Vol. April
184,
No.
BIOCHEMICAL
1, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1992
231-238
Endothelin-1 Stimulates c-fos mRNA Expression and Acts as a Modulator on Cell Proliferation of Rat FRTLS Thyroid Cells Megumi Miyag;:,
Toshio Tsushima’, OsamuIsoxakf’, , Kaxuo Shizume2 and Mariko Arai
Hiroshi
of Clinical Endocrinology, ‘Department of Medicine, Institute Tokyo Women’s Medical College and ‘Institute for Growth Science, 8-l Kawada-cho, Shinjuku-ku, Tokyo, 162, JAPAN Received
February
29,
1992
SUMMARY In FRTL5 thyroid cells, endothelin(EI’)-1 alone had no effect on DNA synthesis but caused a transient increase in c-fos mRNAlevels and stimulated IGF-I induced DNA synthesis and cell proliferation. By contrast, ET-1 inhibited the stimulator-y effects of TSH actions on DNA synthesis, cell proliferation and c-AMP production. 8-Bromo-CAMP-induced DNA synthesis was also inhibited by ET-I, suggesting that ET-1 exerts its inhibitory effects at step(s) involving CAMP production and post CAMP pathway. ET-l-induced suppression of TSH actions were reversed by a C-kinase inhibitor,H-7. These results suggest that the effect of KT on functions of FRIYL5cells is, at least, in part mediated by C-kinase dependent pathway. 0 1992 Academic Press,1°C.
Kndothelin(ET) medium of porcine
was originally aortic
isolated
endothelial
of the most potent vasoconstrictor has been reported
glomerular
thyroid
In addition,
(5).
tells(3),
Furthermore,
via interaction
we investigated
smooth muscle
osteoblastic
tells(4)
we have found that KT
epithelial
with EI’ receptors(6).
the effects
cell
cells
In the present
of ET-1 on growth of a rat
line(FRTL5 cells).
1 modifies growth of FRTL5 cells
it
proliferation
TSH-induced iodine metabolism in porcine thyroid
in culture study,
such as vascular
mesa&al
or Swiss 3T3 fibroblasts inhibits
peptides(1).
cells
culture
and shown to be one
that ET is able to induce cell
in a number of cultured tells(2),
cells
from the
We show here that W-
stimulated
by TSH or insulin-
like growth factor(IGF)-1.
MATERIALSAND METHODS Materials ET-1 and ET-2 were purchased from Peptide Institute Inc,(Osaka, Japan) and other chemicals or hormones were from Sigma Chemical Co.(St.Louis, MO). 0006291X/92
231
$1.50
Vol.
184,
No.
1, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cell culture FRTI.5 thyroid cells were kindly provided by Dr.L.D. Kohn NIH, Bethesda, MD(7) and used between the third and 20th pa&age. Cells were grown in Coom’s modified Ham’s F-12 medium, supplemented with 5% calf serum and a six-hormone mixture which contained bovine TSH(lmU/ml) , insulin (W, cortisol(0.4ng/ml), transferrin(5pg/ml), glycyl-l(l~riW), histidyl-L-1-lysine acetate(lOng/ml) ,and somatostatin(lOng/ml) (8). When cells reached confluence, medium was changed to 5H(without TSH) or 4H(without TSH and insulin) medium depending on the purpose of experiments. All assays were performed in triplicate and were repeated on at least three separate occasions with different batches of cells. 3H-thymidine incorporation into DNA The assay was performed as described previously(9). After replaced in 5H medium for 2 days, test materials and 3H-thymidine(0.1 pCi/well) were added to the culture medium and cultured for 3 days. 3H-thymidine incorporated into TCA-insoluble cellular fractions was counted with a Bcounter. Cell numbers were counted by a coulter counter(Coulter Electronics, Hialeah, FL) _ Assays were performed as previously CAMP levels in cells descrlbed(l0) The medium was shifed to 5OOpl of Hanks’ balanced salt solution’with 0.5 mM 3-isobutyl-1-methyl-xanthine(IBMX) and 20 mM HEPES(pH7.4) containing the indicated concentration of TSH with or without endothelin. After incubation at 37 C for 2 h, the reactions were terminated by adding 5OOpl of 20% TCA. The CAMP content in the extract was determined by RIA using commercial kits(New England Nuclear/ Dupon, Boston, MA) c-fos mRNA levels Total cellular RNA was isolated essentially d b d b Chirgwin et al.(ll). For Northern blot analysis EA ?set w&e electrophoresed in 1% agarose gels containing 0.66 M formaldehyde and blotted on nylon filters. The filters were hybridized to v-fos probe; Oncor(Gaithersburg,MD) (2-4 ~10~ cpm/ml each) and then subjected to autoradiography. Hyblidization and washing were performed as previously described(l2); final washings were carried out at 65 C in 1 x SSPE(20 x SSPE is 2.9H NaCl, 0.2 M NaH,PO,, O.OlM NaOH, and O.OlM EIYl’A). Statistical analysis Statistical evaluation was performed by Student’s t-test when variation of the data was uniform or by Cochran-Cox’s test when variation was not uniform. P values less than 5% were considered significant. Unless otherwise indicated, all values presented represent the meantSD.
