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.
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