Estrogen Stimulates Peptidylarginine Deiminase Biosynthesis in the Primary Culture of Dispersed Rat Anterior Pituitary Cells(Endocrinology)

4 Pages·1991·9.5 MB·English

by  Saburo Nagata

ZOOLOGICAL SCIENCE 8: 729-732 (1991) 1991 Zoological Society ofJapan
[COMMUNICATION]
Estrogen Stimulates Peptidylarginine Deiminase Biosynthesis
in the Primary Culture of Dispersed
Rat Anterior Pituitary Cells
Saburo Nagata and Tatsuo Senshu
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology,
Sakaecho 35-2, Itabashi-ku, Tokyo-173, Japan
—
ABSTRACT Effects ofsex steroids on peptidylarginine deiminase biosynthesis were examined in the
primary culture of dispersed rat anterior pituitary cells. Without steroids, cells from 7 week to 6
month-old rats incorporated ?>S]-amino acids into the immunoprecipitable enzyme protein. No
[
incorporation was detected in cells from 4 week-old rats. The cells responded to 10nM 17^-estradiol
(E:) by an increase in the rate of enzyme biosynthesis regardless o\ sex and age of the rats.
Diethylstilbestrol was as effective as E2, whereas testosterone and progesterone had no effect. Dot
blot hybridization analysis demonstrated an increase in the znzyme mRNA level in the I stimulated
cells. The results support that estrogen up-regulates the peptidylarginine deiminase expression in the
rat pituitary.
acteristics from normal pituitary cells. Therefore,
INTRODUCTION
the present study has been done using primary
Peptidylarginine deiminase (protein-L-arginine cultures of dispersed rat anterior pituitary ceils
iminohydrolase: EC 3.5.3.15) converts arginine The results confirm the data obtained in our pre-
residues in proteinstocitrulline residues (reviewed vious in vitro and in vivo studies [6, 7). suggesting
in [1]). The enzymes reported thus far can be that estrogen up-regulates the expression of pep
classified into 3 types based on the differences in tidylarginine deiminase in the rat pituitary.
their tissue distribution, antigenicity and substrate
specificity [2]. Among them, the muscle type MATERIALS AND METHODS
enzyme distributes in a widest variety of tissues
which differ morphology and function, and has Wistar rats derived from Japan SIX (Shi/uoka.
been best characterized biochemically (2-4]. It is Japan) were bred in the animal facilits Ol the
present in lactotrophs of the mature female rat IokyO Metropolitan Institute Ol ( rerontOlOg) . and
pituitary and its content varies under the influence immature (4 weeks ot age) to mature (4-0 months)
Oi estrogen [5, 6], suggesting possible involvement males and fem.ties were used. Rats were decapi-
of the enzyme in female specific activity of lacto- tated under light ether anesthesia, pituitanes were
trophs. We have previously reported that estrogen rem<»\ed asepticalK . and the anterior lobes were
stimulates the enzyme biosynthesis in a clonal rat isolated under a dissecting miCTOSCOpe, I he cells
pituitary cell line [7]. However, such an estab- were dispersed With trypsin-! I) I A solution (( rlli
lished cell line mav have different functional char- CO, Grand Island. NY) according to the previous
k described method |x|. After washing writh the
Accepted February 20. 1991 medium, the cells Irom 6 10 lobes were rCSUS-
Received December 27. 1990 pended at 4 5X10 cells ml. and dispensed at ;
730 S. Nagata and T. Senshu
ml/well into 6 well plates (Corning Glass Works, 1 2 3
Corning, NY). The culture medium was a phenol
red-free DMEM/F-12 mixture (Sigma, St. Louis,
Mo.) supplemented with 10% normal horse
94
serum, 2.5% fetal bovine serum, 2.85 g/1 of —
NaHC03 and 1.5 g/1 ofglucose. Serawere pretre- 67^
ated with dextran-coated charcoal to deplete free
steroids [7j. After incubation for 4 days in 5% 43
CO2 and95% airat37°C, thecellsweretreatedfor
24 hr with steroids as described previously [7].
