| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
INSERM, U-361, Maternité Port-Royal-Cochin, Université René Descartes, 75014 Paris, France
Address all correspondence and requests for reprints to: M. J. Leroy, Ph.D., INSERM U-361, Pavillon Baudelocque, 123 boulevard Port Royal, 75014 Paris, France. E-mail: leroy-zamia{at}u361.cochin.inserm.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we used a model of human myometrial cells in culture to determine whether an elevated cAMP concentration could influence PDE expression. As in myometrial tissue, high levels of PDE4 activity were detected in these smooth muscle cells. Long term treatment with 8-bromo-cAMP or forskolin resulted in a selective induction of PDE4B and of PDE4D short form messenger RNA variants. Concurrently, an increased immunoreactive signal for the PDE4B- and PDE4D-related isoenzymes was detected. This induction was consistent with an observed significant up-regulation of PDE4 activity.
Accordingly, our results demonstrate that in human cultured myometrial cells, cAMP-elevating agents manipulate PDE4 activity through selective induction of synthesis of PDE4B and PDE4D short forms. Such a mechanism might have physiological importance during pregnancy by dampening hormonal stimulation and could thereby be involved in tolerance to the tocolytic effect of ß-adrenoceptor agonists.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although little information is available concerning the biochemical mechanisms by which human myometrium integrates hormonal stimulation through specific intracellular effectors, it is now well established that smooth muscle relaxation is partly a consequence of elevation in intracellular cyclic nucleotides, i.e. cAMP and cGMP (2). Hence, modulation of rates of such second messenger formation and degradation is considered to play a pivotal role in the uterine contraction/relaxation process.
Of particular interest are changes in cyclic nucleotide phosphodiesterase activity (PDE), the sole mechanism for inactivating cAMP or cGMP through their hydrolysis to 5'-AMP or 5'-GMP, respectively. This enzymatic system provides a powerful means of manipulating the magnitude and duration of the biological response to these signal-transducing molecules (3). Moreover, PDE is readily manipulated pharmacologically (4). Thus, inhibition of PDE offers an important potential in an attempt to modulate the myometrial activity of women in preterm labor.
PDE cyclic nucleotides comprise an abundant group of structurally related isoenzymes, derived from at least seven distinct, but related, gene families. Strongly conserved in evolution, all of the mammalian PDEs that have been isolated contain a putative catalytic domain with approximately 30% amino acid identity. Because they are differentially expressed and regulated, PDE1 to PDE7 families are found in different amounts, proportions, and subcellular locations depending on the cell, tissue, and species (5, 6).
In human myometrium, we previously observed modifications in the kinetic behavior of the cAMP PDE enzyme during pregnancy compared with nonpregnant tissue (7, 8). Five distinct families (PDE1–5) have been identified in human myometrium extracts. The predominant forms of PDE in myometrial tissue belong to the PDE4 isozyme family (9). Also, rolipram, a selective inhibitor of PDE4, has been shown to exert a potent relaxant effect on spontaneous contractions of human myometrial strips (10).
PDE4 enzymes (cAMP-specific PDE gene family) are characterized by their high affinity for cAMP. They are insensitive to cGMP, which is a poor substrate for these enzymes. Four human and rat PDE4 genes (4A, 4B, 4C, and 4D) have been identified. The four human and rat loci present a one to one correspondence and pairwise homolog variants generated by a complex arrangement of the transcriptional units (11, 12). The PDE4D gene is transcribed after at least three upstream promoters with the formation of the so-called long forms of the enzyme, whereas activation of a further intronic promoter produces the short forms (13). Both short and long forms of the enzyme can also be transcribed from PDE4A, PDE4B, and PDE4C genes (14, 15, 16).
Recently, we reported the concurrent expression of the four PDE4 genes, 4A, 4B, 4C, and 4D, in human myometrial tissue. Our studies using a semiquantitative RT-PCR approach suggested high steady state levels of PDE4D and PDE4B messenger RNAs (mRNAs), whereas those of PDE4A and PDE4C were less elevated. Furthermore, we showed an increase in the proportion of only PDE4B short form transcripts in the myometria of pregnant women compared with that in nonpregnant tissue (17).