RESULTS ET-l
alone
replication cultured
0.5%
calf
in
basal
serum).
changed by ETmRNA levels
no
c-fos
1.
However,
between
reported
1O-*c
medium (without
and
lO-*M ET-1 or as shown
significantly
its
232
and with also
isopeptide,
in Fig.
at 30 min after mRNA expression
cell
7M in FRTL5
10.
were
in 120 min. The time course for c-fos
and
TSH, insulin
CAMP levels
mRNA expression
increased
previously
on DNA synthesis
Intracellular
of El’ and disappeared that
effects
at concentrations
cells
increased
had
1.
not El-2,
The
c-fos
the addition is similar by TSH(12).
to
Vol.
184,
No.
1, 1992
BIOCHEMICAL
AND
12
*3c
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
3 4
Fig.1. Effect of endothelin(ET) on c-fos mRNA levels. The cells grown to near confluency were shifted to the medium without TSH and were incubated for 3 days. Then cells were exposed to TSH (lOml’W) or El’(10m8H) for 30 min. Total RNA was isolated and subjected to Northern blot analysis(lOpg/lane) as described in Materials and Methods.
Although expression of
alone
of c-fos,
ET-l
that
ET-1
on
IGF-I
growth is
DNA synthesis
not
affect
a competent of
gene,
F’RTLS cells.
mitogenic
Fig.SA
cells(l4).
did
to
of FRTL5 cells
ET-l
stimulated
a possible
or
with
reflect IGl-I
mitogenic in
the
activity, absence
shown thyroid
able to potentiate
is
by IGF-I
FRTL5 cells presence
or
role
have
porcine
dependent manner. To demonstrate that “H-thymidine really
FR’M,5 cells,
studies
cells(l3)
that
of
suggested Earlier
Fwn5
illustrates
growth
in a doseincorporation
were cultured of
r
increasing
?
T
5
Fig.2. Effect of m-1 on K&I(A) or TSH(B) stimnlated 3Hthymidine incorporation into DNA. Cells were preincubated for 3 days in 4H or 5H medium plus 0.5% calf serum and then exposed to fresh redium containing the indicated concentrations of ET-1 with ICF-I or TSH. 3H-thymidine incorporation into DNA was measured 72 hr later, as described in Materials and Methods. The bars represent the meanfSD of triplicated determines of one of three experiments. * Significantly different from ICF-I alone(P
184,
No.
BIOCHEMICAL
1, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1992
231-238
Endothelin-1 Stimulates c-fos mRNA Expression and Acts as a Modulator on Cell Proliferation of Rat FRTLS Thyroid Cells Megumi Miyag;:,
Toshio Tsushima’, OsamuIsoxakf’, , Kaxuo Shizume2 and Mariko Arai
Hiroshi
of Clinical Endocrinology, ‘Department of Medicine, Institute Tokyo Women’s Medical College and ‘Institute for Growth Science, 8-l Kawada-cho, Shinjuku-ku, Tokyo, 162, JAPAN Received
February
29,
1992
SUMMARY In FRTL5 thyroid cells, endothelin(EI’)-1 alone had no effect on DNA synthesis but caused a transient increase in c-fos mRNAlevels and stimulated IGF-I induced DNA synthesis and cell proliferation. By contrast, ET-1 inhibited the stimulator-y effects of TSH actions on DNA synthesis, cell proliferation and c-AMP production. 8-Bromo-CAMP-induced DNA synthesis was also inhibited by ET-I, suggesting that ET-1 exerts its inhibitory effects at step(s) involving CAMP production and post CAMP pathway. ET-l-induced suppression of TSH actions were reversed by a C-kinase inhibitor,H-7. These results suggest that the effect of KT on functions of FRIYL5cells is, at least, in part mediated by C-kinase dependent pathway. 0 1992 Academic Press,1°C.
Kndothelin(ET) medium of porcine
was originally aortic
isolated
endothelial
of the most potent vasoconstrictor has been reported
glomerular
thyroid
In addition,
(5).
tells(3),
Furthermore,
via interaction
we investigated
smooth muscle
osteoblastic
tells(4)
we have found that KT
epithelial
with EI’ receptors(6).
the effects
cell
cells
In the present
of ET-1 on growth of a rat
line(FRTL5 cells).
1 modifies growth of FRTL5 cells
it
proliferation
TSH-induced iodine metabolism in porcine thyroid
in culture study,
such as vascular
mesa&al
or Swiss 3T3 fibroblasts inhibits
peptides(1).
cells
culture
and shown to be one
that ET is able to induce cell
in a number of cultured tells(2),
cells
from the
We show here that W-
stimulated
by TSH or insulin-
like growth factor(IGF)-1.
MATERIALSAND METHODS Materials ET-1 and ET-2 were purchased from Peptide Institute Inc,(Osaka, Japan) and other chemicals or hormones were from Sigma Chemical Co.(St.Louis, MO). 0006291X/92
231
$1.50
Vol.
