) of the polyGln domain in wild type AR and
the mechanisms by which this expansion leads to neurodegeneration are unknown.
It is important to underline that in the CNS many neurons express AR, while
cell death occurs only in some spinal motoneurons. Interestingly, a polyGln
tract elongation has been found in a variety of proteins responsible for
other neurodegenerative diseases (i.e., Huntington's disease, spinal-cerebellar
ataxia I, etc.) indicating a common neurotoxic mechanism. To determine
the physiopathological funtion(s) of the elongated polyGln tract in the
AR, we have stably transfected immortalized motoneurons (NSC34 cells kindly
provided by Dr. N.L. Cashman) with a number of AR cDNA's. The plasmids
used expressed wild type AR (containing 21 Gln's), an AR devoid of the
polyGln tract (Gln0), and an AR construct containing an elongated polyGln
tract (Gln43). The resulting trasfected cell lines were named NSC/WT-AR,
NSC/Gln0 and NSC/Gln43, respectively. The expression of the recombinant
ARs within the clones of the motoneuronal cells was characterised with
RT-PCR, using primers comprising the polyGln tract of AR. Growth curves
have been obtained on the various cellular clones using MTT analysis, and
cell viability has been tested using LDH assay. The results indicated that
NSC/Gln43 had a reduced growth rate when plated at low density (8000 cells/cm2),
but behaved as NSC/Gln0 or NSC/WT-AR clones when plated at higher density
(more than 15000 cells/cm2). DNA laddering analysis indicated that the
reduced survival observed in motoneuronal clones transfected with AR-Gln43
is probably linked to apoptosis. Interestingly the survival was increased
by testosterone treatment, possibly because of a stabilizing effects of
the hormone on its receptor. These preliminary data indicate that the model
selected may be useful for the study of neurodegeneration linked to polyGln
tract elongationGrants funding TeleThon-Italy (A.096) MURST, ACRO95.00395.PF39,
Aging95.00470PF40, FATMA95.00868PF41, CNR (CT96.03105.CT04 are gratefully
acknowledge
ANDROGENS MAY DIRECTLY ACT ON LH-RH-SYNTHESISING NEURONS.
A. Poletti, A. Coscarella, L. Martini, M. Zanisi.
Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano, Italy.
Modulation of LH-RH secretion is primarly exerted by hormonal steroids
acting on the hypothalamic-pituitary-gonadal axis. This control may occur
either directly via steroid receptors localized within the LH-RH synthesising
neurons and/or indirectly via neurotransmitter/neuropeptide pathways. Immortalized
LH-RH secreting cell lines, like the GT1-1 cells have been shown to possess
androgen binding sites (ABS) and the 5a-reductase (5a-R, the enzyme which
transforms testosterone into the more potent androgen dihydrotestosterone,
DHT). Two isoforms of the 5a-R have been cloned: 5a-R type 1, widely distributed
in the body, and 5a-R type 2, mainly localized in androgen target structures.
The aim of this study has been that of determining whether the ABS observed
in GT1-1 cells represents a 'classical' intracellular androgen receptor
(AR), and whether both 5a-R isoforms are present in GT1-1 cells. The gene
expression of AR and of the two isozymes has been analysed by RT-PCR. The
mRNA coding for the 'classical' AR is highly expressed in GT1-1 cells,
since a specific product of amplification, corresponding to that found
analysing the total RNA from mouse seminal vesicle (used as positive control)
was observed; no transcript was detectable in a mouse neuroblastoma cell
line (N18TG2) used as negative control. Both 5a-R isoforms are expressed
in GT1-1 cells, while only the type 1 isoform was detectable in neuroblastoma
N18TG2 cells and in mouse liver. However, studies on the variation of the
activity of the 5a-R as a fuction of pH in GT1-1 cells have shown a peak
of activity around pH 5.5, which represents the optimum for the 5a-R type
2. In addition, the affinity for testosterone was very similar to that
observed for the recombinant type 2 isozyme expressed in yeasts. These
data indicate that GT1-1 cells express both 5a-R isoforms, but produce
only the type 2 enzyme. The presence of the AR and of the enzyme catalysing
testosterone metabolism support the concept of a direct action of these
steroids on LH-RH synthesising neurons. Grants from MURST, ACRO95.00395.PF39,
Aging95.00470PF40, FATMA95.00868PF41 are gratefully acknowledge
ANDROGEN-ACTIVATING ENZYMES IN THE CENTRAL NERVOUS SYSTEM.
A. Poletti*.
Istituto di Endocrinologia dell'Università degli Studi di Milano,
via Balzaretti 9, 20133 Milano, Italy. E-mail: Angelo.Poletti@unimi.it
In the rat brain several steroids can be converted by specific enzymes
to either more potent compounds or to derivatives showing new biological
effects. One of the most studied enzyme is the 5a-reductase (5a-R), which
acts on 3keto-D4 steroids. In males, testosterone is the main substrate
and gives rise to the most potent natural androgen dihydrotestosterone.
In females, progesterone is reduced to dihydroprogesterone, a precursor
of allopregnanolone a natural anxyolytic/ anesthetic steroid. Other substrates
are some gluco- and minero-corticoids. Two isoforms of the 5a-R, with limited
degree of homology, have been cloned: 5a-R type 1 and type 2. The 5a-R
type 1 possesses low affinity for the various substrates and is widely
distributed in the body with highest levels in the liver; in the brain,
this isoform is expressed throughout life, and it is not controlled by
androgens. Specific 5a-R type 1 mRNA is detectable both in neuronal and
glial cells (including oligodendrocytes), but the translated isozyme is
mainly concentrated in myelin membranes where it might be involved in steroid
catabolism. The 5a-R type 2 shows high affinity for the various substrates,
a peculiar pH optimum at acidic values and is localized in androgen-dependent
structures. In the rat brain, the type 2 isoform is expressed at high levels
only in the perinatal period and is differentially controlled by androgens
in the two sexes, as shown by the different responses to the antiandrogen
flutamide. In adulthood, the type 2 gene is expressed at low levels in
particular brain structures, like the hypothalamus and the hippocampus.
Surprisingly, high levels of 5a-R type 2 mRNA are found in spinal cord
where is confined in motoneurons. The physiological significance of these
and additional data will be discussed. Grants funding ACRO95.00395.PF39,
CNR CT96.03105.CT04, Telethon (Italy) A.096 are gratefully acknowledge.
Not available