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) Protein Kinase A Holoenzyme
Department of Obstetrics and Gynaecology, School of Surgical and Reproductive Sciences, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom
Address all correspondence and requests for reprints to: G. Nicholas Europe-Finner, M.D., Department of Obstetrics and Gynaecology, School of Surgical and Reproductive Sciences, University of Newcastle upon Tyne, 4th Floor Leazes Wing, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom. E-mail: g.n.europe-finner{at}ncl.ac.uk.
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Abstract |
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protein subunit of PKA in the myometrium. This increase in protein expression is also mirrored at the mRNA level, indicating transcriptional control throughout pregnancy, whereas during parturition both transcript and protein are significantly decreased. This increase in RII protein also resulted in increased particulate PKA activity in the myometrium during gestation, which was subsequently decreased during labor. Two specific A kinase anchoring proteins, AKAP95 and AKAP79, which have high binding affinities for RII subunits, were found to form complexes with myometrial RII species employing immunoprecipitation assays, but their levels of expression remained uniform in all myometrial tissue samples investigated. Our findings indicate that increased particulate type II PKA activity occurs throughout pregnancy, therefore directing the cAMP quiescence signal to specific subcellular loci within myometrial smooth muscle cells including the contractile machinery at the cytoskeleton; this effect is then removed during parturition. |
Introduction |
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There is now extensive evidence to indicate that components of the cAMP-signaling pathway are up-regulated in the human myometrium during pregnancy so as to potentiate the maintenance of uterine quiescence until term. These include human chorionic gonadotropin/LH (3) and calcitonin gene-related peptide (4) receptors and the adenylyl cyclase stimulatory G protein Gs (5, 6, 7), whose levels of expression are substantially increased within the myometrium during gestation, resulting in the increased production of cAMP, which is further amplified by the progesterone-induced down-regulation of myometrial cAMP phosphodiesterase activity (8). The effects of cAMP are mediated via protein kinase A (PKA), a heterotetrameric protein complex consisting of two regulatory (R) and two catalytic (C) subunits (9). cAMP binds cooperatively to two sites on the R subunits within the PKA holoenzyme, thereby liberating the C subunits, which are involved in phosphorylation events resulting in inactivation of the contractile machinery or are translocated to the nucleus via a regulated mechanism where they affect myometrial gene expression. Here the C subunits encourage the phosphorylation of cAMP-dependent transcription factors such as cAMP response element-binding protein, cAMP response element modulator protein, and activating transcription factor family, thereby allowing them to interact with cAMP response elements on the control regions of affected genes (10, 11). Once the catalytic C subunit has phosphorylated its target substrate, it must reassociate with the regulatory subunits before PKA can be reactivated by cAMP (Fig. 1
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/RIß and RII/RIIß) that can form homo- and heterodimers and three isoforms of the catalytic C subunit (C, Cß, and C
) (9) that can freely associate with different R subunit homo- and heterodimers (9). Furthermore, PKA holoenzyme type I (RI or RIß) is located within the soluble fraction of cells, whereas PKA holoenzyme type II (RII or RIIß) is particulate and associated with the cytoskeleton and a number of organelles (12, 13). Targeting of PKA to various subcellular loci is mediated by the anchoring of PKA to a family of A kinase-anchoring proteins (AKAPs) and brought about by protein-protein interactions with R subunit species (12, 13, 14, 15). Activation by cAMP of AKAP-bound particulate PKA holoenzymes provokes dissociation of C subunits resulting in increased localized enzyme activity and phosphorylation events. Subsequently C subunits must reassociate with AKAP-bound R subunits for PKA to become sensitive to further stimulation by cAMP. Because total kinase activity is influenced by the rate of activation of catalytic subunits, which are highly susceptible to degradation when unbound to AKAP/R subunit complexes (12, 13, 14, 15) and because the level of expression of R subunits/AKAPs may be critical in determining the sensitivity of particulate PKA to cAMP, the level of expression of R subunits/AKAPs may be critical in defining localized total kinase activity.
