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Biomedical Research Institute (E.K., D.G.), Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; and The Medical School (E.W.H.), University of Leeds, Leeds LS2 9NL, United Kingdom
Address all correspondence and requests for reprints to: Dr. D. Grammatopoulos, Biomedical Research Institute, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.
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Introduction |
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UCN II and UCN III bind exclusively to type-2 CRH receptor (CRH-R2), although the dissociation constant Kd of UCN III is approximately 10-fold lower than the Kd values for UCN I and UCN II (3). CRH-R2 is a G-protein-coupled receptor with distinct pharmacological characteristics compared with CRH-R1, which binds CRH and UCN I with similarly high affinities. UCN I displays high affinity for CRH-R2, whereas CRH has approximately 10-fold lower affinity for this receptor.
To date, it is well established that among reproductive tissues, both CRH and UCN I are expressed in the endometrium, decidua, placenta, fetal membranes, and myometrium (4, 5, 6, 7). During human pregnancy, CRH that is produced from the intrauterine tissues is secreted into the maternal circulation. Extensive studies in our laboratory suggest that during pregnancy CRH actions on the human myometrium initiate signaling cascades that primarily act to prevent uterine contractions (8).
One of the proteins involved in myometrial smooth muscle contractile process is myosin light chain (MLC20). It is generally accepted that the initiation of smooth muscle contraction involves increase of MLC20 phosphorylation at positions Thr 18/Ser19 (9). Both sites are phosphorylated by myosin light chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme. This increased phosphorylation induces a conformational change in MLC20 that enables actin-myosin interaction and cell contraction. Numerous studies have shown that the activity of myosin-filament formation and actin-activated myosin ATPase is regulated by the phosphorylation of Ser19 by MLCK (10). Ser19 is dominantly phosphorylated in vitro, Thr18 is phosphorylated only at a high concentration of MLCK. Smooth muscle relaxation is preceded by dephosphorylation of MLC20 by MLC phosphatase (MLCP) (11).
However, other pathways have now been described that may regulate the contractility of smooth muscle by regulating the phosphorylation of MLC20 independently of a rise in intracellular Ca2+ (12, 13) and involve the monomeric GTP-binding protein RhoA, activation of which leads to activation of RhoA-associated kinase (ROK) (7). ROK is then able to regulate the phosphorylation of MLC20 through inactivation of MLCP through the phosphorylation of the regulatory myosin-binding subunit (11). Therefore the increased actomyosin interaction can be achieved through Ca2+-mediated MLC kinase (MLCK) activation and through 
-dependent, Ca2+/calmodulin-independent inhibition of MLC20 dephosphorylation, both leading to an increase in MLC20 phosphorylation (14). Other second messengers appear to modulate cell contractions. The adenylyl cyclase/cAMP system, for instance, mediates relaxant effects in smooth muscle cells by inhibiting both the Ca2+-mediated MLCK- and the RhoA/ROK-mediated pathways controlling MLC20 phosphorylation (14).
The aim of this study was to evaluate the localization and gene expression of UCN II in human myometrial tissues and investigate its potential to modulate MLC20 phosphorylation in human pregnant myometrial cells in vitro.
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Materials and Methods |
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Total RNA extraction and cDNA synthesis
Total RNA was prepared from individual samples using RNeasy total RNA kit (QIAGEN, Crawley, UK) according to the manufacturer’s guidelines. First-strand cDNA synthesis was performed using RNase reverse transcriptase (Gibco BRL, Paisley, UK).
PCR
All PCRs were carried out using Taq DNA polymerase (Gibco BRL) with 200 ng of cDNA for each amplification, as previously described (11). Briefly, myometrial cDNAs were amplified at 94 C (45 sec), 58 C (45 sec), and 72 C (1 min) in a total of 30 cycles with a final extension step at 72 C for 10 min. The set of primers for the amplification of UCN II, CRH-R2, and ß-actin were as follows: UCN II sense 5'-ACCAGGTGTGCTCTGCTGTT-3', antisense 5'-GATAGGACAATGCGCGAGCC-3'; CRH-R2 sense 5'-TCAGCCGTGAGGAAGAGGTG-3', antisense 5'-GGCCGTCTGCT TGATGCTGT-3'. Ten microliters of the reaction mixture were subsequently electrophoresed on a 1.8% agarose gel and visualized by ethidium bromide, using a 1-kb DNA ladder (Gibco BRL) to estimate the band sizes. As a negative control for all of the reactions, distilled water was used in place of the cDNA. The resultant PCR products were sequenced in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Centre for Biotechnology Information.
