From the Allergan, Inc., Irvine, California 92612
Received for publication, January 30, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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Connective tissue growth factor (CTGF)1 is also a cysteinerich, CCN family protein. CTGF is a fibrogenic cytokine, a growth factor that is required for the fibrotic response mechanism in tissues (3). Unlike Cyr61, it functions as an autocrine growth factor, which acts on the same cells that produce it, causing the cells to proliferate, differentiate, and produce more collagen (4). Thus, both Cyr61 and CTGF are multifunctional, extracellular matrix-associated signaling proteins that directly regulate cell adhesion, migration, proliferation, survival, and differentiation.
Cyr61 and CTGF may also play a role as secreted proteins involved in tissue
remodeling. This may be of some importance for understanding the underlying
mechanisms involved in ocular hypotension produced by certain drugs used as
glaucoma therapy. The ocular hypotensive effects prostaglandins, prostamides,
and 2-adrenoreceptor agonists (exemplified by brimondine)
involve an increase in uveoscleral outflow of aqueous humor
(58).
Increases in uveoscleral outflow result from a remodeling of the ciliary body
such that aqueous humor outflow is increased through widened interstitial
spaces between ciliary muscle bundles
(912).
Prostaglandins are the products of cyclooxygenase-catalyzed metabolism of
arachidonic acid (13).
Prostaglandin receptors have been classified into eight subtypes, FP, DP, IP,
TP, EP1, EP2, EP3, and EP4. FP,
TP, and EP1 receptors trigger Gq protein-coupled
mechanisms involving Ca2+ signaling, inositol 1,4,5-trisphosphate
turnover, and activation of protein kinase C
(14), whereas EP2,
EP3, EP4, DP, and IP receptors trigger
Gs protein coupled to adenylate cyclase and activation of
protein kinase A. FP or EP2 receptors activate different
intracellular mechanisms and result in the reduction of intraocular pressure
in experimental animal models
(15,
16).
In addition to prostaglandin FP and EP2 receptor selective ligands, prostamides, a novel class of compounds where the -COOH typical of prostaglandins is replaced by an amide group, were identified as highly efficacious ocular hypotensives in animal models and human subjects (17, 18). Prostamides are formed from anandamide via metabolic transformation catalyzed by cyclooxygenase-2 (19). To date, the physiological actions of prostamides have not been fully investigated, and little is known about their mechanisms of actions. The activities of prostamides at prostaglandin receptors have been investigated, but, compared with their corresponding natural prostaglandins, have been shown to exert only very weak activity (17, 20, 21). Experimental evidences suggest that prostamides may interact with their own receptors (17, 21).
Using a gene chip technology, we first identified that
PGF2 dramatically up-regulated Cyr61 and CTGF mRNA
expression. Because both Cyr61 and CTGF play important roles in ECM
remodeling, we further compared the effects of prostaglandin FP receptor
stimulation (PGF2
), an EP2-selective agonist
(Butaprost), and a synthetic prostamide analog (Bimatoprost) on the regulation
of Cry61 and CTGF mRNA expression. In this study, we report differential
regulation of Cyr61 and CTGF mRNA expression following PGF2
,
Butaprost, or Bimatoprost treatments in cultured human primary trabecular
meshwork (TM) and ciliary smooth muscle (SM) cells, which are cells critical
in aqueous humor drainage from the eye.
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MATERIALS AND METHODS |
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Human ciliary SM cells were isolated from a 69-year-old male donor eye (National Disease Research Interchange, Philadelphia) and cultured in DMEM with 10% fetal bovine serum and 0.5% penicillin/streptomycin according to the method reported previously by Wolde-Mussie et al. (24).
Human TM cells were a gift from Dr. J. Polansky (University of California, San Francisco, CA). The human TM cells were derived from a 30-year-old male donor eye and cultured in DMEM with 10% fetal bovine serum and 0.5% penicillin/streptomycin in humidified 8% CO2, 92% air at 37 °C. Both human primary TM and SM cells were grown to confluence before addition of the appropriate compounds.
Stock solutions of PGF2, Butaprost, and Bimatoprost were
prepared in Me2SO. The treated cells were incubated with graded
concentrations of PGF2
, Butaprost, or Bimatoprost, and the
control cells received equivalent vehicle treatment.
