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Induction of c-Fos by Prostaglandin F
2a
in Human Ciliary
Smooth Muscle Cells
James D. Lindsey, Hoang D. To, and Robert N. Weinreb
Purpose. To evaluate the induction of the proto-oncogene c-fos in cultured human ciliary
muscle cells by prostaglandin F
2a
(PGF
2a
) and by 17-phenyltrinor-PGF
2a
.
Method. Human ciliary muscle cells were grown to confluency in monolayer cell culture,
placed in medium containing 1% fetal bovine serum for 5 days, treated by addition of PGF
2a
or
the trinor derivative, fixed, and then immunocytochemically stained using an antibody to
c-Fos, the protein product of the translation of c-fos.
Results. After treatment with either agonist, the mean induction score (proportion of brightly
immunostained nuclei) increased to a maximal level during the first hour and returned to basal
levels 4 to 8 hours after treatment. Increasing the agonist concentration increased the maximal
level, but had no effect on the time course of the response. The dose responses after 1 hour of
treatment with PGF
2o
or 17-phenyltrinor-PGF
2a
increased similarly between 1.6 X 10~
9
M and
2 X 10~
7
M. When treated with 1 X 10~
6
M of either agonist, however, the induction was half
that obtained at 2 X 10"
7
M.
Conclusion. Exposure of ciliary smooth muscle cells to either PGF
2a
or 17-phenyltrinor-PGF
2o
induces an immediate early gene expression response that is similar to c-Fos induction in other
cell systems. These results establish the basis for future investigations evaluating the potential
role of c-fos induction in mediating the effects of PGF
2a
on uveoscleral outflow. Invest Ophthal-
mol Vis Sci. 1994;35:242-250
1 opical instillation of prostaglandin F
2a
(PGF
2
J has
been shown to reduce intraocular pressure in normal
human subjects and glaucoma subjects.
1
'
2
This re-
sponse appears to be due to a marked increase in
uveoscleral outflow facility without any change in con-
ventional outflow facility or in aqueous humor flow.
3
Although the precise biologic mechanism mediating
this effect has not been established, experimental evi-
dence suggests that remodeling of ciliary muscle ex-
tracellular matrix may be involved.
4
Recently, we have found that incubation of human
ciliary muscle cells in vitro with PGF
2a
produces a
From the Glaucoma Center and Research Laboratories, University of California at
San Diego, Lajolla, California.
Presented in part at the Annual Meeting of the Association for Research in Vision
and Ophthalmology, Sarasota, Florida, May 1992.
Supported in part by NIH grant EY05990 (RNW).
Submitted for publication August 4, 1992; revised July 19, 1993; accepted July
21, 1993.
Proprietary interest catagory: N.
Reprint requests: Robert N. Weinreb, Glaucoma Center and Research Laboratories,
UCSD/0946, Lajolla, CA 92093.
dose-dependent calcium efflux from intracellular
stores to the exterior of the cells.
5
Mobilization of cal-
cium contained in intracellular stores in other smooth
muscle types has been associated with activation of the
proto-oncogene c-fos.
6
-
7
The protein product of c-fos,
designated c-Fos, has been shown to play a key role in
the regulation of promoters and enhancers associated
with a number of eukaryotic genes.
8
"
10
Thus, its induc-
tion in ciliary smooth muscle cells may provide a sensi-
tive marker for the activation of gene regulatory
events possibly associated with modulation of uveos-
cleral outflow resistance by PGF
2a
.
In other cell types, specific responses to PGF
2a
stimulation appear to be mediated by activation of the
FP-receptor, a prostanoid receptor subtype with mod-
erately preferential sensitivity to PGF
2a
.
n
~
13
Support
for a role of this receptor in mediating PGF
2a
-induced
hypotension is derived from the observation that topi-
cal application of certain PGF
2a
analogs can produce
ocular hypotension with greater potency than
PGF
2a
.
14
'
15
However, the results of other studies have
raised questions about the possible role of the FP-re-
242
Investigative Ophthalmology & Visual Science, January 1994, Vol. 35, No. 1
Copyright © Association for Research in Vision and Ophthalmology
Prostaglandin F
2a
Induces c-Fos in Ciliary Muscle Cells 243
ceptor.
1617
For example, 17-phenyl-18,19,20-trinor
PGF
2a
(17-phenyltrinor PGF
2a
) appears to be a selec-
tive FP-receptor agonist
1819
, but it fails to reduce intra-
ocular pressure when topically applied to rabbits.
16
Thus, to evaluate the possible role of the FP-receptor
in PGF
2a
-mediated c-Fos induction, this response
should be carefully compared to c-Fos induction by
similar treatments with 17-phenyltrinor PGF
2a
.
In the current study, we investigated the induction
of c-Fos in ciliary smooth muscle cells by PGF
2a
. Evalu-
ations included the time course of the response and
the relationship between treatment dose and the maxi-
mal response. To address the potential role of the FP-
receptor, we also examined the induction of c-Fos by
17-phenyltrinor PGF
2a
.
METHODS
Cell Cultures
Eyes from four persons (aged 53, 59, 61, and 64 years;
obtained from the San Diego Eye Bank) were enucle-
ated within 6 hours of death, and stored at 4°C for less
than 24 hours. All procedures in this study followed
UCSD guidelines for the use of human tissue in re-
search. Human ciliary muscle cells were cultured as
described previously.