Numerous AKAPs have now been identified, each having a distinct subcellular location (12, 13, 14, 15). In most cases AKAPs interact exclusively with RII subunits (12, 13, 14, 15). This interaction is mediated by a small amphipathic helical segment in the AKAPs that bind to the N-terminal dimerization domain of the RII subunit (16). Some AKAPs also act as scaffolds for assembling multiprotein complexes. For example, AKAP 79 localizes to the actin cytoskeleton (17) and not only interacts with PKA but with protein kinase C and calcium/calmodulin-dependent protein phosphatase 2B (18). Recent data strongly suggest that specific AKAPs have differing binding affinities for RII and RIIß protein subunits. For instance, AKAP 79 binds RII subunits with 3-fold higher affinity than RIIß species, whereas AKAP 95 exclusively binds RII (12). Therefore, in addition to the distinct tissue distribution of PKA type I and II holoenzymes, isozymes with RII and RIIß subunits may be localized within differing regions of the cell, depending on the presence of specific AKAPs as has been observed in the Golgi-centrosomal area (19).
At present the pattern of expression of both the R and C subunits of PKA and the AKAPs that modulate the cAMP signal in the human myometrium during fetal maturation and parturition remains to be elucidated. In the studies described here, we used specific antibodies in Western blotting/immunoprecipitation, RT-PCR, and functional assays employing the PKA-specific substrate kemptide to determine the PKA holoenzymes/AKAPs that are expressed in the myometrium throughout pregnancy and labor. We report that as early as the second trimester of pregnancy, there is a substantial increase in expression of the regulatory RII protein subunit of PKA in comparison with nonpregnant (NP) controls. This increase is also mirrored at the mRNA level indicating transcriptional control during pregnancy, whereas at the onset of labor, both transcript and protein levels of RII are decreased to values approaching those observed in tissue samples obtained from NP women. Moreover, this increase in RII protein also gives rise to increased particulate type II PKA catalytic activity in membranes prepared from term nonlaboring myometrium in comparison with laboring and NP membrane preparations. These data indicated that increased particulate type II PKA activity occurs during gestation, which would appear to be involved in directing the cAMP quiescence signal to specific subcellular loci within myometrial cells including the contractile machinery at the cytoskeleton; this effect is then removed during parturition. These findings implicate an important role for the differential expression of myometrial PKA isoforms in the maintenance of uterine relaxation during pregnancy and the switch to a contractile state at the onset of labor. Our studies also indicated that there appeared to be no topographical distribution of R and C subunits of PKA between the upper and lower myometrial segments because in all cases uniform expression was observed. Two specific AKAPs, AKAP95 and AKAP79, which have high binding affinities for RII subunits, were observed to form complexes with myometrial RII species by employing immunoprecipitation assays, but their levels of expression remained uniform in all myometrial tissue samples investigated.
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Materials and Methods |
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All electrophoretic reagents for proteins, DNA, and RNA were of the highest grade available and obtained from Bio-Rad Laboratories, Inc. (Hemel Hempstead, UK), National Diagnostics (Atlanta, GA), and Life Technologies, Inc. (Gaithersburg, MD). Antibodies to PKA R subunits [RIß (sc-907), RII (sc-908] and PKA C subunits [C (sc-903), Cß (sc-904), and C (sc-905), AKAP95 (sc-10766), and AKAP79 (sc-10764)] were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to PKA R subunits RI (Ab-1611) and RIIß (Ab-1614) were from Chemicon Ltd. (Harrow, UK). Additional RII- (catalog no. 539234) and RIIß (catalog no. 539235)-specific antibodies were from Calbiochem (La Jolla, CA). Goat antirabbit IgG-linked horseradish peroxidase was obtained from DAKO Corp. (High Wycombe, UK). PCR primers and Superscript reverse transcriptase were from Invitrogen Ltd. (Paisley, UK). Taq polymerase was from Promega Corp. (San Luis Obispo, CA). The Vectastain ABC kit was obtained from Vector Laboratories Ltd. (Peterborough, UK). The [-32P] ATP (3000 Ci/mmol) and the enhanced chemiluminescence (ECL) assay system were obtained from Amersham International (Aylesbury, UK). The cAMP analog 8, 4-chlorophenylthio (CPT)-cAMP (CPT-cAMP) and RII inhibitor RpII (Rp-CPT-cAMPS) were from Sigma (Poole, Dorset, UK).
Selection of patients and tissue collection
All women were recruited from the Department of Obstetrics and Gynaecology at the Royal Victoria Infirmary. This study received approval from the Newcastle and North Tyneside Health Authority Ethics Committee, and all patients gave informed consent. The division of the uterus into upper and lower uterine segments is not possible in the nonpregnant uterus because the lower uterine segment has not yet been formed. Therefore, to allow topographical comparisons in this study, samples referred to as upper uterine segment samples in the term pregnant (P) and term spontaneously laboring (SL) populations correspond topographically with upper corpus samples in the NP populations; equally samples referred to as lower uterine segment correspond to lower corpus samples (well clear of the cervix). All temporal comparisons made in this study are made between tissues of a corresponding topographical location.