Interestingly, the PDE4 affinity constant for cAMP corresponds closely to the level of intracellular cAMP observed after hormonal regulation. Thus, it has been suggested that according to the cell-specific environment, PDE4 isoenzymes are essential modulators of cAMP concentration during hormonal stimulation (18). Furthermore, it became obvious that these forms are regulated by their own substrate (cAMP) both at the gene expression level and in the posttranslational modifications (19, 20, 21). With such mechanisms, PDE4 isozymes can provide both a short and a long term feedback regulation of cAMP content.
In light of this information, the present studies were conducted to determine whether the elevated intracellular cAMP content that occurs after stimulation of adenylate cyclase by numerous factors throughout pregnancy could modulate the PDE4 expression pattern in human myometrium and thereby influence the rate of its own hydrolysis. For this purpose, we used a model of human myometrial cells in culture, under conditions supporting the maintenance of a smooth muscle differentiated state (22). We explored the effects of long term treatment of cAMP-elevating agents on cellular expression of the different PDE4 isozymes. We show that such treatments lead to a marked increase in PDE4 activity, with changes occurring selectively in the expression of variant products of the PDE4B and PDE4D genes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-smooth muscle actin, myosin heavy chain isoforms
(SM1, SM2), and desmin (22). For this particular experimental procedure, cells were then incubated for various times with vehicle, 8-bromo-cAMP (8Br-cAMP; 0.1–2 mM) dissolved in water, or forskolin (1–50 µM) dissolved in ethanol at a 0.1% final concentration. We controlled that the presence of 0.1% alcohol had no effect on PDE activity. When actinomycin D or cycloheximide was used, it was dissolved in water and added together with the tested substances at the start of the incubation period.
cAMP-PDE assays
After 8Br-cAMP or forskolin treatment, media were removed,
and cells (5 x 105/35-mm dish·treatment) were
washed twice in cold PBS. Cells were then harvested by scraping in
ice-cold homogenization buffer. The homogenization buffer comprised 100
mM Tris-HCl (pH 7.4), 2 mM MgSO4, 2
mM EDTA, 10% glycerol, and 1 mM
ß-mercaptoethanol and was supplemented with a protease inhibitor
cocktail: leupeptin (1 µM), aprotinin (10 µg/ml),
Pefabloc (25 µg/ml) (Interchim, Montluçon, France),
benzamidine (130 µg/ml) and soybean trypsin inhibitor (50 µg/ml).
After sonication, samples were immediately stored at -20 C until
use.
cAMP PDE activity was determined using the Kincaid and Manganiello method (23). Activities were measured in high affinity conditions with 1 µM cAMP as substrate in the presence or absence of the different selective inhibitors added 10 min before the beginning of the reaction and are expressed as specific activity (picomoles per min/mg protein). All assays were carried out in the linearity conditions with respect to time and protein concentration. PDE4 activity was gauged as the fraction of total cAMP PDE activity that was inhibited by 10 µM rolipram; non-PDE4 activity was estimated as the remaining cAMP PDE activity. Protein concentrations were determined using the Bio-Rad modified Bradford (24) protein assay (Bio-Rad Laboratories, Inc., Richmond, CA) with BSA as a standard.
cAMP assay
To measure cAMP content, cells (5 x 105/35-mm
dish·treatment) that were previously incubated with drugs were
exposed to ice-cold trichloroacetic acid (10%) to stop the reaction
(25) and then were scraped and stored at -20 C. At the time of cAMP
assays, cell preparations were thawed on ice, and the precipitated
proteins were separated from the soluble extracts by centrifugation at
300 x g for 10 min at 4 C. Trichloroacetic acid was
removed from the sample by four successive extractions with
water-saturated ethyl ether. cAMP content was measured after
acetylation using a commercially available RIA kit (Biotrak,
Amersham, Aylesbury, UK). cAMP content was expressed as
picomoles of cAMP per 106 cells (26).
RT-PCR analysis
Total RNA was extracted from myometrial cells using the Trizol
reagent method (Life Technologies, France).
Briefly, scraped cells (107) were resuspended in 1 ml
Trizol and homogenized by repeated pipetting. RNA preparations were
recovered by phenol/chloroform extraction, isopropanol precipitation,
and ethanol washing, according to the manufacturer’s instructions.