520
Briefly, the globe was bi-
sected between the ora serrata and equator. The ante-
rior segment was placed in a dish with the corneal epi-
thelium facing down. Under a dissecting microscope,
the lens was removed and the iris disinserted. The cili-
ary body was gently removed from the sclera and
placed in a sterile dish. Against the dark background
of the pigmented epithelium, the muscle was easily
identified as a broad, pale, circular band. The outer-
most portion of the muscle was dissected free. Strips
of the muscle then were explanted into 35 mm culture
dishes (Falcon, Lincoln Park, NJ) filled with Dul-
becco's modified Eagle's medium and Ham's F12 nu-
trient mixture (DMEM/F12, University of California
San Diego Cell Culture Facility) supplemented with
10% fetal bovine serum (J.R. Scientific, Woodland,
CA) and 1 ng/ml recombinant human basic fibroblast
growth factor (R & D Systems, Inc., Minneapolis, MN).
The cultures were incubated in a humidified atmo-
sphere of 95% air, 5% CO
2
. This growth medium was
changed every 3 to 4 days. Primary cell explant cul-
tures reached confluency within 30 days. These and
subsequent confluent cultures were trypsinized and
subcultured at a ratio of 1:3. The subsequent passage
cultures reached confluency within 7 to 10 days. Cells
in these cultures express smooth muscle actin and a
moderate amount of desmin similar to ciliary smooth
muscle cells in vivo.
5
In addition, electron microscopic
evaluation of the cultures in the same study reveal par-
allel microfilament bundles in the cultured cells like
those found in ciliary muscle cells in vivo.
Experimental Treatments of the Cultures
Second- or third-passage cells were plated onto glass
coverslips and allowed to grow to confluency. Because
serum contains agents that also activate c-Fos expres-
sion,
21
the cells were transferred to medium with the
serum concentration reduced to 1% before treatment
with the test agonists. A standard time for this prein-
cubation was based on the time required for the c-Fos
scores to reduce to basal plateau (see Results).
Stock solutions of 10 mM of PGF
2a
and 17-phen-
yltrinor-PGF
2a
(Cayman Chemical Company, Ann Ar-
bor, MI) were prepared in ethanol and diluted to ap-
propriate test concentrations with DMEM/F12 nu-
trient mixture. The vehicle control was DMEM/F12
nutrient mixture containing 0.1% ethanol (the same
concentration of ethanol as present in the highest ago-
nist concentration experimental test media). The me-
dium exchange control contained DMEM/F12 nu-
trient mixture alone. All conditions within the same
experiment were completed with triplicate parallel
cultures and repeated at least once. Treatments were
initiated by changing to the test media and terminated
by fixation with 4% formaldehyde in phosphate-buff-
ered saline.
Immunocytochemical Characterization
of c-Fos Expression
c-Fos expression was assessed by immunostaining the
cultures using a rabbit polyclonal immunoglobulin G
that recognized the N-terminal domain of human p62
c-Fos (Ab-2, Oncogene Science, Manhasset, NY; di-
luted 1:100). After treatments with 0.3% Triton X-100
and 10% goat serum to facilitate antibody access to the
nucleus and to block nonspecific staining, the cultures
were exposed to the primary antibody for 90 minutes,
washed, exposed to rhodamine-conjugated goat anti-
rabbit immunoglobulin G (Cappel, Durham, NC; di-
luted 1:40), washed, and mounted in gelvatol.
Cultures were viewed using a fluorescence micro-
scope equipped with appropriate filters for rhoda-
mine. Staining in the cell nuclei was either very low
(basal) or moderately bright. Within each of these two
groups the staining intensity was highly consistent. For
each culture, induction of c-Fos was scored by deter-
mining the proportion of cells with brightly immunos-
tained nuclei. The proportion of cells with brightly
stained nuclei was scored by evaluating the first 200 to
240 cells on each coverslip and was expressed as a
percentage. For each coverslip, scoring was done
through a single viewing until complete. This mini-
mized possible variations arising from light adaptation
occurring with intermittent viewing of the fluores-
cence field. In trial experiments, the difference in the
scores of the same coverslips obtained by two investi-
gators (JL and HT) averaged less than 2%. For the sake
of consistency, all of the counts reported in this study
244 Investigative Ophthalmology & Visual Science, January 1994, Vol. 35, No. 1
were performed by one of the investigators (HT). The
mean and standard deviation of the triplicate percent
scores from each experimental condition were ob-
tained. Statistical comparisons of the results were
made using Student's t test. For experiments in which
several different treatment results were compared to a
common control, the Bonferroni t test was used.
22
A P
value of less than 0.05 was considered significant.
Dosage Considerations for Dose-Response
Analysis
The test concentration range employed for assessing
the c-Fos induction responses to PGF
2a
and to 17-
phenyltrinor-PGF
2a
was based on the observed con-
centration of PGF
2a
in aqueous humor after topical
PGF
2a
administration in monkeys, and a minimal ocu-
lar hypotensive dose of PGF
2a
in humans. Aqueous
humor collected after topical treatment of monkey
eyes with 250 ng of PGF
2a
contained 28 ng/ml of
PGF
2a
(7.8 X 10"
8
M).