NP myometrium
Paired upper and lower corpus samples of myometrium were obtained from NP premenopausal women (n = 56; age, 32–46 yr; median, 39 yr) undergoing hysterectomy for benign gynecological conditions. The samples were taken in the theater immediately following removal of the uterus. The uterus was incised longitudinally and samples of myometrium were taken from the middle of the uterine wall, with care being taken to allow generous clearance margins from the serosal and endometrial surfaces. Samples obtained in both the follicular and luteal phase of the cycle were used.
First and second trimester myometrium
Samples of myometrium were obtained from patients undergoing elective first trimester suction termination of pregnancy (n = 12; age, 15–39 yr; median, 21 yr; gestation, 7–13 wk; median, 9 wk) or second trimester dilatation and evacuation (n = 12; age, 17–38 wk; median, 21 wk; gestation, 13–20 wk; median, 15 wk) under general anesthesia. Samples from the upper corpus of the uterus were taken after evacuation of the uterus using Wolf biopsy forceps. Under ultrasound guidance, the forceps were introduced into the myometrium and four separate biopsies taken, avoiding the site of the placental bed as determined by ultrasound before termination.
Term pregnant myometrium
Paired upper and lower uterine segment myometrial samples were obtained from healthy women undergoing elective cesarean section at term (n = 86; age, 16–43 yr; median, 27 yr; gestation, 37–40 wk; median, 38 wk). The indications for section were breech presentation, previous cesarean section, or bad previous obstetric outcome. Excluded from this group were women whose cervixes had dilated beyond 2 cm or who were experiencing regular painful uterine contractions. Patients who had had prostaglandin gel administered or whose amniotic membranes were not intact were also excluded. The samples were obtained immediately following the delivery of the placenta and membranes before the closure of the uterine cavity. Samples from the upper uterine segment were taken under direct vision using Wolf biopsy forceps introduced into the uterine cavity through the incision. The forceps were pushed through the decidual layer and into the myometrium. Eight separate myometrial biopsies were taken from individual patients, each from a nonplacental bed site (as determined by manual palpation before the delivery of the placenta). Samples from the lower uterine segment were taken from the upper lip of the incision through the lower uterine segment using toothed biopsy forceps to grasp the myometrium from between its serosal and decidual layers and then curved scissors to sample it.
Term laboring myometrium
Paired upper and lower segment myometrial samples were obtained from women admitted in spontaneous labor (as defined as the onset of painful, regular uterine activity resulting in the progressive and serial dilatation of the cervix beyond 3 cm) undergoing emergency cesarean section at term (n = 50; age, 16–41 yr; median, 28 yr; gestation, 37–42 wk; median, 40 wk). Indications for section were failure to progress and fetal distress. Women who had their labor induced or augmented before reaching 3 cm were excluded from the study. Upper- and lower-segment biopsies were obtained in a similar manner to those from women undergoing elective cesarean section. All myometrial samples were snap frozen at the time of collection using liquid nitrogen-cooled isopentane and then stored at -70 C.
Preparation of myometrial tissue homogenates and membranes
All procedures were carried out on ice. Tissue samples were mechanically homogenized in 25 mmol/liter Tris buffer (pH 7.6), containing 0.25 mol/liter sucrose and 1 mmol/liter ethylenediamine tetraacetate in the presence of a protease inhibitor mixture from Sigma (St. Louis, MO) that contained 4-(2-aminoethyl) benzenesulfonyl fluoride, transepoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64), bestatin, leupeptin, aprotinin, and sodium ethylenediamine tetraacetate, used at a 1:10 dilution. Homogenates were then centrifuged at 1,000 x g to remove tissue debris and supernatants stored at -70 C. Membranes were prepared as described by Europe-Finner et al. (5). Briefly, homogenates were prepared as detailed above and tissue debris removed and the supernatant centrifuged at 40,000 x g for 1 h to pellet membranes that were resuspended in the above buffer and then aliquoted and stored at -70 C. Note that this type of preparation contains membranes from all cellular organelles including the nucleus. Homogenate and membrane proteins were assayed in triplicate using the DC protein assay (Bio-Rad Laboratories, Inc.) with BSA as standard.