Cells were biosynthetically labeled with 35S]- 30
[ jm
amino acids as described previously [7]. Briefly,
monolayered cells in each well were incubated for
20.1*
lhr with 3.7 MBq Tran 35S-Label (ICN, Costa
Mesa, Ca.) in 1 ml methionine-free DMEM/F-12 Fig. 1. Immunoprecipitation of 35S]-labeled peptidy-
medium. After incubation, the cells were lysed in larginine deiminase, GH and P[RL. Anteriorpituit-
1% NP-40, ImM PMSF, 150mM NaCl, 10mM arycellsfrom6-month oldfemalesculturedwithout
Tris-HCl, pH7.2, andthe lysateswere centrifuged Paedpdteiddysltaerrgoiindinweerdeeilmaibnealseed w(ilatnhe[315)S,]-GaHmin(olaanceids2.)
for 20min at 10000Xg. The supernatants were and PRL (lane 3) in the cell extractwere immunop-
normalized for radioactivity incorporated into recipitated and those from about 2X105 cells (lane
TCA-precipitable proteins and then immunopre- 1) or 1X104 cells (lanes 2 and 3) were analyzed by
cipitated with rabbit antisera against rat muscle SDS-PAGE. Molecular weight of marker proteins
were given as kd.
peptidylarginine deiminase, growth hormone
(GH) orprolactin (PRL) andproteinA-Sepharose
CL-4B (Pharmacia, Uppsala, Sweden). The im- (24kd). Whencellswere incubatedwith 10nME2
munoprecipitates were analyzed by SDS- or DES, incorporation oflabeled amino acids into
polyacrylamide gel electrophoresis (SDS-PAGE) the enzyme and RRL increased as compared with
followed by fluorography [7]. the control culture (Fig. 2). Testosterone had no
Total RNA fractions from cultured cells were appreciable effect, and progesterone seemed
prepared by the guanidinium/CsCl method and slightly suppressive on the incorporation into both
analyzed by dot blot hybridization technique as the enzyme and PRL. None of these steroids
described previously [9]. Probes used for hybri- affected the incorporation into GH. Theobserved
dization were peptidylarginine deiminasse cDNA effects ofsteroids on PRL biosynthesis are consis-
[4] and chicken actin cDNA (Oncor, Gaith- tent with those reported previously [10].
ersburg, Md.) which were labeled using [32P]- Effects of E2 on the peptidylarginine deiminase
dCTP (New England Nuclear, Boston, Ma.) and biosynthesis were examined similarly in cells from
the random priming DNA labeling kit (Boehrin- female and male rats at various ages (Fig. 3).
ger, Mannheim, Germany). Without E2, no enzyme biosynthesis could be
detected in cells from 4 week-old males and
females. However, the enzyme biosynthesis was
RESULTS AND DISCUSSION
detectable in these cells upon E2 stimulation. The
First, anterior pituitary cells from 6 month-old enzyme biosynthesis was detectable in cells from7
females were cultured without steroid and labeled week-and4month-oldratsofbothsexes, although
with 35S]-amino acids. Figure 1 shows a fluoro- its activity was slightly lower in male cells than in
[
graph of immunoprecipitates prepared from the female cells. The E2 treatment increased the
cell extract, demonstrating incorporation of enzyme biosynthesis in these cells. It caused 5 to
labeled amino acids into peptidylarginine deimi- 10-foldincrease intherateofenzymebiosynthesis,
nase (75 kilodalton (kd)), GH (22kd) and PRL as estimated by liquid scintillation counting ofthe
Rat Pituitary Peptidylarginine Deiminase 731
1 2 3 4 5 immunoprecipitates (data not shown)
—4t<*— To sec the effect ofestrogen on the peptidylargi-
A •• nine deiminase mRNA level, anterior pituitary
•
cells from 5 to 6 month-old females were cultured
B
for 24 hr with or without 10 nM F: and the total
RNA
C fractions were analyzed by dot blot hybri-
dization. Figure 4 shows about 4 to 8-fold increase
I Effects of various steroids on biosynthesis of in the enzyme mRNA level by F; treatment.