23
Trials with normotensive hu-
mans found significant intraocular pressure reduc-
tions after single 62.5 /*g doses of PGF
2a
.
24
The results
of subsequent human trials using multiple treatment
regimens suggest that even lower doses, and by exten-
sion, peak aqueous humor concentrations, can effec-
tively produce ocular hypotension.
25
Thus, to investi-
gate doses that encompass the range of expected
aqueous concentrations following established PGF
2a
treatments that lower intraocular pressure, compari-
son of PGF
2a
and 17-phenyltrinor PGF
2a
treatment
was made using concentrations ranging from 4 X 10~
9
M to 1 X 10-
6
M.
Dosage Considerations for Comparison of
Response Time Course After PGF
2a
or
17-phenyltrinor-PGF
2a
Treatment
For these comparisons of c-Fos induction, a low ago-
nist concentration was used to avoid additional re-
sponses that occur at high concentrations, which
could lead to errors in interpretation. The first of
these additional responses, receptor desensitization,
has been observed after PGF
2a
treatment of bovine iris
smooth muscle cells at concentrations higher than 1 X
10"
6
M.
26
The second additional response, PGF
2a
-
mediated calcium release, is often associated with con-
traction and has a threshold in human ciliary smooth
muscle cells between 1 X 10"
8
M and 1 X 10~
7
M.
5
In
the experiments reported here, 1-hour exposure to 8
X 10"
9
M PGF
2a
produced a highly significant increase
c-Fos induction when compared to vehicle control.
Because this concentration is less than would be likely
to produce either receptor desensitization or calcium
release, it was chosen to compare the time course of
c-Fos elevation produced by treatment with PGF
2a
or
V
FIGURE 1. Phase image of third-passage human ciliary
smooth muscle cells cultured from the eye of a 59-year-old
donor. The cells formed a monolayer of aligned spindle-
shaped cells that grew in a characteristic "hill-and-valley"
pattern, that is, with condensed regions and sparse regions.
These regions are labeled H and V, respectively. Unstained;
Magnification X 145.
17-phenyltrinor-PGF
2a
in cultures of different pas-
sage number and donor origin.
RESULTS
The cultured cells grew to confluency in 5 to 7 days
forming monolayers of spindle shaped cells with the
characteristic hill-and-valley pattern found in previous
cultures of this cell type
520
(Fig. 1). There were no
major differences in the growth or structure of the
cells from the different donors. Immunocytochemical
staining for c-Fos in untreated cultures revealed
mostly faint nuclear staining and very faint back-
ground staining of the cytoplasm (Fig. 2A), Treatment
with PGF
2a
or 17-phenyltrinor-PGF
2a
produced a dra-
matic increase in the the proportion of cells with
brightly stained nuclei (Fig. 2B). Thus this response
was quantified by determining the proportion of cells
with bright nuclei in each culture.
Reduction of c-Fos Scores After Switch to 1%
Serum
The medium of confluent cultures in growth medium
was exchanged to fresh maintenance medium contain-
ing 1% serum. Triplicate wells from parallel cultures
were fixed at daily intervals during the next 5 days.
During the first 4 days, c-Fos induction scores gradu-
ally dropped from 38% to between 6% and 9% (Fig. 3).
At 5 days after refeeding, the induction score was es-
sentially the same as on day 4. Because of the similar
Prostaglandin F
2n
Induces c-Fos in Ciliary Muscle Cells
245
FIGURE 2. Fluorescence image of third-pas-
sage ciliary smooth muscle cells from the
eye of a 59-year-old donor treated for 1
hour with either vehicle control (A) or with
2X10~
7
M prostaglandin F
2o
, (B) and immu-
nocytochemically stained with an antibody
to c-Fos. Note that the intensity of the nu-
clear staining is elevated in the treated cul-
tures. Magnification X 350.
scores at 4 and 5 days, this level of induction was con-
sidered for the purpose of this investigation to be "ba-
sal." To ensure that c-Fos expression in the cultures
had achieved a "basal" status before treatment with
PGF
2a
or 17-phenyltrinor PGF
2a
, the cultures were
transferred to maintenance medium for 5 days before
initiation of experimental testing.
Induction of c-Fos by PGF
2a
Treatment of parallel confluent ciliary muscle cell cul-
tures with 8 X 10-
9
M, 4 X 10"
8
M, and 2 X 10"
7
M
PGF
2a
produced a sharp increase in the mean induc-
tion score during the first hour (Fig. 4). During the
second hour, the proportion of cells with bright nuclei
dropped by 50%. During the subsequent 8 hours, the
response decreased back toward the starting (basal)
scores. The magnitude of the maximal response (ie,
the maximal proportion of cells responding) was a di-
rect function of agonist concentration. In contrast,
the time course of the response appeared to be same
regardless of agonist concentration. Treatment of par-
allel cultures with vehicle control medium resulted in
no change of c-Fos expression. The response of third-
passage cells was insignificantly different from the re-
sponse of the second-passage cells at each time point
measured (data not shown).