Cat Iris Tissue Bath StudyCat eyes were enucleated immediately following intravenous euthanization with 1 ml of Eutha-6 (a mixture of barbiturates) and placed on ice. Eyes were then hemisected along the ora serata to expose the posterior chamber of the anterior segment. After removing the lens by clipping the zonules, the iris was peeled away from the sclera, cut equally into two pieces (for treatment and control), and placed in a 10-ml jacketed organ bath containing Krebs buffer (NaCl 118 mM, KCl 4.7 mM, KH2PO4 1.2 mM, CaCl2 1.9 mM, MgSO4 1.18 mM, NaHCO3 25 mM, glucose 11.7 mM, Indomethacin 1 µM). Krebs buffer was gassed with 95% O2/5% CO2 to obtain a pH of 7.4, and the temperature was maintained at 37 °C. After an equilibration period of 15 min, the tissue specimens were incubated for 6 h in the presence or absence of test compounds. All animals were managed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) resolution on the Use of Animals in Ophthalmic and Vision Research.
Plasmids and Luciferase Reporter AssayA DNA fragment containing the CTGF promoter region (-2047 to +65) (25) was cloned from human genomic DNA (Clontech). The fragment was subcloned into a pGL3 luciferase expression vector (Promega), creating pGL3-CTGF-LUC plasmid. The Cyr61 promoter region (-885 upstream of translation start site (ATG) to -31 from ATG) (26) was isolated from human genomic DNA and subcloned into pGL3 luciferase expression vector (Promega) at KpnI/XhoI site to create a pGL3-Cyr61-LUC plasmid.
Luciferase reporter plasmids were transfected into HEK 293/EBNA cells
stably expressing human FP receptors using the FuGENE 6 transfection method
(Roche Diagnostics), according to manufacturer's instruction. In brief, the
cells were washed twice and resuspended in 1 ml of DMEM. 0.2 µg of plasmid
DNA in 1 ml of DMEM containing 0.6 µl of FuGENE 6 solution was mixed with
the cell suspension, and the cells were cultured for 24 h at 37 °C.
PGF2 or Bimatoprost at concentrations ranging from
10-11 to 10-6 M were added to the culture,
and 6 h later, the cells were harvested and lysed in 100 µl of lysis buffer
(Promega). 20 µl of soluble extracts were assayed for the luciferase
activity. The luciferase assay was performed with a Promega assay kit at room
temperature using an Autolumat LB 953 (EG&, Berthold, Germany). Luciferase
content was measured by calculating the light emitted during the initial 10 s
of the reaction. Relative luciferase activity was expressed as -fold values of
ratio compared with control. The luciferase assay results shown in figures are
representative of experiments independently repeated at least three times.
RNA Preparation and Northern Blot AnalysisTotal RNA was isolated from cells and cat iris tissue using an RNase kit (Qiagen) according to its manufacturer's instruction. RNA concentrations were determined by a UV spectrophotometer (Beckman DU640) at A 260 nm, and storied at -80 °C.
Total RNA (10 µg) was denatured at 65 °C in RNA loading buffer
(Ambion, Inc.) for 15 min and separated on 1.2% agarose gels containing 0.66
M formaldehyde. RNA loading was assessed by ethidium bromide
staining of 28 and 18 S ribosomal RNA bands. The relative intensities of 28
and 18 S ribosomal RNA bands were used as internal controls to normalize the
hybridizations. Human 0.4-kb CTGF (+899 to +1326; GenBankTM accession
number M92934
[GenBank]
) or 1.4-kb Cyr61 (+60 to +1459; GenBankTM accession number
AF003594
[GenBank]
) gene-specific DNA fragment was radiolabeled using an
[-32P]dCTP and Klenow (Ambion, Inc.). The blots were
hybridized with the gene-specific probes in 50% formamide, 4x SSC,
1x Denhardt's solution, 50 mM sodium phosphate, pH 7.0, 1%
SDS, 50 µg/ml yeast tRNA, and 0.5 mg/ml sodium pyrophosphate at 42 °C
overnight and washed with 2x SSC and 0.1% SDS twice at 42 °C and
0.1x SSC and 0.1% SDS twice at 42 °C. The hybridized blots were
exposed to phosphor screens, and the exposed screens were analyzed in a
PhosphorImager (Amersham Biosciences).