Western immunodetection of myometrial proteins
One hundred micrograms myometrial homogenate or membrane protein were solubilized in sample buffer, resolved on 12.5% polyacrylamide gels containing 0.0625% bisacrylamide for 5–6 h at 45 mAmp, and then electrotransferred onto nitrocellulose at 90 V for 2 h before antibody immunodetection. Nitrocellulose blots were first blocked for 90 min in 5% nonfat milk in PBS. All primary antibodies used were at a 1:2000 dilution in the presence of 3% nonfat milk and 0.05% Tween 20 in PBS for 90 min at 4 C. Primary antisera were removed and blots washed three times for 10 min in PBS. Blots were then reincubated with goat antirabbit IgG coupled to horseradish peroxidase at 1:1000 dilution for 60 min at room temperature. The blots were washed in PBS three times for 10 min before ECL was added. All blots were reprobed using the Gß SW/1 primary antibody, recognizing an epitope common to the five different Gß-subunits, at 1:1000 dilution in PBS for 1 h to confirm equal protein loading in each lane as described previously (6, 20, 21). Data were obtained under conditions in which a linear relationship existed between the amount of protein loaded and the intensity of the ECL signal from the immunoblots. Immunodetected bands were scanned using a UMAX PS 2400 scanner coupled to the Intelligent Quantifier software package (BioImage, St. Neots, Cambridgeshire, UK). Data were compared using one-way ANOVA with Bonferroni post test, with P less than 0.05 considered statistically significant.
Immunoprecipitation of myometrial membrane proteins
Myometrial membrane preparations were dialyzed against a 1,000x volume of 1x PBS, 0.5% deoxycholic acid, 0.1% SDS, 0.1% Triton X-100 (all from Sigma) overnight at 4 C. This was precleared to minimize nonspecific reactions with the precipitating primary serum by incubation with 5 µg normal IgG from the same species as the antibody and 20 µl protein A agarose suspension with agitation at 4 C for 30 min. The agarose was pelleted by microcentrifugation at 3,000 rpm for 5 min, and the supernatant was transferred to a fresh tube. Five micrograms of the precipitating primary antibody (AKAP95, AKAP79, and RII) were added per milligram of total protein and incubated at 4 C for 1 h with agitation. Fifteen microliters of the protein A agarose suspension was then added, and incubation continued as above overnight. The sample was centrifuged as above, and the pellet washed with 1 ml cold PBS, followed by another centrifugation step. This was repeated four times, after which the precipitated protein was eluted from the A agarose by incubating with an equal volume of 2x Laemmli buffer at 80 C for 5 min. The sample was microcentrifuged at 13,000 rpm for 5 min to pellet the agarose, and the eluted protein subjected to SDS-PAGE and Western blotting analysis as above. To demonstrate specificity and ensure adequate preclearing, experiments were done in parallel with similar control assays with the only difference being the omission of the precipitating primary antibody.
Immunohistochemistry
Immunohistochemistry was performed on frozen sections of the myometrial samples using a Vectastain ABC kit (Vector Laboratories). Sections were allowed to come to room temperature, washed in Tris-buffered saline (TBS), and then quenched with hydrogen peroxide (5.8 ml water to 0.2 ml hydrogen peroxide). Normal horse serum blocking serum replaced the TBS before the primary antibody or control rabbit sera were added at a 1:1000 dilution for 90 min. This was removed and the slide washed with TBS twice for 5 min before a biotinylated secondary antibody was added for 60 min. This was removed and the slide washed with TBS twice for 5 min. An avidin peroxidase complex replaced the TBS for 30 min before the slide was washed twice for 5 min with TBS. The slide was then stained with aminoethylcarbazole and counterstained with Hemalum.