Fluorescent in situ hybridization (FISH)
Paraffin-embedded sections of human myometrial tissues were dewaxed and dehydrated by successive washes through ethanol and air dried. Specific 40-mer synthetic sense and antisense oligonucleotide probes with fluorescein conjugated at their 5'-ends for UCN II were used in this study. Hybridization solution (100 µl) containing 1 ng/µl of the probe was allowed to hybridize at 37 C overnight as previously described (15). Slides were then placed in preheated (45 C) 2x standard saline citrate buffer, in which they were washed twice, followed by another 10-min immersion in 0.1x standard saline citrate (45 C). The tissue sections were rinsed with PBS for 5 min, and the cell nuclei were visualized as previously described (15).
Immunofluorescence
Fixed myometrial cells were washed in PBS and incubated with 3% BSA for 1 h before incubation with the first primary polyclonal UCN II (Phoenix Pharmaceuticals, Belmont, CA) antibody for 60 min, which was used at a 1:200 dilution. All dilutions were made in 3% BSA in PBS. After three washes with PBS, specimens were incubated for 1 h with the secondary antigoat IgG conjugated with fluorescein isothiocyanate. Specimens were washed thoroughly, and the results were viewed under a fluorescent microscope using appropriate filters.
Chemical cross-linking and SDS-PAGE
Myometrial tissues were weighed and homogenized in 6 ml Dulbecco’s PBS as previously described (15). The homogenate was centrifuged at 3000 rpm for 30 min at 4 C. The supernatant was then transferred and centrifuged at 19,000 rpm for an additional 60 min at 4 C. The resultant pellet was washed, resuspended in extraction buffer, and centrifuged at 19,000 rpm for an additional 60 min at 4 C. The final pellet was resuspended in extraction buffer using homogenizer for 20 sec. Human myometrial membranes were incubated with human [125I]UCN II (1 nM) for 2 h in 300 µl buffer (50 mmol/liter Tris-HCl, 2 mmol/liter EGTA, 10 mmol/liter MgCl2, 1.5 g/liter BSA, pH 7.2) at 22 C to reach equilibrium in the presence or absence of cold UCN II (1 µM) and anti-sauvagine-30 (1 µM). Ten microliters of disuccinimidyl suberate were added to the preparation to give a final concentration of 1.5 mmol/liter. The reaction was then allowed to proceed for 10 min at 22 C before termination with 1 ml ice-cold extraction buffer and centrifugation at 12,000 rpm for 10 min. The pellets were then washed, solubilized, and subjected to SDS-PAGE as previously described (16). The gel was then dried and exposed to Fuji x-ray film at -70 C for 3 d.
Myocyte culture
Myometrial biopsies, weighing approximately 3 g were collected from pregnant women. Primary myometrial cell cultures were established as described by Phaneuf et al. (17). Cells were resuspended in DMEM, containing 10% fetal calf serum, 0.2% L-glutamine, 10,000 U/ml penicillin G, and 7610 IU/ml streptomycin. Myometrial cells were plated into 25-cm2 culture flasks at a density of 0.5–2 x 104 cells/cm2 and stored at 37 C in humidified atmosphere (95% air and 5% CO2) for up to 72 h.
RhoA translocation
Myometrial cell cultures were treated with UCN II (100 nM), followed by the addition of 1 ml ice-cold extraction buffer containing 10 mM MgCl2, 2 mM EGTA, 0.15% BSA (wt/vol), 0.15 mM bacitracin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2, in Dulbecco’s PBS. The lysate was then centrifuged at 3000 rpm for 30 min at 4 C to precipitate out any cell debris/impurities. The supernatant was then further centrifuged at 19,000 rpm for an additional 60 min at 4 C, and the cytosolic and pellet (membrane-bound) fractions were collected. Both fractions were solubilized in SDS-PAGE sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 50 mM dithiothreitol.