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RESULTS |
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Multiple Signal Transduction Pathways Are Involved in the
PGF2- and Butaprost-induced Up-regulation
of Cyr61 and CTGF mRNA ExpressionTo clarify the signal
transduction pathways associated with PGF2
- and
Butaprost-induced Cyr61 and CTGF mRNA expression, pathway-specific inhibitors
were utilized to distinguish the intracellular mechanisms. Both hFP- and
hEP2-HEK 293/EBNA cells were pretreated with each of these
inhibitors (Toxin B 1 ng/ml, GF 109203X 2.5 µM, PD 98059 20
µM, BAPTA 2.5 µM) for 30 min, and the incubation
was continued with 10-7 M PGF2
or
Butaprost for an additional 6 h. A Rho inhibitor (Toxin B) completely blocked
PGF2
-induced Cyr61 mRNA up-regulation
(Fig. 2a), whereas a
protein kinase C inhibitor (GF 109203X), a MAP kinase inhibitor (PD 98059),
and a Rho inhibitor (Toxin B) partially inhibited
PGF2
-induced CTGF mRNA up-regulation
(Fig. 2b). These
results suggested that PGF2
-induced Cyr61 mRNA upregulation
is via the Rho pathway, but PGF2
-induced CTGF mRNA
expression is through multiple pathways that involved the activation of
protein kinase C, MAP kinase, and activation of small G protein, Rho.
Butaprost-induced Cyr61 mRNA upregulation appears different from that of
PGF2
(Fig.
3). Both MAP kinase and Rho inhibitors attenuated
Butaprost-induced up-regulation of Cyr61 mRNA expression, suggesting not only
the Rho pathway but also MAP kinase involvement.
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Novel Transcript Synthesis Is Required for the Up-regulation of Cyr61
and CTGF mRNA ExpressionUp-regulation of Cyr61 and CTGF mRNA in
PGF2- and Butaprost-treated hFP- and hEP2-HEK
293/EBNA cells may be because of an increase in the rate of synthesis, a
decrease in the rate of degradation, or a combination of both. To test these
possibilities, a transcription inhibitor, actinomycin D (10 µg/ml; actinD),
was used to pretreat the cells for 30 min with continued incubation with
10-7 M PGF2
or Butaprost for 6 h.
ActinD, at a concentration of 10 µg/ml, completely prevented
PGF2
-induced up-regulation of Cyr61 and CTGF mRNA expression
(Fig. 4, a and
b). ActinD not only prevented Butaprost-induced
up-regulation of Cyr61 mRNA expression but also significantly decreased Cyr61
mRNA level below the basal line (Fig.
4c). Thus, it is most likely that the rates of Cyr61 and
CTGF mRNA transcription are increased by PGF2
and
Butaprost.
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To determine whether PGF2- and Butaprost-induced
up-regulation of Cyr61 and CTGF mRNA requires de novo protein
synthesis, a protein synthesis inhibitor, cycloheximide (10 µg/ml), was
used to pretreat the cells for 30 min with continued incubation with
10-7 M PGF2
or Butaprost for 6 h.
Cycloheximide did not block PGF2
- or Butaprost-induced
up-regulation of Cyr61 and CTGF mRNA expression
(Fig. 4), suggesting that
PGF2
- and Butaprost-induced up-regulation of Cyr61 and CTGF
mRNA expression did not require de novo protein synthesis.
Differential Up-regulation of Cyr61 and CTGF mRNA Expression Following
PGF2, Butaprost, and Bimatoprost
Treatments in Cat Iris Tissue and Cultured Human Trabecular Meshwork and
Ciliary Smooth Muscle CellsUsing Cyr61 and CTGF mRNA as markers,
we further studied the mechanisms of PGF2
and Bimatoprost at
the gene expression level in cat iris. This is an isolated tissue preparation
that is sensitive to prostamides but is advantageous in that it also responds
to FP receptor agonists (17).