Comparison of Treatment Regimens
Because the response begins a rapid decline in the
second hour after treatment, the action of available
PGF
2a
after the peaking of c-Fos expression may influ-
ence the time course of the response or the ability of
the cells to respond to subsequent treatments. To ex-
amine this issue, parallel cultures were treated with
one of four regimens in which the exposure to 2 X
10~
7
M PGF
2ff
was varied. Response was evaluated 4, 8,
and 16 hours after the beginning of the experiment.
In the first regimen, added PGF
2a
remained on the
cells throughout the experiment. In the second regi-
men, the cultures were exposed to PGF
2a
for 1 hour
and then the medium was changed to medium without
PGF
2a
taken from parallel untreated cultures for the
remainder of the test periods. To evaluate the viability
of the cells during the test periods, a third group of
cultures was not treated initially, but was treated 1
hour before the end of each test period. To evaluate
the possible effect of activation on viability, a fourth
group of cultures was exposed to PGF
2a
for 1 hour,
changed to medium without PGF
2a
for 14 hours, and
then treated a second time. These cultures were fixed
1 hour after the second treatment.
As shown by the open bars in Figure 5, the re-
sponse to the simple addition of PGF
2a
was the same as
is shown in Figure 4. The response to only 1 hour of
PGF
2a
exposure, shown by the solid bars, was essen-
tially the same at each time point as with continuous
exposure. The viability of the cultures throughout the
various test periods was confirmed by the large re-
sponses (shaded bars) in the cultures that were treated
1 hour before the test periods were over. Activation
appeared to have little effect on viability because the
cultures treated during the initial hour responded to a
second treatment 14 hours later (crosshatched bar)
similarly to the cultures that had not received the ini-
tial treatment (shaded bar, 16 hours).
246 Investigative Ophthalmology & Visual Science, January 1994, Vol. 35, No. 1
50
(0
0)
o
<* -
o
c
0)
o
o
Q.
V)
O
LJL

O
0)
a>
LU
Q
(0
+1
c
(0
p
40 "
30 -
20 H
10 -
2nd Passage
3rd Passage
80
Days After Change to 1% FBS
FIGURE 3. Daily decline in the proportion of cells with ele-
vated c-Fos after changing from medium with 10% fetal bo-
vine serum to medium with 1% fetal bovine serum. Cultures
were second- and third-passage cells from the eye of a 61-
year-old donor. Note that further decline is not observed
between the fourth and fifth days after the medium change.
Triplicate cultures were evaluated for each data point. In all
subsequently described experiments, the cells were incu-
bated for 5 days in medium with 1% fetal bovine serum be-
fore starting.
Comparison of Dose-Response to PGF
2a
and to
17-phenyltrinor-PGF
2a
Because maximal c-Fos induction was observed at 1
hour after PGF
2a
treatment, comparison of the dose-
response relationship after PGF
2a
or 17-phenyltrinor-
PGF
2a
exposure was assessed 1 hour after treatment
(Fig. 6). Between 1.6 X 10"
9
M and 2 X 10"
7
M, both
PGF
2a
and the trinor derivative produced dose-depen-
dent increases in the percentage of cells expressing
elevated c-Fos. Differences between the two agonist
responses at each test concentration in this range were
insignificant. The observed half-maximal response was
approximately 3 X 10~
8
M. At 1 X 10~
6
M, the number
of cells responding to either agonist was less than at 2
X 10~
7
M. The responses to the various concentrations
of the two agonists were very similar between the sec-
ond- and third-passage cells from the 59-year-old eyes
(not shown). In contrast the responses of the second-
passage cells from the 59-year-old eyes were roughly
50, 35, and 15% less at 8 X 10"
9
M, 4 X 10"
8
M, and 2 X
10~
7
M, respectively, than the 1-hour responses of the
60 -
"3 6
o
l
o> re
u3 g
20-
2x10"' M
Hours After Treatment
FIGURE 4. Time course of c-Fos induction (changes in the
proportion of brightly stained nuclei) after treatment of sec-
ond-passage ciliary smooth muscle cells from the eye of a
64-year-old donor with various doses of PGF
2a
. Note that
the percentage of cells with elevated nuclear c-Fos quickly
rises during the first hour and then returns back to basal
scores during the next few hours. Similar results were ob-
tained in a duplicate of this experiment using third-passage
cells from the same donor.
o5! M
SI S
D Simple Addition
• Treat 1 hr at Start
Treat 1 hr at End
Treat at Start & End
n
1 6
Hours After Initial Treatment
FIGURE 5. Time course of c-Fos induction after various treat-
ment regimens with 2 X 10~
7
M PGF
2o
. Cultures were
treated by simple addition of PGF
2o
for the entire test pe-
riod, by exposure to PGF
2a
only during the initial hour, and
in controls either in the last hour of incubation only, or in
both the initial and last hour of the incubation. Significant
differences at each time point between the cultures treated
by simple PGF
2a
addition and the other experimental condi-
tions is indicated with an asterisk. See text for further de-
tails. The cultures were third-passage cells from a 64-year-
old donor. Similar results were obtained in a duplicate of
this experiment using second-passage cells from the same
donor.
Prostaglandin F
2a
Induces c-Fos in Ciliary Muscle Cells
80
100 1000
Concentration of Agonist
( nM )
FIGURE 6. Dose-response analysis of c-Fos induction in sec-
ond-passage ciliary smooth muscle cells from a 59-year-old
donor after 1 hour of treatment with either PGF
2a
or 17-
phenyltrinor-PGF
2a
. Note that the response to either pros-
tanoid is very similar. In addition, this response appears to
be quite similar between second- and third-passage cells.