peptidylarginine deiminase (A), PRL (B) and GH However, no detectable change was found in the
(C). Anterior pituitary cells from 6-month old mRNA
actin levels.
female rats were preincubated for 24h in the
medium alone (lane l) orwith ID nM ofE: (lane 2). These results agree with our previous data in-
DES (lane 3), testosterone (lane 4) or progesterone dicating that estrogen stimulates peptidylarginine
(Lane 5), were labeled with ("S]-amino acids, and deiminase biosjnthesis m a clonal pituitary cell line
the immunoprocipitates were analyzed by SDS- [7]. It has been well established that the plasma
PAGE.
estrogen level variesduring the estrous cycle ofthe
12 3 4 female rat [11). Therefore, variation of the en-
zymecontentsofthe pituitary [6| and the uterus [5]
A *» during the estrous cycle may be, at least in part,
brought about by the direct action oi estrogen on
B
-•-- these tissues. From the present and our previous
C studies [7] the stimulatory action of estrogen on
the enzyme synthesis ma\ be most likck mediated
Fig 3. Effects of E: on peptidylarginine deiminase by increased transcription in a subset of cells.
biosynthesisin anteriorpituitarycellsfrom maleand However, it may be also mediated in part by
females rats at various ages. Cells were obtained
fromfemales(lanes 1 and2)ormales(lanes3and4) selective proliferation ofa heterogeneous pituitary
at 4 weeks (A). 7 weeks (B) and 4 months (C) of cell population.
ages and incubated for 24hr with (lanes 2 and 4) or Our previous study have shown that both male
without (lane 1 and 3) 10nM E2 The cells were and female rat pituitariescontain barely detectable
labeled and peptidylarginine deiminase was im- amounts of peptidylarginine deiminase at 3 weeks
munoprecipitated for SDS-PAGE analysis.
ofage [6]. The enzyme content of female pituitar-
RNA ies markedly increases b\ 3 months showing an
1 2 1 2 (M9) estrous cycle-related variation, whereas it remains
• • • 5 low in males. The present findings on the enzyme
biosynthesis in E^untreated cells are consistent
• •
• 2.5 with this ontogenic appearance of the enzyme in
• • • 1 .3 the female pituitary. However, cells from 7 week-
and 4 month-old rats were found to synthesize
• • 0.6 substantia] amounts ot the enzyme regardless ol
A » B 0.3 sex I his apparent difference suggests some male
specific control may be operating //; vivo to sup-
Fig tm4.oR6NEAfmfoenlcettvshe-loofolfdEa;nfoteenmraitloheresppiwetpeutiritedaryyliancrcegulilbsnaitne(edeldlfesoirmfir2no4amsher5 preWsasttahneabeenzyeimealex[pl>r|eshsaivoen rbeycepnittlujitarre)pocreltlesd. an
wtoittahl(lRaNneA2)forracwtiiotnhsoutwe(rleaneanla)ly1z0endMb\1 ,doatndbtlhote ninitneeredsetiimnignavsaeriamtRioNnApaltetveerlninoffetmhaelepetpattipdiytluairtgaii
hybridization. Blots were first hybridized with a ies around the estrous cycle I he mRNA level
[,:P]-labeledpeptidylargininedeiminasecDNA (A), starts increasing at metesterus attaining th<
and then the probe was stripped by immersing for 3 imum level at diestrus. while the en/\me
min in boiling water for reprobing with I labeled
chicken actin cDNA (B) attains its maximum level between DTOeStrUS .md
732 S. Nagata and T. Senshu
estrus [6]. This shows, a sharp contrast to the in REFERENCES
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