PGF2
or Bimatoprost at 10-7 M were
added to the isolated cat iris tissue bath and incubated for 6 h. Cyr61 and
CTGF mRNA analysis revealed that both PGF2
and Bimatoprost
up-regulated Cyr61 mRNA expression in the cat iris
(Fig. 5a). Only
PGF2
, but not Bimatoprost, up-regulated CTGF mRNA expression
in the cat iris (Fig.
5b). In further contrast to prostaglandin FP receptor
stimulation (Gq-coupled), activation of the prostaglandin
EP2 receptor (Gs-coupled) with Butaprost also
up-regulated Cyr61 (Fig.
5c) but not CTGF mRNA expression in the cat iris (data
not shown). Bimatoprost and Butaprost seemed to be very similar in the pattern
of Cyr61 and CTGF mRNA induction, although they caused opposing action in the
cat iris contraction assay. Therefore, Bimatoprost appears to interact with a
unique receptor that is neither FP nor the EP2 receptor in cat
iris.
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TM and ciliary SM cells are thought to be the major target cells in the
aqueous humor outflow pathway for glaucoma treatments. To further compare the
mechanisms of PGF2, Bimatoprost, and Butaprost in human
ocular tissues, each of the compounds at a concentration of 10-7
M was used to treat the cultured human trabecular meshwork and
ciliary smooth muscle cells for 6 h. Northern blot analysis of Cyr61 and CTGF
mRNA expression revealed that Cyr61 and CTGF mRNA inductions by
PGF2
, Bimatoprost, and Butaprost in human ciliary SM cells
are very similar to the cat iris (major cell type of cat iris is smooth muscle
cells). PGF2
, but not Butaprost and Bimatoprost,
dramatically induced up-regulation of CTGF mRNA expression in human ciliary SM
cells (Fig. 6d),
whereas PGF2
, Butaprost. and Bimatoprost all up-regulated
Cyr61 mRNA expression (Fig.
6b). PGF2
and Bimatoprost are very
similar in potency for inducing Cyr61 mRNA up-regulation, whereas Butaprost is
much less potent (Fig.
6b, bottom panel). PGF2
, but
not Bimatoprost and Butaprost, up-regulated Cyr61 and CTGF mRNA in human
trabecular meshwork cells (Fig. 6,
a and c), suggesting that the properties of TM
cells are much different from ciliary SM cells at the level of gene
transcriptional regulation. All together, we conclude that the effects of
PGF2
, Bimatoprost, and Butaprost on the Cyr61 and CTGF mRNA
expression in human ciliary SM cells are very similar to those observed in the
cat iris but are different in trabecular meshwork cells.
PGF2
, Bimatoprost, and Butaprost differentially regulated
Cyr61 and CTGF mRNA expression in smooth muscle cells, implying that
Bimatoprost might exert its pharmacological actions through a unique mechanism
in human ocular tissues, which is different from PGF2
(FP)
and/or Butaprost (EP2).
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Functional Analysis of Cyr61 and CTGF Promoter in Response to
PGF2, Butaprost, and Bimatoprost
Treatments in hFP-HEK 293/EBNA cells and
hEP2-HEK 293/EBNA Cells To
further study the transcription mechanisms of FP- and EP2-mediated
Cyr61 and CTGF mRNA expression, Cyr61 and CTGF promoters were isolated from a
human genomic DNA library and subcloned in a luciferase reporter plasmid,
pGL3. Cyr61 promoter-luciferase reporter plasmids were transfected
into hFP-HEK 293/EBNA cells and then treated with PGF2
or
Bimatoprost at concentrations ranging from 10-11 to 10-6
M. PGF2
, but not Bimatoprost, activated the Cyr61
promoter in a dose-dependent manner (Fig.
7a). This result suggested that Bimatoprost-induced
up-regulation of Cyr61 mRNA expression (see
Fig. 5a and
Fig. 6b) is not
because of the activation of the prostaglandin FP receptor but a different
receptor. In the CTGF promoter-direct reporter assay, PGF2
,
but not Bimatoprost, induced CTGF promoter activity in a dose-dependent manner
(Fig. 7c). In a
comparison study, Cyr61 or CTGF promoter was transfected into
hEP2-HEK293/EBNA cells and then treated with Butaprost at
concentrations ranging from 10-11 to 10-6 M.
Butaprost activated Cyr61 promoter (Fig.