Myometrial tissue RNA isolation
RNA was isolated using a Poly (A) pure mRNA isolation kit (Ambion, Inc., Austin, TX), according to its published protocol. Briefly, samples of NP, term pregnant nonlaboring (P), and SL myometrium were homogenized on ice in 10 ml lysis solution per gram of tissue. Dilution buffer, twice the volume of the homogenate, was added, with cellular debris removed by centrifugation at 12,000 x g, 4 C for 15 min. One hundred milligrams of oligo (dT) cellulose per gram of tissue were added and incubated for 60 min at room temperature on a gyro rocker. The mRNA-oligo (dT) cellulose macromolecules were subsequently isolated by centrifugation at 4,000 x g at room temperature for 3 min and the supernatant discarded. Nonspecifically bound material was removed by resuspending the mRNA-oligo (dT) cellulose macromolecules in 10 ml binding buffer and repelletted and washed by centrifugation three times. Ribosomal RNA was washed away by resuspending in 10 ml wash solution and repelleted and washed by centrifugation three times. The mRNA-oligo (dT) cellulose macromolecules were then resuspended in 0.6 ml wash buffer and transferred to a spin column. Columns were then centrifuged at 4,000 x g room temperature to allow binding of mRNA-oligo (dt) cellulose macromolecules to the spin column membrane. The spin column, with membrane, was then transferred to a new microfuge tube and the mRNA-oligo (dT) cellulose macromolecules eluted with 200 µl elution buffer at 67.5 C. RNA was precipitated by adding 40 µl ammonium acetate, 2 µl glycerol, and 1100 µl ethanol and RNA quantified at 260 nm, where 1 absorbance unit equals 40 µg single-stranded RNA per milliliter.
RT-PCR with RII-specific primers
Reverse transcription was carried out employing 1 µg mRNA isolated from NP, P, and SL myometrial samples and 1 µl oligo dt primer, 2.0 µl of 10 mM deoxynucleotide triphosphate nucleotide mix, 10 µl first-strand reaction buffer, 1 µl of 0.1 M dithiothreitol, 1 µl RNase inhibitor, 0.5 µl Superscript reverse transcriptase, and 50 µl nuclease-free water added to a 1.5-ml eppendorph and incubated in a 37 C water bath for 90 min. The cDNA synthesized in the reverse transcription reaction was then used as a template in a PCR reaction employing 0.5 µl of the forward human RII-specific primer 5'-CCTAGCAGATTTAATAGACG-3' and 0.5 µl of the reverse human RII-specific primer 5'-ATCATCTCCTTGGTCAATGA-3', 0.2 µl taq polymerase, 1.0 µl of 10 mM deoxynucleotide triphosphate nucleotide mix, 5 µl reaction buffer, 4 µl cDNA template, 3 µl magnesium chloride, and 35.8 µl nuclease-free water. PCR was performed under standard conditions with 25 cycles, involving 30 sec at 94 C denaturing, 30 sec at 56 C annealing, and 60 sec at 72 C extension per cycle. Reactions were terminated in the exponential phase. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific oligonucleotide primers were also used as control primers in RT-PCR reactions with cDNA templates. DNA sequences for the housekeeping gene GAPDH primers were forward 5'-CTGCCGTCTAGAAAAACC-3' and reverse 5'-CCACCTTCGTTGTCATACC-3'. PCR products were resolved on 2% agarose gels containing ethidium bromide with ultraviolet light exposure and photography. RII primers and GAPDH primers gave PCR products of 371 bp and 210 bp, respectively. The 371-bp RII product was cloned into the TOPO-TA cloning vector (Invitrogen) and sequenced to ensure fidelity with the human RII sequence +460 bp to +831 bp (accession no. NM004157). PCR product bands were scanned using a UMAX PS 2400 scanner coupled to the Intelligent Quantifier software package from BioImage. Data were compared using one-way ANOVA with Bonferroni post hoc test, with P less than 0.05 considered significant.
PKA activity/Kemptide phosphorylation assay
PKA activity was assayed by the method of Roskoski (22), with minor modifications. SignaTECT cAMP-dependent PKA assay system (Promega Corp.) was used according to the manufacturer’s protocol. Briefly, the reaction mix was preincubated in a water bath at 30 C for 5 min and consisted of 5 µl PKA biotinylated kemptide peptide substrate, 5 µl 0.025 mM cAMP, or 0.025 mM of the cAMP analog CPT-cAMP, 5 µl PKA 5x assay buffer [consisting of 200 mM Tris-HCl (pH 7.4), 100 mM MgCl2, 0.5 mg/ml BSA], and 5 µl ATP mix consisting of 5 µl 0.5 mM ATP and 0.05 µl [32P]ATP (3000 Ci/mmol). The reaction was initiated by the addition of 5 µl (5–40 µg membrane protein) of membranes prepared from NP, P, and SL tissues and incubated at 30 C for 20 min (the optimal time). The reaction was then terminated by adding 12.5 µl of 7.5 M guanidine hydrochloride. Ten microliters of each terminated reaction was spotted onto the streptavidin matrix of a SAM2 biotin capture membrane. The membrane was then washed: once for 30 sec with 200 ml of 2 M NaCl, three times for 2 min with 200 ml of 2 M NaCl, four times for 2 min with 200 ml of 2 M NaCl in 1% H3PO4, and then twice for 30 sec with 100 ml deionized water. Membranes were air dried for 60 min before being transferred to a 1.5-ml eppendorph, to which 1 ml ß-plate scintillation fluid (Wallac, Inc., Turku, Finland) was added, before 32P counting in a MicroBeta 1450 liquid scintillation counter (Wallac, Inc.). Protein concentration (5–40 µg of membrane protein) and incubation time (5–50 min) were used in the linear range, and counts per minute were translated into picomoles 32P incorporated into the biotinylated kemptide by using an unwashed membrane as a control, and PKA enzyme activity expressed as picomoles ATP per minute per microgram of protein.