The first strand of complementary DNA (cDNA) was generated from 8 µg total RNA using random hexamers to prime the RT in a total reaction volume of 50 µl. Essentially, total RNA was denatured by heating at 72 C for 10 min and cooling immediately on ice. This preparation was then incubated with 800 U murine reverse transcriptase (Life Technologies) in the presence of 10 mM dithiothreitol, 20 µM random hexamers, and 20 U ribosomal RNasin ribonuclease inhibitor (Promega Corp., Madison, WI) for 60 min at 39 C. The reaction was stopped by heating at 95 C for 5 min followed by cooling. RT products were stocked at -20 C. Preparations achieved without reverse transcriptase were routinely used as a control of each RNA sample. No PCR product was detected in the absence of reverse transcriptase during the RT step, indicating that the RNA preparations were free from intact genomic DNA.
Amplification was performed in 1 x PCR buffer (50 mM
KCl and 20 mM Tris-HCl, pH 8.3) in a 25-µl total reaction
volume. This contained 200 µM of each deoxy-NTP and 1–2
mM MgCl2 together with 1 µM of
each primer, sense and antisense, 1.25 U Taq DNA polymerase
(Life Technologies), and 3 µl of the RT product (480 ng
cDNA). The amplification profile consisted of denaturation at 94 C for
1 min, annealing for 1 min at the specific temperature (Table 1
), and extension at 72 C for 1 min, with
a final extension at 72 C for 10 min. The primers for each studied PDE4
(Table 1) were designed from the reported primary sequences (12, 27, 28, 29, 30) deposited with the GenBank database. After the indicated
subsaturating cycles of denaturation and extension, a 15-µl aliquot
from each reaction mixture was resolved by electrophoresis on a 3%
Nusieve agarose gel and visualized by ethidium bromide staining under
UV light. The DNA molecular mass standards ladder consists of fragments
between 100-1500 bp in multiples of 100 bp; the 600-bp band is brighter
than the other ladder bands (100-bp DNA ladder, Life Technologies). Additional validity control was achieved by a
Southern blot analysis of the PCR product as previously described (17)
(data not shown).
|
The intensities of the bands on Polaroid (European Imap Scientist, Massy, France) pictures of the ethidium bromide-stained gels were analyzed densitometrically using a computer-linked scanner and the NIH Image 1.60 software package (NIH, Bethesda, MD). The results are expressed in arbitrary densitometric units (ADU) as the mean ± SEM.
Immunodetection of PDE4 isozymes
Immunoblotting was carried out using monoclonal antibodies
designed to be specific for particular PDE4 isozymes (donated by Dr. K.
Ferguson, ICOS Corp., Seattle, WA). They were raised against
glutathione-S-transferase fusion proteins formed from a
portion of the C-terminal end found to be unique to each of the PDE4
isozymes and which appear to be common to all active proteins produced
by a particular PDE4 family isoform. The monoclonal antibodies were the
PDE4B-specific species 96G7A, which can recognize amino acids
529–558 in clone TM72 (GenBank accession no. L20966) found in the
common C-terminus of PDE4B isoforms, and the PDE4D-specific species
61D10E, which can recognize amino acids 618–634 in clone PDE43
(GenBank accession no. L20970) found near the C-terminus of all known
active PDE4D splice variants, but not in any other PDE4 classes (20, 36).
After their exposure to the various agents, cells were harvested and sonicated in the homogenization buffer described above. They were immediately frozen and stored at -80 C until use. Samples (50 µg of proteins/lane) were dissolved (vol/vol) in Laemmli buffer (37) and boiled for 5 min before electrophoresis on a 8% SDS-PAGE. Then proteins were transferred to a nitrocellulose membrane (Amersham) in a Bio-Rad Transblot apparatus. Blots were dried for 1 day and then blocked for 1 h in 10% nonfat dried milk powder in TBS-T (Tris 10 mM, NaCl 150 mM, and Tween-20 0.1%, pH 7.6) at room temperature. Blocked membranes were washed three times with TBS-T. The blots were then incubated overnight at 4 C with a 1:10,000 dilution of the primary antibody (in TBS-T containing 1% nonfat dried milk powder). After three washes with TBS-T, blots were incubated for 45 min with horseradish peroxidase-linked antimouse IgG whole antibody from sheep (DAKO Corp., Glostrup, Denmark) diluted 1:2,000 and washed five times with TBS-T. Immunoreactive proteins were detected by chemiluminescence (Amersham ECL reagents). The intensities of the bands were analyzed densitometrically by the NIH software package and expressed in ADU as the mean ± SEM.