7b) but not CTGF promoter in hEP2-HEK 293/EBNA
cells (Fig. 7d). These
data are matched to Northern blot analysis (see
Fig. 5b and
Fig. 6, c and
d).
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DISCUSSION |
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Activation of FP receptors initiated by ligand binding triggers
Gq protein-coupled mechanisms involved intracellular
Ca2+ signaling, IP turnover, and activation of protein kinase C
(28). Butaprost is a synthetic
prostaglandin analog that interacts with EP2 receptors; it triggers
G
s protein-coupled mechanisms involved in activation of
adenylate cyclase and initiation of the cAMP pathway with resultant activation
of protein kinase A (29).
Despite different intracellular mechanisms, it has been shown that activation
of prostaglandin FP or EP2 receptors resulted in lowering
intraocular pressure (15,
16). The precise mechanisms by
which PGF2
and Butaprost reduce intraocular pressure (IOP)
are not yet clear. The effects of PGF2 and Butaprost on intraocular
pressure lowering appear to be mediated through increasing uveoscleral outflow
of aqueous humor by alteration of ciliary muscle bundles and remodeling
extracellular matrix. Changes of gene expression following
PGF2
or Butaprost treatment may be attributed to the
molecular basis of prostaglandin actions.
To identify changes in gene expression that are regulated in response to
PGF2 or Butaprost stimulation, both hFP-HEK293/EBNA and
hEP2-HEK 293/ENBA cells were used to compare gene expression
profiles in unstimulated cells versus PGF2
- or
Butaprost-stimulated cells. It is shown herein that, with respect to
activation of CCN early response genes, recombinant FP and EP2
receptors expressed in HEK 293/EBNA cells are coupled to the same signal
transduction pathways as in smooth muscle cells
(30), fibroblast cells
(31), and ocular tissues
(3234).
In unstimulated hFP-HEK 293/EBNA cells, PGF2
rapidly induced
both Cyr61 and CTGF expression within 30 min of stimulation. The expression
levels of both genes reached a maximum 6 h after stimulation and stayed
elevated for at least 24 h. This time course is similar to the kinetics of
Factor VIIa and thrombin induction of human Cyr61 and CTGF expression in human
fibroblasts (35), serum
induction of murine Cyr61 expression in fibroblasts
(36), TGF-
induction of
CTGF expression in human fibroblasts
(37), and muscarinic receptor
induction of rat Cyr61 in rat primary neurons and brain
(38). Only Rho inhibition
completely blocked PGF2
-induced Cyr61 expression, whereas
PGF2
-induced up-regulation of CTGF expression could be
attenuated by the pre-incubation of Rho, protein kinase C, and MAP kinase
inhibitors. The signal mechanisms of FP-mediated up-regulation of Cyr61 and
CTGF expression are different from mechanisms initiated by other stimulators.
Protein kinase C and Ca2+ are two principal signaling mechanisms
that couple Cyr61 expression to muscarinic receptor activation
(38). Factor VIIa and thrombin
up-regulated Cyr61 through different signal mechanisms; Factor VIIa induced
Cyr61 expression via phospholipase C and activation of MAP kinase pathways,
whereas thrombin did not (35).
Lysophosphatidic acid, serotonin, and TGF-
-induced CTGF up-regulation
was dependent of the Rho pathway but independent of the MAP kinase protein
kinase C pathways (39). In
comparison with the Gq-coupled FP receptor, activation of the
Gs-coupled EP2 receptor by Butaprost also induced Cyr61
expression in a similar kinetic manner as PGF2
, but
Butaprost did not induce CTGF expression. EP2-mediated Cyr61
expression is via the Rho and MAP kinase pathways. Signal transduction
pathways of FP- and EP2-mediated Cyr61 and CTGF mRNA expression are
summarized in a diagram in Fig.
8. Rho is a common signal mechanism by which FP and EP2
activation is coupled to Cyr61 expression. Rho, protein kinase C, and MAP
kinase pathways were coupled to FP-mediated CTGF expression, suggesting
multiple mechanisms are involved in. Both Cyr61 and CTGF belong to the CCN
gene family and regulate ECM remodeling through activation of matrix
metalloproteinases (2). FP
receptor activation up-regulates both Cyr61 and CTGF, whereas EP2
receptor stimulation causes only Cyr61 up-regulation, which may reflect
different mechanisms of FP- and EP2 receptor-mediated ECM
remodeling. This result further allowed us to use CTGF and Cyr61 gene
expression as markers to further differentiate down stream signaling after
treatment with Bimatoprost versus PGF2
.