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Results |
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, RII, C, and Cß protein species were the only PKA subunits expressed in the myometrium and that RIß, RIIß, and C PKA species were not detected in any samples investigated. Comparison of P myometrium sampled from the upper- and lower-uterine segment indicated uniform levels of cellular RI, RII, C, and Cß proteins indicating no topographical differences for these PKA species (Fig. 2, A, C, E, and G). This was also observed in NP and SL tissues (data not shown). Note that in each case protein loading was the same as demonstrated by the Gß loading control (Fig. 2, B, D, F, and H). Because according to the manufacturer (Santa Cruz Biotechnology, Inc.) the C, Cß, and C antibodies have partial cross-reactivities for each other, the possibility exists that the same catalytic C species is being detected by all three antibodies. However, C appears to be expressed only in human testis (23) and no bands were observed employing the C antibody in Western blotting of myometrial tissues (data not shown), thus indicating the absence of this catalytic species in the human myometrium as well as the specificity of the antibody because no cross-reactivity with 40 kDa C/Cß proteins was found. In contrast, both C and Cß antibodies, respectively, stained myometrial proteins with expected molecular masses of 40 kDa, which may indicate that each antibody is specific. However, cross-reactivity cannot be ruled out; consequently, the data presented for C/Cß expression in the myometrium should be taken to represent total cellular catalytic C species levels, which remain uniformly expressed between the upper and lower uterine segments.
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). Fig 3, A and C, demonstrate that levels of cellular C and Cß (i.e. total catalytic C) subunits remained equivalent among NP, P, and SL tissues, indicating that no temporal change in expression occurred during pregnancy or parturition. Similar results were observed for RI species (Fig. 4A). However, expression of cellular levels of RII proteins were observed to be 2- to 3-fold higher in P homogenates, compared with NP and SL myometrial homogenates (Fig. 4C). A similar increase in particulate RII protein expression was also observed in P membrane preparations, compared with NP and SL membranes as detailed in Fig. 5A, thus indicating that RII expression was also reflected at a functional level by increased binding of RII to AKAP (i.e. AKAP95/79, see below) species during gestation. This increase in particulate RII protein levels was also associated with an increase in total particulate catalytic C/Cß levels in P membranes, compared with NP and SL membrane preparations as detected by the C antibody in Western immunostaining (Fig. 5C).
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expression and lack of detection of RIIß species in the myometrium were respectively confirmed using specific antibodies from Calbiochem (data not shown). Immunohistochemistry employing the RII antibody (Santa Cruz Biotechnology, Inc.) confirmed localization of this PKA subunit to myometrial cells and indicated a decrease in expression in SL, compared with P tissues (Fig. 6, A and B), thus supporting the Western blotting data. Similarly, RI and C/Cß species were also found to be primarily expressed in myometrial cells (data not shown).
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subunits were also observed at the mRNA level using specific primers in semiquantitative RT-PCR. This is demonstrated in Fig. 7A in which a 2- to 3-fold increase in RII transcript in P tissues was observed over NP and SL myometrial tissues in comparison with uniform expression of the housekeeping control GAPDH mRNA in all three tissue types (Fig. 7B).
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subunits first occurred, upper corpus myometrial samples were compared between nonpregnant, first trimester and second trimester populations. Figure 8A
indicates that a small increase in expression of cellular RII proteins was observed as early as the first trimester of pregnancy, although a more statistically significant increase was seen in the second trimester. The order of magnitude of this increase was similar to that found in term pregnant nonlaboring myometrium (Fig. 4C).