Another set of antibodies (donated by Dr. M. Conti, Stanford University, Stanford, CA) was used for additional controls: K118, rabbit polyclonal antibodies raised against PDE4B, and M3S1 murine monoclonal antibodies directed against PDE4D (38). They were used as described above.
Statistical analysis
The nonparametric Wilcoxon-Mann-Whitney test for paired samples
was applied for comparison of the cAMP PDE activities of cultured
myometrial cells. All results were expressed as the mean ±
SEM. The difference was considered significant when
P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, addition of
isobutylmethylxanthine (IBMX), a nonselective PDE inhibitor, resulted
in a concentration-dependent inhibition of total homogenate cAMP PDE
activity in these smooth muscle cells. Even using high doses of IBMX,
10% of PDE activity remained; hence, we could not exclude the
expression of PDE7 isoforms, an IBMX-resistant PDE family for which no
specific inhibitor is yet available. The selective PDE1 inhibitor,
8-methoxy-IBMX, decreased cAMP PDE activity by about 20%, suggesting
the presence of a subtype of this family. Zaprinast, a PDE5 inhibitor,
led to the same weak, but significant, decrease in cAMP PDE activity.
cGMP present at low concentration was responsible for about 20%
inhibition of the activity. The degree of inhibition was similar when
using cilostamide, an inhibitor selective for the cGMP-inhibited PDE3
family, indicating contributions of PDE3 isoforms. cGMP-stimulated PDE2
appeared to be a minor component of the activity, with no significant
effect of EHNA, described as a PDE2 inhibitor. In contrast, rolipram, a
selective inhibitor of PDE4, caused a 50% significant decrease in cAMP
PDE activity. Together, these results indicated that although cAMP PDE
activity inhibition was seen with PDE1, PDE3, and PDE5 inhibitors, PDE4
was responsible for the major cAMP hydrolysis activity in human
myometrial cells. Because no difference was observed in the inhibition
degree when using 10 or 50 µM of rolipram, we defined
PDE4 activity as the cAMP PDE activity inhibited by 10 µM
rolipram in the experiments which followed.
|
, both of these treatments led to a
slight, significant increase in the cAMP PDE activity of these cells.
The PDE4 activity increased dramatically after both treatments, whereas
rolipram-resistant PDE activity (non-PDE4 activity) remained identical
in control cells vs. treated cells.
|
, the
drug produced a 40-fold increase in cAMP content within 1 h of
incubation. After 5 h of treatment, the cAMP content was still
greater than that of control cells, but did not differ from that
observed after a 1-h exposure. Addition of rolipram, which prevents
degradation of cAMP due to the PDE4 family, had no effect on basal cAMP
content, but during forskolin treatment, it permitted a time-dependent
accumulation of the cyclic nucleotide, reaching a 100-fold rise within
5 h.
|
). After exposing the cells to 1
mM of the cAMP analog, PDE4 activity rose during the first
5 h, reaching a value significantly different from that of control
cells. The significant increase in PDE4 activity was still seen at
18 h of treatment. The ability of 8Br-cAMP to raise PDE4 activity
was also concentration dependent (Fig. 3B). We observed a significant
rise in PDE4 activity from the 0.5 mM cAMP analog, and a
2-fold induction was obtained with 2 mM 8Br-cAMP compared
with untreated cells.
|
show the
time course for the PDE4 activity induction in response to forskolin
treatment. When challenged with 10 µM forskolin, PDE4
activity increased rapidly within 2 h and reached values
significantly different from those of control cells after 5 and 18
h of of treatment. Examination of the PDE4 activity in cells incubated
for 5 h with a range of 1–50 µM forskolin revealed
a significant concentration-dependent increase. The rise attained a
maximum with 10 µM forskolin (Fig. 4B).