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Bimatoprost represents a novel class of anti-glaucoma compounds where the
-COOH typical of prostaglandins is replaced by an amide group
(17). It has been clinically
proven to be a very efficacious IOP lowering drug
(18). The mechanisms of its
actions have not been fully investigated, and little is known about its
intracellular signal transduction pathways and its receptor system. Although
the activities of prostamides at prostaglandin receptor(s) have been
investigated, they have been shown to exert no meaningful activity
(17). In this and previous
isolated cat iris contraction studies, we found that both
PGF2 and Bimatoprost triggered cat iris smooth muscle
contraction at very similar EC50
(17). Thus, the isolated cat
iris sphincter was used as a prostamide-sensitive pharmacological preparation
and a model for comparison of gene expression for all three drug classes. Gene
expression studies revealed that PGF2
, Bimatoprost, and
Butaprost up-regulated Cyr61 mRNA expression in the cat iris.
PGF2
, but not Bimatoprost, up-regulated CTGF mRNA expression
in the cat iris. Similar data were also observed in the human primary ciliary
smooth muscle cells treated with PGF2
and Bimatoprost.
Therefore, PGF2
and Bimatoprost appear to interact with
different receptors in cat iris and human ocular tissues. Bimatoprost failed
to stimulate up-regulation of Cyr61 in human TM cells, suggesting either that
these cells are lacking Bimatoprost-sensitive receptors or an insufficiency of
signal mechanisms coupled to Cyr61 expression.
Actinomycin D blocked Cyr61 and CTGF mRNA expression in response to
PGF2 and Butaprost treatments, which implied that
transcription mechanisms were involved in PGF2
- and
Butaprost-induced up-regulation of Cyr61 and CTGF expression. Human Cyr61 and
CTGF promoters (hCyr61 and hCTGF) have been identified previously
(25,
26) to be functional
promoters. hCyr61 promoter located at -885 bp to -31 bp of Cyr61 gene have
been shown to contain a TATA box, CEBP-
, CREB, and CA repeat elements
(26). In hCyr61 promoter
reporter gene assays, PGF2
and Butaprost, activated Cyr61
promoter in a dose-dependent manner in hFP and EP2-HEK 293/EBNA
cells. Bimatoprost did not activate Cyr61 promoter, because it did not
activate either FP in HEK 293/EBNA cells
(Fig. 7a). Bimatoprost
may interact with its own receptor in human ciliary smooth muscle cells and
mediate Cyr61 expression. The hCTGF promoter contained TATA box, AP-1, C/EBP,
and Smads binding elements
(25). In hCTGF reporter gene
assays, PGF2
activated CTGF promoter in a dose-dependent
manner in hFP-HEK 293/EBNA cells, but Butaprost did not activate the CTGF
promoter in hEP2-HEK 293/EBNA Cells. These data suggested that
activation of FP receptor directly initiated and regulated CTGF expression
through promoter-directed transcriptional mechanisms, and EP2
receptor-mediated pathways may not couple to CTGF expression. The reporter
gene assays are consistent with the Northern blot analysis in this study, and
the data further support the contention that Bimatoprost does not interact
with FP receptor but rather a different receptor.
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FOOTNOTES |
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To whom Correspondence should be addressed: Dept. of Biological Science,
Allergan, Inc., 2525 Dupont Dr., Irvine, CA 92612. Tel.: 714-246-5490; Fax:
714-246-5578; E-mail:
Woodward_David{at}Allergan.com.
1 The abbreviations used are: CTGF, connective tissue growth factor;
PGF2, prostaglandin F2
; ECM, extracellular
matrix; TM, trabecular meshwork; SM, smooth muscle; HEK, human embryonic
kidney; DMEM, Dulbecco's modified Eagle's medium; MAP, mitogen-activated
protein; actinD, actinomycin D; FP, prostaglandin FP receptor; EBNA,
Epstein-Barr nuclear antigen.
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REFERENCES |
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