Similar results were obtained in a duplicate of this experi-
ment using third-passage cells from the same donor.
second-passage cells from the 64-year-old eyes shown
in Figure 4.
Comparison of Response Time Course After
PGF
2a
or 17-Phenyltrinor-PGF
2a
Experiments were conducted with second- and third-
passage cells from a 59-year-old donor and second-
passage cells from a 53-year-old donor. After 8 X 10~
9
M treatment with either agonist, the scores increased
threefold to fivefold to peak 1 hour after treatment,
and returned to basal expression within the next 6
hours. Within each experiment, standard deviations of
the mean responses after 1 hour of exposure to either
agonist were less than 9%. The responses to each ago-
nist were insignificantly different at every time point
measured. However, the differences between either
agonist and the vehicle control at 1 hour were highly
significant (P < 0.01). The averages and standard de-
viations of the mean responses from each experiment
at each time point are shown in Figure 7. Neither the
treatment with vehicle control nor with the medium
247
change control induced c-Fos expression to rise signifi-
cantly above the basal expression observed at the be-
ginning (0 time point) of the experiment.
DISCUSSION
These data demonstrate that PGF
2a
induces c-Fos in
human ciliary smooth muscle cells. This response con-
sists of a rapid increase of the proportion of cells with
elevated nuclear c-Fos content. The response was
maximal at 1 hour after stimulation and was followed
by reduction to basal proportions during the next sev-
eral hours. Control experiments indicate that the drop
in the score after 1 hour reflects an internal regulatory
mechanism rather than any change in the viability of
the cells during the course of the experiment. The
magnitude of the response increases with increasing
agonist concentration between 4 X 10~
9
M and 2 X
10~
7
M. The response was similar in time course and
magnitude in second- and third-passage cells from the
same donor. The time course of the responses in cells
generated from different donors also were similar.
However, there was some variation in the magnitude
of the response of cells from different donors.
40
C
e
l
l

n
t

o
f
P
e
r
e
c
<n
O
ii
6
s
v
a
t
e
i
t
h

E
l
«
w
•H
m
e
a
r
a
n

o
f
30
20
E 10-
PGF
2u
17-PTPGF2a
Vehicle Control
Medium Change Control
Hours After Treatment
FIGURE 7. Time course analysis of c-Fos expression after
treatment with 8 X 10~
9
M of prostaglandin F
2
« or of 17-
phenyltrinor-prostaglandin F
2
«. This low concentration of
prostaglandin F
2a
was chosen to avoid the induction of ei-
ther receptor desensitization or contraction (see text). Cul-
tures analyzed include second- and third-passage cells from
a 59-year-old donor, plus second-passage cells from a 53-
year-old donor. The mean of the mean responses at each
time point in these three studies is shown. The differences of
the mean responses to the two agonists was statistically insig-
nificant (P > 0.05) at each of the time points examined.
248
Investigative Ophthalmology & Visual Science, January 1994, Vol. 35, No. 1
The transient elevation of c-Fos in ciliary smooth
muscle cells exposed to PGF
2a
is similar to c-Fos in-
duction observed in rat vascular smooth muscle cells
exposed to vasopressin.
27
Moreover, it is consistent
with the faster rise and fall of c-fos messenger RNA
observed in vascular smooth muscle cells treated with
platelet-derived growth factor, endothelin-1, angioten-
sin II, or thrombin.
28
"
31
Similar rapid time courses for
c-fos induction followed by transient elevation of c-Fos
has been observed in fibroblasts stimulated by various
mitogens.
20
In vivo, rapid induction of c-fos has been
observed in the paraventricular neurons of rats after
immobilization stress onset,
32
and in rat adrenal me-
dula after subcutaneous injection of capsaicin.
33
Thus,
the time course of c-Fos induction in the ciliary
smooth muscle cells exposed to PGF
2a
appears to be
similar to c-Fos induction patterns observed with
other smooth muscle types and agonists, as well as with
other cell types and treatments both in vitro and in
vivo.
As a further control, freshly dissected ciliary mus-
cle tissue was incubated overnight in culture medium
with 1% fetal bovine serum, treated with 2 X 10~
7
M
PGF
2a
for 1 hour, and then fixed. Immunocytochemis-
try for c-Fos was performed on frozen sections of the
muscle. Cells on the surface of treated muscle strips
contained brightly immunostained nuclei. However,
cells within the tissue did not respond (data not
shown). These results may be due to delayed penetra-
tion of the agonist into the explanted muscle during
the 1-hour treatment. An important consideration is
that the diffusion of medium into explanted ciliary
muscle tissue is probably substantially different from
the flow of aqueous humor through the muscle in situ
by the uveoscleral pathway. Nevertheless, it does pro-
vide support for the relevancy of the results to the
response of ciliary smooth muscle cells to PGF
2a
in
vivo.
In several different cell systems, c-fos activation
has been associated with increased biosynthesis of en-
zymes that are involved in extracellular matrix degra-
dation. For example, c-fos activation in platelet-de-
rived growth factor treated 3T3 fibroblasts and nerve
growth factor treated PC 12 pheochromocytoma cells
has been linked to subsequent transcription of an
RNA transcript called transin.