ERK1/2 activation and MLC20 phosphorylation
ERK1/2 detection after UCN II stimulation of myocytes was carried out as previously described (18). Briefly, myometrial cells were cultured in six-well dishes and when 80% confluency was reached, cells were cultured overnight in serum-free media, followed the next day by the addition of the agonists/antagonists. Cells were then lysed by the addition of 300 µl SDS-PAGE sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 50 mM dithiothreitol. The solubilized material was then removed from the dishes, sonicated for 20 sec, heated for 5 min at 100 C, and cooled on ice. Before electrophoresis, the extracts were centrifuged at 4000 rpm for 5 min to remove insoluble material.
Western blotting
Samples were separated on an SDS-12.5% polyacrylamide gel, and the proteins were electrophoretically transferred to a nitrocellulose filter at 250 mA for 16–18 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. The filter was then blocked in PBS containing 0.1% Tween 20 and 5% dried milk powder (wt/vol), for 2 h at room temperature. After three washes with PBS-0.1% Tween, the nitrocellulose filters were incubated with primary antibody for the ERK1/2 (New England Biolabs, Beverly, MA) and phospho-MLC20, MLC20, and RhoA (Santa Cruz Biotechnology, Santa Cruz, CA). In some experiments, antibodies that recognized both phospho- and unphosphorylated forms of MLC20 (Sigma Chemical Co., St. Louis, MO) were also used The primary antisera were used at a 1:1000 dilution in PBS-0.1% Tween overnight at 4 C. The filters were washed thoroughly for 30 min with PBS-0.1% Tween before incubation with the secondary horseradish peroxidase-conjugated Igs (1:2000) for 1 h at room temperature and additional washing for 30 min with PBS-0.1% Tween. Antibody complexes were visualized as previously described (15).
Statistical analysis
Data are shown as the mean ± SEM of each measurement. In each case, results were evaluated between groups by using two-tailed Student’s t test, with significance determined at the level of P < 0.05. The relative density of the bands was measured by optical density scanning using the software Scion Image-ß 3b for Windows (Scion Corp., Frederick, MD).
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, inset).
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, A and B), whereas in the pregnant state, strong staining was detectable in smooth muscle cells (Fig. 1, C and D). A similar pattern of staining emerged from cultured pregnant myometrial cells, where cytoplasmic localization of the UCN II was evident across single myocytes (Fig. 1, E and F). In the same cells, results also showed localization of the mRNA signal for CRH-R2 (Fig. 1, G and H).
In addition, immunofluorescent analysis using a UCN II-specific antibody confirmed protein expression of this peptide in human myometrial cells in vitro. UCN II appeared to be expressed as intense granular staining throughout the cytoplasm of the smooth muscle cells (Fig. 2A).
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). Similar results were obtained when anti-sauvagine-30 (a specific CRH-R2 antagonist) was used as the displacing agent (data not shown).
UCN II-induced phosphorylation of MLC20 in human myometrial cells
We then assessed the effect of UCN II on the phosphorylation of MLC20 in vitro, using cultured human myometrial cells that had been obtained from lower-segment myometrial biopsies at term. In preliminary experiments, the degree of MLC20 phosphorylation was evaluated by using antibodies that recognize either phospho-MLC20 (mono- and diphosphorylated forms), unphosphorylated MLC20, or both forms (Fig. 3, inset). UCN II was able to induce phosphorylation of MLC20 at Ser19 and at Thr18 (Fig. 3A). Despite increased levels of diphosphorylation of MLC20, the total amount MLC20 remained unchanged (data not shown). Time course experiments showed that Ser19/Thr18 phosphorylation of MLC20 was maximal after 5 min of treatment and returned to basal levels after 45 min of treatment. This effect appeared to be dose dependent, with maximal effect achieved at 100 nM (Fig. 3B). UCN II action on MLC20 phosphorylation was abolished by coincubation with anti-sauvagine-30 (1 µM), demonstrating the involvement of CRH-R2 in mediating UCN II actions. Interestingly, anti-sauvagine-30 exerted a small but statistically significant inhibitory effect (10–15%) on basal MLC20 phosphorylation levels, suggesting potential tonic regulation of MLC20 phosphorylation by myometrially synthesized UCN II (data not known).