|
). The stimulation induced by forskolin
was partially blocked by both inhibitors. When used alone, actinomycin
D or cycloheximide caused a slight, but nonsignificant, decrease in
PDE4 activity compared with control cells.
|
). As illustrated in Fig. 6, A and B, the four PCR amplifications
yielded fragments of the appropriate size, as assessed by the mol wt
standard migrations, in control and treated cells. We verified
successful normalization of RNA amounts by obtaining equivalent
intensity for the ß2-microglobulin band in control
vs. treated cells. When control cells were treated with
actinomycin D alone, the signals for PDE4A decreased slightly as did
those for PDE4B and PDE4D, whereas no change was seen for PDE4C or
ß2-microglobulin. In the RT-PCR preparations from
8Br-cAMP- and forskolin-exposed cells, no difference in signal
intensity was seen for the PDE4A and PDE4C products, whereas a slight
elevation of the band intensity for the PDE4D fragment and a notable
increase in the PDE4B PCR signal were observed. Incubation of the cells
with 8Br-cAMP or forskolin in the presence of actinomycin D prevented
the increase in signals for PDE4B and PDE4D.
|
|
), signals for the short form of PDE4B, namely PDE4B2
(567 bp), and long forms of PDE4B (674-bp fragment present in PDE4B1
and PDE4B3) were revealed by ethidium bromide (Fig. 7, A and B). We were able to detect in
untreated cells, a signal for the long forms of PDE4D, i.e.
a fragment of 642 bp found in PDE4D3, 4D4, and 4D5. Signals for the
short forms of PDE4D, a 530-bp fragment present in PDE4D1 and a 456-bp
fragment present in PDE4D2, were also obtained (Fig. 7).
|
Effect of long-term exposure to 8Br-cAMP or forskolin on PDE4B and
PDE4D immunoreactivities
To further characterize the PDE4B and PDE4D isoforms induced by
cAMP-elevating agents, we conducted Western blot analyses using
antibodies raised against specific epitopes of PDE4B and PDE4D
proteins.
An immunoblot representative of results using the anti-PDE4B monoclonal
antibodies (96G7A) is shown in Fig. 8, A
and B. In control cells, we observed a 76-kDa signal and two
immunoreactive species of 98 and 105 kDa, respectively. Upon treatment
with 8Br-cAMP or forskolin, the intensity of the signals corresponding
to the latter two PDE4B isoforms decreased slightly, whereas the
intensity of the 76-kDa band was stronger. The 76-kDa signal had the
same migration on SDS-PAGE as the short form PDE4B2 detected in human
monocytes (20). The 105- and 98-kDa signals might have corresponded to
the PDE4B long forms 4B3 and 4B1, even though some differences existed
in molecular mass estimation compared with human PDE4B species
expressed in COS-7 cells (39). These three immunoreactive bands were
also detected when using polyclonal PDE4B-specific K118 antibodies,
raised against a different PDE4B-specific epitope (data not shown).
|
, A and B).
The 67-kDa signal in untreated cells was more intense than the 72-kDa
signal. In 8Br-cAMP- or forskolin-treated cells, the signals detected
at 72 and 67 kDa were stronger than those in control cells, and the
band at 93 kDa almost disappeared, whereas the intensity of the 105-kDa
signal did not change. The 72- and 67-kDa signals had the same
migration behavior as the PDE4D short forms 4D1 and 4D2, previously
described in human peripheral mononuclear cells (32). This antibody
also recognized PDE4D species that have identical behavior on SDS-PAGE,
i.e. 105 and 93 kDa, and that are identified as the long
forms, PDE4D5 and PDE4D3, respectively, in HeLa cells (36). Similar
results were obtained with M3S1 monoclonal antibodies directed toward a
different PDE4D-specific epitope (data not shown). Several bands of molecular masses lower than 50 kDa were also present on the immunoblots and probably resulted from partial proteolysis (40).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We first established a pharmacological profile of the cAMP PDE activity based on the effects of family-selective inhibitors on total cAMP hydrolysis (41). The observed predominance of the PDE4 family agrees with our previous data in human myometrial tissue, in which PDE4 contributes to the major cAMP catalytic activity (9). Like whole myometrium (8), cultured myometrial cells presented additional minor cAMP activities of PDE1, PDE3, and PDE5 families, and only PDE2 was not recovered in the cells. This difference, also observed in other smooth muscle tissues and their corresponding primary cell culture, might have been due to the presence of nonsmooth muscle cellular constituents in the whole tissue or else was inherent to the cellular model (41). Nevertheless, it is evident that the strong contribution of PDE4 in our cell model, as in the tissue, leads to the recognition that PDE4 isoenzymes play a pivotal role in regulating cAMP turnover in the human myometrium.