34
'
35
Transin has re-
cently been shown to code for a protein that is the
equivalent in rat to human stromelysin.
36
This enzyme
can degrade proteoglycans, fibronectin, and laminin
in extracellular matrix.
3738
In addition, c-fos induction
has been shown to lead to transcriptional activation of
collagenase.
39
Together, stromelysin and collagenase
has been shown to mediate normal extracellular ma-
trix turnover in a number of different cell systems in-
cluding trabecular meshwork cells.
40
"
43
Hence, in-
creased secretion of these enzymes in response to c-
Fos induction could account for the remodeling of the
ciliary muscle extracellular matrix observed in PGF
2a
-
treated monkeys.
4
It remains to be determined if
PGF
2a
increases c-Fos in trabecular meshwork cells or
other cell types facing the uveoscleral outflow path, or
if PGF
2a
treatment stimulates stromelysin and collage-
nase secretion at these sites.
Activation of factors in addition to c-Fos may also
play important roles in outflow regulation. For exam-
ple, because c-Fos interacts with c-Jun to modulate
collagenase expression
44
, changes in c-Jun expression
could also be important. Ultimately, changes in extra-
cellular matrix deposition will reflect the net effect of
changes in the synthesis and assembly of the matrix
components themselves, changes in the synthesis of
degradative enzymes such as collagenase and strome-
lysin, and changes in the production of agents that
regulate the activity of these enzymes such as tissue
inhibitor of metalloprotease.
45
Careful examination of
each of these points will be important to the establish-
ment of the complete mechanism of PGF
2a
action.
The striking similarities in the responses to PGF
2a
and to 17-phenyltrinor-PGF
2a
, are consistent with the
possibility that they are mediated by the FP receptor.
This is based on evidence that 17-phenyltrinor-PGF
2a
appears to be a highly specific ligand for the FP-recep-
tor.
1618
Autoradiography of frozen human eye sec-
tions exposed to various labeled ligands demonstrate
specific PGF
2a
binding sites in the human ciliary mus-
cle tissue.
46
While quantitative displacement analysis
of this binding with various prostanoid analogs indi-
cates that much of the PGF
2a
binding to the sections is
to EP
2
receptors, differences in the maximum binding
site results for PGE
2
and PGF
2a
indicate that the FP
receptor could also be present.
47
Because the resolu-
tion of these autoradiographic studies was insufficient
to resolve blood vessels and there is evidence of EP
2
receptors on human vascular endothelial cells
48
and
on human macrophages,
49
it is possible that a portion
of the PGF
2a
binding to EP
2
receptors in these sections
was to nonciliary smooth muscle cell types. Thus, al-
though the current study supports a role of the FP
receptor on ciliary smooth muscle cells in PGF
2a
-
mediated c-Fos induction, further work will be neces-
sary to evaluate its possible role in PGF
2a
-mediated
ocular hypotension. It should be noted that activation
of other prostanoid receptor types within the eye, such
as the DP receptor, may also mediate changes in intra-
ocular pressure.
161750
However, as has been empha-
sized by Bito,
51
comparisons across species should be
done with caution because conflicting findings may
merely reflect species differences.
Key Words
c-Fos, prostaglandin, ciliary muscle, immunocytochemistry,
glaucoma
Prostaglandin F
2a
Induces c-Fos in Ciliary Muscle Cells 249
Acknowledgments
The authors thank Dr. Judy L. Meinkoth of the UCSD De-
partment of Medicine, for helpful advice about c-Fos immu-
nocytochemistry.
References
1. Giuffre G. The effects of prostaglandin F
2o
in the hu-
man eye. Graefes Arch Clin Exp Ophthalmol.
1985;222:139-141.
2. Aim A, Villumsen, J. Effects of topically applied PGF
2a
and its isopropylester on normal and glaucomatous
human eyes. In: Bito, LZ, Stjernschantz J, eds. The
Ocular Effects of Prostaglandins and Other Eicosanoids.
New York: Alan R Liss; 1989:447-458.
3. Gabelt BT, Kaufman PL. Prostaglandin F
2a
increases
uveoscleral outflow in the cynomolgous monkey. Exp
Eye Res. 1989;49:389-402.
4. Tamm E, Lutjen-Drecoll E, Rohen JW. Age related
changes of the ciliary muscle in comparison with
changes induced by treatment with prostaglandin F
2a
.
An ultrastructural study in rhesus and cynomolgus
monkeys. Mech Ageing Dev. 1990;51:101-120.
5. Weinreb RN, Kim DM, Lindsey JD. Propagation of
ciliary smooth muscle cells in vitro and effects of pros-
taglandin F
2a
on calcium efflux. Invest Ophthalmol Vis
Sci. 1992;33:2679-2686.
6. Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander
RW, Nadal-Ginard, B. Angiotensin II induces c-fos
mRNA in aortic smooth muscle. Role of Ca
2+
mobili-
zation and protein kinase C activation. J Biol Chem.
1989;264:526-530.
7. Panettieri RA, Yadvish PA, Kelly AM, Rubinstein NA,
Kotlikoff MI: Histamine stimulates proliferation of
airway smooth muscle cells and induces c-fos expres-
sion. AmfPhysiol. 1990;259:L365-L371.