Because AKAP95 and AKAP79 have been reported to have the highest affinity for RII species, coimmunoprecipitation experiments were carried out to determine whether these AKAP species complexed with RII proteins in the myometrium. Immunoprecipitation with the AKAP95 antibody and subsequent Western immunodetection with the RII antibody (Santa Cruz Biotechnology, Inc.) indicated that AKAP95 and RII species interacted (Fig. 9A). This was further ratified by immunoprecipitating with the RII antibody and immunostaining with the AKAP95 antibody (Fig. 9B). Similarly, immunoprecipitation with the AKAP79 antibody and subsequent Western immunodetection with the RII antibody indicated that AKAP79 and RII species also formed complexes (Fig. 9C). This was also confirmed by immunoprecipitating with the RII antibody with subsequent immunostaining with the AKAP79 antibody (Fig. 9D).
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proteins, the level expression of these proteins in NP, P, and SL myometrial membrane preparations were also determined in Western blotting experiments. Uniform expression of both AKAP95 and AKAP79 were observed in all myometrial membrane preparations (see Fig. 10, A and C). Expression of these AKAPs also remained uniform in NP, P, and SL tissue homogenates (data not shown).
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protein expression during pregnancy, PKA phosphorylation assays were carried out employing the PKA-specific peptide kemptide with myometrial membranes prepared from NP, P, and SL myometrial tissues. Data from these assays indicated a significant increase in PKA catalytic activity occurred on stimulation with cAMP (as well as CPT-cAMP, data not shown) using membranes prepared from P, compared with NP and SL tissues as detailed in Fig 11A. Note that basal activity in the absence of cAMP also demonstrated increased activity in P, compared with NP and SL membranes (Fig 11B). In this context basal PKA activity in SL membranes was reduced to levels observed in NP membranes, whereas in the presence of cAMP, PKA activity in SL membranes still remained significantly higher than that in NP membranes. This discrepancy remains to be defined but could be due to the limitations of the experimental protocol or possibly as a result of other factors affecting PKA activity in the laboring condition, which becomes more apparent on stimulation with cAMP. Incubations with the RII inhibitor RpII (Rp-CPT-cAMPS) negated PKA activity in membranes prepared from term pregnant nonlaboring tissues, thus ensuring PKA activity was associated with only particulate type II PKA activity and not soluble type I PKA activity (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, RII, and C/Cß (total catalytic C) protein species of PKA were expressed in the human myometrium. RI, RII, and C species have been shown to be expressed in all tissue and cell types (24), whereas Cß, RIß, and RIIß isoforms are predominantly expressed in brain and gonadal tissues (24), although low levels of expression have been observed in a wide range of human tissues (25). The presence of RI and RII isoforms in the myometrium also demonstrated that this tissue contained both soluble type I and particulate type II PKA holoenzymes. Similarly, evidence was obtained indicating no topographical distribution of myometrial PKA isozymes between the upper and the lower uterine regions during pregnancy and labor in common with expression of the adenylyl cyclase stimulatory GTP-binding protein Gs (20), an up-stream component of the cAMP pathway. It is interesting to note that the uniform expression of both particulate PKA and Gs in both the upper and lower uterine regions suggests that cAMP exerts equivalent relaxatory effects throughout the whole organ, even though contraction are known to be propagated from the fundal region. A major novel finding from this study was a substantial increase in cellular/membrane-bound RII protein and mRNA levels during pregnancy, which were subsequently decreased during labor to values observed in nonpregnant controls. This increase in RII species was also mirrored by an increase in both basal and cAMP-stimulated particulate type II catalytic activity as assayed with the specific PKA peptide kemptide, which was also decreased during parturition. No apparent increase in total cellular levels of C/Cß protein subunits was observed in any of the myometrial tissue homogenates studied, which may indicate no change in expression of C/Cß transcripts during pregnancy and labor. However, the increase in particulate PKA activity in the term nonlaboring myometrium was associated with an increase in particulate membrane levels of both RII and total C/Cß proteins as required by the stoichiometry of the heterotetrameric structure of the PKA complex.