PDE4 activity in cultured myometrial cells is significantly increased upon treatment with either 8Br-cAMP, a cell-penetrant analog of cAMP, or forskolin, a direct activator of adenylate cyclase. Change in the cAMP breakdown is PDE4 family selective, inasmuch as the procedures employed led to identical values for rolipram-resistant activity, whereas rolipram-sensitive activity was markedly enhanced. Furthermore, activation of the cAMP-mediated pathway by forskolin was clearly involved during this process, as a rise in cellular cAMP content preceded or accompanied the up-regulation of PDE4 activity.
Concurrently, our findings reveal that long-lived elevation of cAMP levels in cultured myometrial cells specifically manipulates the expression of at least two genes, PDE4B and PDE4D. We have shown that the four PDE4 genes are expressed in these smooth muscle cells, and interestingly, distinct changes in the pattern of PDE4B and PDE4D PCR products were detected after treatment with 8Br-cAMP and with forskolin. When focusing more precisely on both long and short products of these two genes, we demonstrated that only signals for the short forms of these PDE4 subtypes were enhanced in treated myometrial cells. Such results are correlated with our immunoreactivity analyses of PDE4B and PDE4D expressions. Treatment of myometrial cells with 8Br-cAMP or forskolin enhances immunoreactive PDE4B and PDE4D signals, which are detected within an apparent molecular mass range of 67–76 kDa, classically described as short form variants of PDE4B and PDE4D subtypes (11). It thus appears that long term activation of the cAMP-mediated pathway in myometrial cells induces up-regulation of cAMP hydrolysis, through an increase in specific PDE4B and PDE4D protein variants, providing a way of adapting PDE4 activity to changes in intracellular cAMP content.
The slow increase in PDE4 activity as well as the elevated steady state level of PDE4B and PDE4D subtype mRNAs in treated cells imply that regulation at the transcriptional or posttranscriptional level may occur in myometrial cells. Indeed, the rise in PDE4 activity induced by 8Br-cAMP was completely abolished by actinomycin D. The increase in PDE4B and PDE4D short forms must therefore be due in part to an increase in transcription from both of these genes. Previously reported data on the variant products of PDE4A, 4B, 4C, and 4D subtypes demonstrated that the four genes can generate long and short form variants. This strongly suggests similar organizations of the transcriptional units of these four genes (14, 15, 16). A mechanism of regulation of PDE4D short form expression is now well documented. The two PDE4D short forms, 4D1 and 4D2, which differ from each other by only a short intron sequence, are initiated from the same start site in an intronic promoter (15). The activation of this transcription initiation site is clearly cAMP dependent. Run-off assays indicated that the rate of transcription of rat PDE4D1 mRNA was substantially increased by treatments with (Bu)2cAMP or forskolin in myoblasts (42). It has been shown that exposure of Sertoli cells to (Bu)2cAMP, an analog of cAMP, leads to specific induction of synthesis of PDE4D short forms (19). Conti and co-workers have begun to address this rat PDE4D promoter. The presence of cAMP- and hormone-inducible promoter in the rat PDE4D gene, which controls the transcription of mRNAs encoding for a PDE short form variant of 68–72 kDa, has been demonstrated (43). In accordance with such mechanisms of regulation, we suggest that direct activation of PDE4B and PDE4D gene transcription may also act in myometrial cells in culture via the interaction of a cis-element present in a distinct promoter of PDE4 subtypes with trans-factors of the cAMP-dependent pathway.
Interestingly, an additional posttranslational mechanism of regulation of cAMP hydrolysis appears to occur in forskolin-treated myometrial cells. The up-regulation of PDE4 activity induced by forskolin was not completely blocked by actinomycin D or cycloheximide. Although the concentrations of these drugs might be insufficient to produce a total inhibitory effect, it is more likely that forskolin can activate PDE4 independently of new protein synthesis. In this way, PKA-mediated phosphorylation has been demonstrated to result in activation of a preexisting PDE4 (11).