184,
No.
1, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cell culture FRTI.5 thyroid cells were kindly provided by Dr.L.D. Kohn NIH, Bethesda, MD(7) and used between the third and 20th pa&age. Cells were grown in Coom’s modified Ham’s F-12 medium, supplemented with 5% calf serum and a six-hormone mixture which contained bovine TSH(lmU/ml) , insulin (W, cortisol(0.4ng/ml), transferrin(5pg/ml), glycyl-l(l~riW), histidyl-L-1-lysine acetate(lOng/ml) ,and somatostatin(lOng/ml) (8). When cells reached confluence, medium was changed to 5H(without TSH) or 4H(without TSH and insulin) medium depending on the purpose of experiments. All assays were performed in triplicate and were repeated on at least three separate occasions with different batches of cells. 3H-thymidine incorporation into DNA The assay was performed as described previously(9). After replaced in 5H medium for 2 days, test materials and 3H-thymidine(0.1 pCi/well) were added to the culture medium and cultured for 3 days. 3H-thymidine incorporated into TCA-insoluble cellular fractions was counted with a Bcounter. Cell numbers were counted by a coulter counter(Coulter Electronics, Hialeah, FL) _ Assays were performed as previously CAMP levels in cells descrlbed(l0) The medium was shifed to 5OOpl of Hanks’ balanced salt solution’with 0.5 mM 3-isobutyl-1-methyl-xanthine(IBMX) and 20 mM HEPES(pH7.4) containing the indicated concentration of TSH with or without endothelin. After incubation at 37 C for 2 h, the reactions were terminated by adding 5OOpl of 20% TCA. The CAMP content in the extract was determined by RIA using commercial kits(New England Nuclear/ Dupon, Boston, MA) c-fos mRNA levels Total cellular RNA was isolated essentially d b d b Chirgwin et al.(ll). For Northern blot analysis EA ?set w&e electrophoresed in 1% agarose gels containing 0.66 M formaldehyde and blotted on nylon filters. The filters were hybridized to v-fos probe; Oncor(Gaithersburg,MD) (2-4 ~10~ cpm/ml each) and then subjected to autoradiography. Hyblidization and washing were performed as previously described(l2); final washings were carried out at 65 C in 1 x SSPE(20 x SSPE is 2.9H NaCl, 0.2 M NaH,PO,, O.OlM NaOH, and O.OlM EIYl’A). Statistical analysis Statistical evaluation was performed by Student’s t-test when variation of the data was uniform or by Cochran-Cox’s test when variation was not uniform. P values less than 5% were considered significant. Unless otherwise indicated, all values presented represent the meantSD.
RESULTS ET-l
alone
replication cultured
0.5%
calf
in
basal
serum).
changed by ETmRNA levels
no
c-fos
1.
However,
between
reported
1O-*c
medium (without
and
lO-*M ET-1 or as shown
significantly
its
232
and with also
isopeptide,
in Fig.
at 30 min after mRNA expression
cell
7M in FRTL5
10.
were
in 120 min. The time course for c-fos
and
TSH, insulin
CAMP levels
mRNA expression
increased
previously
on DNA synthesis
Intracellular
of El’ and disappeared that
effects
at concentrations
cells
increased
had
1.
not El-2,
The
c-fos
the addition is similar by TSH(12).
to
Vol.
184,
No.
1, 1992
BIOCHEMICAL
AND
12
*3c
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
3 4
Fig.1. Effect of endothelin(ET) on c-fos mRNA levels. The cells grown to near confluency were shifted to the medium without TSH and were incubated for 3 days. Then cells were exposed to TSH (lOml’W) or El’(10m8H) for 30 min. Total RNA was isolated and subjected to Northern blot analysis(lOpg/lane) as described in Materials and Methods.
Although expression of
alone
of c-fos,
ET-l
that
ET-1
on
IGF-I
growth is
DNA synthesis
not
affect
a competent of
gene,
F’RTLS cells.
mitogenic
Fig.SA
cells(l4).
did
to
of FRTL5 cells
ET-l
stimulated
a possible
or
with
reflect IGl-I
mitogenic in
the
activity, absence
shown thyroid
able to potentiate
is
by IGF-I
FRTL5 cells presence
or
role
have
porcine
dependent manner. To demonstrate that “H-thymidine really
FR’M,5 cells,
studies
cells(l3)
that
of
suggested Earlier
Fwn5
illustrates
growth
in a doseincorporation
were cultured of
r
increasing
?
T
5
Fig.2. Effect of m-1 on K&I(A) or TSH(B) stimnlated 3Hthymidine incorporation into DNA. Cells were preincubated for 3 days in 4H or 5H medium plus 0.5% calf serum and then exposed to fresh redium containing the indicated concentrations of ET-1 with ICF-I or TSH. 3H-thymidine incorporation into DNA was measured 72 hr later, as described in Materials and Methods. The bars represent the meanfSD of triplicated determines of one of three experiments. * Significantly different from ICF-I alone(P