Coimmunoprecipitation experiments with antibodies to RII, AKAP95, and AKAP79 demonstrated that RII proteins precipitated with both AKAP95 and AKAP79 proteins. These data indicated that in the human myometrium, RII species formed complexes with both AKAP95 and AKAP79, although it must be realized that other AKAP species not tested in this investigation might also have some binding affinity for myometrial RII subunits. However, the increase in particulate type II PKA holoenzyme was not associated with an increase in AKAP95 and AKAP79 moieties because expression of these proteins remained constant during pregnancy and labor. This may therefore suggest that sufficient AKAP95 and AKAP79 proteins are expressed in the myometrium during fetal maturation to accommodate the increase in levels of RII protein subunits observed as early as the first and second trimester of pregnancy. With respect to detection of AKAP79 observed in this study, an AKAP species similar to AKAP79 was also detected in the immortalized pregnant human myometrial cell line PHM1–41 employing an AKAP overlay assay (26).
There is now increasing evidence that one of the actions of PKA in regulating myometrial activity involves modulation of the phosphatidylinositide/calcium signaling pathway (26, 27, 28, 29). Recently Yue et al. (28) have shown that phosphorylation of phospholipase Cß3 by PKA inhibits stimulation by Gq in several cell lines and this is localized at the plasma membrane through association with an 86-kDa AKAP protein, having high homology to AKAP79 (30, 31), in the myometrial cell line PHM1–41 (26). In the rat model, PKA was found to immunoprecipitate with a 150-kDa AKAP found in rat myometrial plasma membranes (29). This rat AKAP species is thought to be homologous to the human AKAP79 protein (30), which has been shown to bind not only PKA but also protein phosphatase 2B, protein kinase C, calcineurin, phosphatidylinositol-4,5-bisphosphate, and calmodulin (18, 31, 32) and thus acts a scaffold protein to integrate several signaling proteins that affect specific cellular events. Dodge et al. (29) indicated that PKA concentration and activity decreased in rat myometrial plasma membranes on d 21, compared with d 19 of pregnancy. This decrease in PKA concentration was not due to a decrease in cellular PKA levels but as a result of a decrease in localization of PKA to the plasma membranes via binding to AKAP150 possibly because of displacement by protein phosphatase 2B (PP2B) (29). These data suggested that PKA localized to the plasma membrane via binding to AKAP150 was necessary for the cAMP inhibitory effect on myometrial phosphatidylinositide/calcium signaling pathways during gestation. Consequently the possibility exists that a similar mechanism occurs in the human myometrium during gestation and labor.
The decrease in particulate type II PKA activity observed during parturition may therefore be brought about by a decrease in not only RII expression but also PKA localization via displacement by other proteins that can complex with AKAP95 and AKAP79. However, if this were the case, to account for the consistency in the observed correlation between enzyme activity and protein concentration, there would have to exist an equivalent displacement of PKA by other proteins occurring in the nonpregnant state to that observed during labor. Moreover, if the above processes were to occur, it would be difficult to experimentally differentiate them from the changes in PKA-AKAP binding brought about by the differential expression of myometrial RII proteins. In the human this mechanism may well predominate over the processes observed in the rat for localizing PKA activity so as to maintain myometrial quiescence during gestation and the subsequent switch to contractions at the onset of parturition.
In conclusion, we provide novel evidence that the increase in myometrial cAMP levels during fetal maturation brought about by up-regulation of several upstream components of the cAMP signaling pathway (3, 4, 5, 6, 7) including Gs is targeted to discreet loci within myometrial cells via an increase in RII protein levels and particulate type II PKA activity. These loci are likely to include the cytoskeleton, in which inactivation of the contractile machinery may occur as well as the nucleus in which regulation of myometrial gene expression may be brought about via activation of members of the cAMP-dependent basic region/leucine zipper transcription factor family. Down-regulation of RII species and particulate PKA activity before or at the onset of parturition may remove this inhibitory effect on the contractile machinery, thus allowing development of regular coordinate contractions and progress to labor. Results from this investigation also strongly suggest that expression of RII in the human myometrium is transcriptionally regulated during pregnancy and labor, and further studies are envisaged to elucidate the molecular processes by which this occurs.
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Footnotes |
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M.W.J.M. and G.N.E.-F. contributed equally to this study.
Abbreviations: AKAP, A kinase-anchoring protein; C, catalytic subunit; CPT, 8, 4-chlorophenylthio; ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NP, nonpregnant; P, term pregnant; PKA, protein kinase A; R, regulatory subunit; SL, term spontaneously laboring; TBS, Tris-buffered saline.
Received November 26, 2002.
Accepted January 23, 2003.
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