Furthermore, we noted that rolipram permitted a time-dependent accumulation in cAMP content when the cells were challenged with forskolin, which indicates that PDE4 isoenzymes play an increasing role, whereas cAMP content is elevated through hormonal stimulation. These observations reflect the ability of PDE4 subtypes to hydrolyse cAMP within a concentration range higher than that of the other cAMP PDE families (11). Interestingly, in cells treated with forskolin alone, cAMP content attained an apparent steady state level that might be due to an increase in cAMP degradation by PDE4 subtypes rather than to a decrease in adenylate cyclase activity, inasmuch as cAMP content rose during the forskolin-rolipram combined treatment. This observation emphasizes the role of certain inducible PDE4 subtypes under hormonal stimulation.
In light of these observations, we conclude that the increased transcription rate is the primary cause of the increase in PDE4B and PDE4D mRNA steady state levels, but additional regulations are apparent at both the messenger and protein levels. Such intricate adjustments point out the complexity of cAMP breakdown catalyzed by PDE4 isoenzymes. Over the simplistic view of a mechanical means for removing cyclic nucleotides in excess, it is now clear that complex expression of PDE4 isoenzymes finely modulates cAMP catalysis and is clearly implicated in hormonal signalization.
As a device to control the responsiveness of cells to hormones, long term regulation of PDE4 activity by its own substrate is now widely described (20, 44, 30). The fact that forskolin mimicked 8Br-cAMP effects on PDE4 expression in our cell model strongly suggests that endogenous activators of adenylate cyclase, such as epinephrine, PGE2, and prostacyclin, or exogenous activators, e.g. ß-adrenoceptor agonists such as salbutamol, can influence rates of cAMP hydrolysis in myometrial tissue. This latter phenomenon has clinical implications in obstetrics. Indeed, ß-mimetics are commonly used as a treatment in preterm labor to produce myometrial relaxation through elevation of cAMP content. Application of these agonists is repeatedly questioned because of their loss of efficacy over the long term (45). The results of this study give a preliminary indication that up-regulation of PDE4 activity could be involved in tolerance to the tocolytic effect of ß-adrenoceptor agonists and thus lessen the therapeutic usefulness of these drugs.
In conclusion, our results demonstrate that in human myometrial cells, PDE4B and PDE4D short forms are up-regulated by cAMP at the level of transcription, with subsequent modifications in the protein level. Such a feedback loop provides a mechanism by which cells might adapt to chronic stimulation of the cAMP-dependent pathway and obtain a protection from overstimulation. During pregnancy, numerous hormones that are known to act through modulation of the intracellular cAMP level, e.g. catecholamines and prostanoids, are implicated in the growth and differentiation of the uterus as well as in determining its contractile state (1). The culture of human myometrial cells provides a convenient myometrial tissue model whose hormonal environment can be modulated. This would enable further examination of the physiological relevance of hormonal regulation of the cAMP degradation process in human uterine smooth muscle, especially during pregnancy.