8. Curran T, Franza BR Jr. Fos and Jun: The AP-1 con-
nection. Cell. 1988,55:395-397.
9. Ney PA, Sorrentino BP, McDonough KT, Nienhuis
AW. Tandem AP-1-binding sites within the human
beta-globin dominant control region function as an
inducible enhancer in erythroid cells. Genes Dev.
1990;4:993-1006.
10. Lee ME, Dhadly MS, Temizer DH, Clifford JA, Yoshi-
zumi M, Quertermous T. Regulation of endothelin-1
gene expression by Fos and Jun. J Biol Chem.
1991;266:19034-19039.
11. Coleman RA, Humphrey PPA, Kennedy I. Prostanoid
receptors in smooth muscle: Further evidence for a
proposed classification. Trends Autonom Pharmacol.
1982;3:35-49.
12. Coleman RA, Humphrey PPA, Kennedy I, Lumley P.
Prostanoid receptors: The development of a working
classification. Trends Pharmacol Sci. 1984;5:303-306.
13. Gardiner PJ. Classification of prostanoid receptors.
Adv Prostaglandin, Thromboxane Leukot Res. 1990;
20:110-118.
14. Villumsen J, Aim A. PHXA34—a prostaglandin F
2a
analogue—effect on intraocular pressure in patients
with ocular hypertension. Brf Ophthalmol. 1992; 76:
214-217.
15. Resul B, Stjernschantz J, No KY, et al. Phenyl-substi-
tuted prostaglandins—potent and selective antiglau-
coma agents. / Med Chem. 1993; 36:243-248.
16. Woodward DF, Burke JA, Williams LS, et al. Prosta-
glandin F
2a
effects on intraocular pressure negatively
correlate with FP-receptor stimulation. Invest Ophthal-
mol Vis Sci. 1989;30:1838-1842.
17. Waterbury LD, Eglen RM, Faurot GF, Cooper GF.
EP
3
, but not EP
2
, FP, orTP prostenoid-receptor stimu-
lation may reduce intraocular pressure. Invest Ophthal-
mol Vis Sci. 1990;31:2560-2567.
18. Miller WL, Weeks JR, Lauderdale JW, Kirton KT. Bio-
logical activities of 17-phenyl-18,19,20-trinorprosta-
glandins. Prostaglandins. 1975;9:9-18.
19. Powell WS, Hammerstrom S, Samuelson B. Interac-
tions between prostaglandin analogues and a receptor
in bovine corpora lutea. Eurf Biochem. 1975;59:271-
276.
20. Tamm E, Flugel C, Baur A, Lutjen-Drecoll E. Cell cul-
tures of human ciliary muscle: Growth, ultrastructural
and immunocytochemical characteristics. Exp Eye Res.
1991;53:375-387.
21. Mehmet H, Rozengurt E. Regulation of c-fos expres-
sion in Swiss 3T3 cells: An interplay of multiple signal
transduction pathways. Br Med Bull. l991;47:76-86.
22. Glantz SA. Primer of Biostatistics. Third Edition. New
York: McGraw Hill; 1992.
23. Camras CB, Bhuyan KC, Podos SM, Bhuyan DK, Mas-
ter RWP. Multiple dosing of prostaglandin F
2a
or epi-
nephrine on cynomolgus monkey eyes. Invest Ophthal-
mol Vis Sci. 1987;28:921-926.
24. Lee P-Y, Shao H, Xu L, Qu C-K. The effect of prosta-
glandin F
2o
on intraocular pressure in normotensive
human subjects. Invest Ophthalmol Vis Sci. 1988; 29:
1474-1477.
25. Camras CB, Siebold EC, Lustgarten JS, et al. Main-
tained reduction of intraocular pressure by prosta-
glandin F
2a
-l-isopropyl ester applied in multiple doses
in ocular hypotensive and glaucoma patients. Ophthal-
mology. 1989;96:1329-1336.
26. Yousufzai SY, Tachado SD, Carter KD, Abdel-Latif
AA. Short-term desensitization of prostaglandin F
2o
receptors increases cyclic AMP formation and reduces
inositol phosphates accumulation and contraction in
the bovine iris sphincter. Curr Eye Res. 1989; 8:1211-
1220.
27. Nambi P, Watt R, Whitman M, et al. Induction of c-fos
protein by activation of vasopressin receptors in
smooth muscle cells. FEBS Lett. 1989;245:61-64.
28. Sachinidis A, Schulte K, Ko Y, et al. The induction of
early response genes in rat smooth muscle cells by
PDGF-AA is not sufficient to stimulate DNA-synthe-
sis. FEBS Lett. 1993;319:221-224.
29. Koide M, Kawahara Y, Tsuda T, Ishida Y, Shii K,
Yokoyama M. Endothelin-1 stimulates tyrosine phos-
phorylation and the activities of two mitogen-acti-
vated protein kinases in cultured vascular smooth
muscle cells, f Hypertens. 1992; 10:1173-1182.
30. Lyall F, Dornan ES, McQueen J, Boswell F, Kelly M.
Angiotensin II increases proto-oncogene expression
and phosphoinositide turnover in vascular smooth
250 Investigative Ophthalmology & Visual Science, January 1994, Vol. 35, No. 1
muscle cells via the angiotensin II ATI receptor./Hy-
pertens. 1992; 10:1463-1469.