|
Acknowledgments |
|---|
Received September 3, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  V. Dostaler-Touchette, F. Bedard, C. Guillemette, F. Pothier, P. Y. Chouinard, and F. J. Richard Cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase is functional in bovine mammary gland J Dairy Sci, August 1, 2009; 92(8): 3757 - 3765. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Heng, R. Ivell, P. Wagaarachchi, and R. Anand-Ivell Relaxin signalling in primary cultures of human myometrial cells Mol. Hum. Reprod., October 1, 2008; 14(10): 603 - 611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Giembycz and R. Newton Beyond the dogma: novel {beta}2-adrenoceptor signalling in the airways. Eur. Respir. J., June 1, 2006; 27(6): 1286 - 1306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Rouget, O. Barthez, F. Goirand, M.J. Leroy, M. Breuiller-Fouche, Z. Rakotoniaina, P. Guerard, E.J. Morcillo, C. Advenier, P. Sagot, et al. Stimulation of the ADRB3 Adrenergic Receptor Induces Relaxation of Human Placental Arteries: Influence of Preeclampsia Biol Reprod, January 1, 2006; 74(1): 209 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Belmonte, C. Ticconi, S. Dolci, M. Giorgi, A. Zicari, A. Lenzi, E. A. Jannini, and E. Piccione Regulation of Phosphodiesterase 5 Expression and Activity in Human Pregnant and Non-pregnant Myometrial Cells by Human Chorionic Gonadotropin Reproductive Sciences, December 1, 2005; 12(8): 570 - 577. [Abstract] [PDF]
|
|
|
|
 |
|
 D. G. Tilley and D. H. Maurice Vascular Smooth Muscle Cell Phenotype-Dependent Phosphodiesterase 4D Short Form Expression: Role of Differential Histone Acetylation on cAMP-Regulated Function Mol. Pharmacol., September 1, 2005; 68(3): 596 - 605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Rouget, M. Bardou, M. Breuiller-Fouche, C. Loustalot, H. Qi, E. Naline, T. Croci, D. Cabrol, C. Advenier, and M. J. Leroy {beta}3-Adrenoceptor Is the Predominant {beta}-Adrenoceptor Subtype in Human Myometrium and Its Expression Is Up-Regulated in Pregnancy J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1644 - 1650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Soloff, Y.-J. Jeng, M. Ilies, S. L. Soloff, M. G. Izban, T. G. Wood, N. I. Panova, G. V.N. Velagaleti, and G. D. Anderson Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector Mol. Hum. Reprod., September 1, 2004; 10(9): 685 - 695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Oger, C. Mehats, M. S. Barnette, F. Ferre, D. Cabrol, and M.-J. Leroy Anti-Inflammatory and Utero-Relaxant Effects in Human Myometrium of New Generation Phosphodiesterase 4 Inhibitors Biol Reprod, February 1, 2004; 70(2): 458 - 464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Bartsch, B. Bartlick, and R. Ivell Phosphodiesterase 4 Inhibition Synergizes with Relaxin Signaling to Promote Decidualization of Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 324 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Oger, C. Mehats, E. Dallot, F. Ferre, and M.-J. Leroy Interleukin-1{beta} Induces Phosphodiesterase 4B2 Expression in Human Myometrial Cells through a Prostaglandin E2- and Cyclic Adenosine 3',5'-Monophosphate-Dependent Pathway J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5524 - 5531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. R. Le Jeune, M. Shepherd, G. Van Heeke, M. D. Houslay, and I. P. Hall Cyclic AMP-dependent Transcriptional Up-regulation of Phosphodiesterase 4D5 in Human Airway Smooth Muscle Cells. IDENTIFICATION AND CHARACTERIZATION OF A NOVEL PDE4D5 PROMOTER J. Biol. Chem., September 20, 2002; 277(39): 35980 - 35989. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mehats, G. Tanguy, E. Dallot, D. Cabrol, F. Ferre, and M.-J. Leroy Is Up-Regulation of Phosphodiesterase 4 Activity by PGE2 Involved in the Desensitization of {beta}-Mimetics in Late Pregnancy Human Myometrium? J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5358 - 5365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Méhats, G. Tanguy, B. Paris, B. Robert, N. Pernin, F. Ferré, and M.-J. Leroy Pregnancy Induces a Modulation of the cAMP Phosphodiesterase 4-Conformers Ratio in Human Myometrium: Consequences for the Utero-Relaxant Effect of PDE4-Selective Inhibitors J. Pharmacol. Exp. Ther., February 1, 2000; 292(2): 817 - 823. [Abstract] [Full Text]
|
|
|
|
|
|
H. Liu, D. Palmer, S. L. Jimmo, D. G. Tilley, H. A. Dunkerley, S. C. Pang, and D. H. Maurice Expression of Phosphodiesterase 4D (PDE4D) Is Regulated by Both the Cyclic AMP-dependent Protein Kinase and Mitogen-activated Protein Kinase Signaling Pathways. A POTENTIAL MECHANISM ALLOWING FOR THE COORDINATED REGULATION OF PDE4D ACTIVITY AND EXPRESSION IN CELLS J. Biol. Chem., August 18, 2000; 275(34): 26615 - 26624. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