31. Berk BC, Taubman MB, Griendling KK, Cragoe EJ Jr,
Fenton JW, Brock TA. Thrombin-stimulated events in
cultured vascular smooth-muscle cells. Biochem J.
1991;274:799-805.
32. Imaki T, Shibasaki T, Hotta M, Demura H. Early in-
duction of c-fos precedes expression of corticotropin-
releasing factor messenger ribonucleic acid in the
paraventricular nucleus after immobilization stress.
Endocrinology. 1992; 131:240-246.
33. Pelto-Huikko M, Dagerlind A, Ceccatelli S, Hokfelt T.
The immediate- early genes c-fos and c-jun are differ-
entially expressed in the rat adrenal gland after capsai-
cin treatment. Neurosci Lett. 1991; 126:163-166.
34. Kerr LD, Holt JT, Matrisian LM. Growth factors regu-
late transin gene expression by c-fos-dependent and
c-fos-independent pathways. Science. 1988; 242:1424-
1427.
35. Machida CM, Rodland KD, Matrisian L, Magun BE,
Ciment, G. NGF induction of the gene encoding the
protease transin accompanies neuronal differentia-
tion in PCI2 cells. Neuron. 1989;2:1587-1596.
36. Nicholson R, Murphy G, Breathnach R. Human and
rat malignant-tumor-associated mRNAs encode stro-
melysin-like metalloproteinases. Biochemistry. 1989;
28:5195-5203.
37. Okada Y, Nagase, H, Harris ED Jr. A metalloprotein-
ase from human rheumatoid synovial fibroblasts that
digests connective tissue matrix components: Purifica-
tion and characterization. J Biol Chem. 1989; 261:
14245-14255.
38. Wilhelm SM, Collier IE, Kronberger A, et al. Human
skin fibroblast stromelysin: Structure, glycosylation,
substrate specificity, and differential expression in
normal and tumorigenic cells. Proc Natl Acad Sci USA.
1987;84:6725-6729.
39. Schonthal A, Herrlich P, Rahmsdorf HF, Ponta H.
Requirement [or fos gene expression in the transcrip-
tional activation of collagenase by other oncogenes
and phorbol esters. Cell. 1988; 54: 325-334.
40. Wu JJ, Lark MW, Chun LE, Eyre DR. Sites of strome-
lysin cleavage in collagen types II, IX, X, and XI carti-
lage. J Biol Chem. 1991; 266:5625-5628.
41. Okada Y, Konomi H, Yada T, Kimata K, Nagase, H.
Degradation of type IX collagen by matrix metallo-
proteinase 3 (stromelysin) from human rheumatoid sy-
novial cells. FEBSLett. 1989;224:473-476.
42. Alexander JP, Bradley JM, Gabourel JD, Acott TS. Ex-
pression of matrix metalloproteinases and inhibitor by
human retinal pigment epithelium. Invest Ophthalmol
Vis Sci. 1990;31:2520-2528.
43. Alexander JP, Samples JR, Van Buskirk EM, Acott TS.
Expression of matrix metalloproteinases and inhibitor
by human trabecular meshwork. Invest Ophthalmol Vis
Sci. 1991; 32:172-180.
44. Auble DT, Brinckerhoff CE. The AP-1 sequence is nec-
essary but not sufficient for phorbol ester induction
of collagenase in fibroblasts. Biochemistry. 1991 ;30:
4629-4635.
45. Emonard H, Grimaud J-A. Matrix metalloproteinases:
A review. Cell Mol Biol. 1990;36:131-153.
46. Matsuo T, Cynader MS. Localisation of prostaglandin
F
2a
and E
2
binding sites in the human eye. BrJ Ophthal-
mol. 1992;76:210-213.
47. Matsuo T, Cynader MS. The EP
2
receptor is the pre-
dominant prostinoid receptor in the human ciliary
muscle. Br J Ophthalmol. 1993;77:110-114.
48. Shirinsky VP, Sobolevsky AV, Grigorian GY, Danilov
SM, Tararak EM, Tkachuk VA. Agonist-induced poly-
phosphoinositide breakdown in cultured human endo-
thelial and vascular smooth muscle cells. Health Psy-
chol 1988;7(suppl):61-74.
49. Beusenberg FD, Van Amsterdam JG, Hoogsteden
HC, et al. Stimulation of cyclic AMP production in
human alveolar macrophages induced by inflamma-
tory mediators and beta-sympathicomimetics. Eur J
Pharmacol. 1992; 228:57-62.
50. Crawford KS, Kaufman PL, Hubbard WC, Woodward
DF: The DP-receptor agonist SQ27986 raises but
does not lower intraocular pressure in ocular normo-
tensive monkeys./ Glaucoma. 1992; 1:94-99.
51. Bito LZ, Camras CB, Gum GG, Resul B. The ocular
hypotensive effects and side effects of prostaglandins
on the eyes of experimental animals. In: Bito LZ,
Stjernschantz J, eds. The Ocular Effects of Prostaglan-
dins and Other Eicosanoids, New York: Alan R Liss;
1989:349-368.

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