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). Similar results were obtained with UCN I, which was used as a positive control, because we have shown previously that UCN activates the ERK1/2 signaling cascade in human myometrial cells (data not shown) (18). Despite increased levels of phospho-ERK1/2, the total amount of immunoreactive ERK1/2 was unchanged (Fig. 4A).
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). Once again this effect was time and concentration dependent. ERK1/2 phosphorylation was maximal after 5–10 min of treatment, returning to basal levels after 60 min and was found to be significant only at UCN II concentrations greater than 10 nM, reaching a maximum effect at 100 nM. Higher concentrations did not produce any additional stimulation, and there was no increase in the total amount of ERK1/2 immunoreactivity (data not shown).
In attempting to assess the involvement of this pathway in the phosphorylation of MLC20, we have used U0126 and bisindolylmaleimide to assess their effect on the UCN II-induced phosphorylation in cultured pregnant myometrial cells. Treatment with U0126 totally inhibited MLC20 phosphorylation by UCN II (Fig. 5A). In contrast, the protein kinase C (PKC) inhibitor bisindolylmaleimide caused a 75% decrease on the UCN II effect (Fig. 5B). Collectively, these data suggest that the UCN II-induced phosphorylation of MLC20 is modulated via the ERK1/2 pathway in human pregnant myometrial cells.
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), an effect that was found to be dose dependent and reached maximal translocation at 5 min (data not shown). It is well documented that in various types of smooth muscle cells, activation of RhoA/ROK inhibits MLCP activity, thereby leading to an increase in MLC20 phosphorylation and contractile force (19). To test whether a similar mechanism mediates UCN II actions, we measured MLC20 phosphorylation in UCN II-treated cells using the kinase inhibitor Y27632 and the RhoA inhibitor exoenzyme C3. UCN II-induced increase in MLC20 phosphorylation was completely abolished by preincubation for 30 min with the inhibitors Y27632 (1 µM) and exoenzyme C3 (2 µg/ml) (Fig. 6, B and C), indicating that UCN II actions involve activation of the RhoA/ROK pathway.
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), suggesting the involvement of MEK1/ERK1/2 in the RhoA translocation.
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, B and C). The total amount of immunoreactive ERK1/2 was also unchanged upon treatment with Y27632 and exoenzyme C3 (data not shown). |
Discussion |
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A number of studies using knockout mice have conclusively demonstrated that in various physiological systems CRH and CRH-R2-specific agonists such as UCN II play distinct roles mediated via activation of R1 and R2 types of CRH-R, respectively (1, 2, 21). In the human myometrium, CRH has been proposed to be involved in the maintenance of myometrial relaxation during pregnancy, an effect mediated via activation of the Gs/adenylyl cyclase/cAMP/ protein kinase A system (8). Interestingly, the present study demonstrated that UCN II-mediated activation of myometrial CRH-R2s leads to increased MLC20 phosphorylation, a key signaling event involved in the interaction of actin and myosin and development of myometrial contractile response. This is the first demonstration of UCN II/CRH-R2 potential to modulate MLC20 functional activity in myometrial smooth muscle cells, a signaling cross-talk that might be also important in other cellular systems. Our studies identified two signaling cascades, the MEK/ERK1/2 and the small GTP-binding protein RhoA p21/RhoA-associated kinase (ROK), that appear to play critical roles in UCN II/MLC20 interactions. Inhibition of ERK1/2 phosphorylation (by U0126), translocation and activation (by exoenzyme C3), or ROK activity (by the pyrimidine derivative Y27632) totally abolished UCN II-induced MLC20 phosphorylation. Activation of ERK1/2 has been proposed to be involved in the uterotonin-induced activation of myometrial contractility (22, 23) through mechanisms potentially involving direct phosphorylation of the Ca2+/calmodulin-dependent MLCK (24). Furthermore, we have previously shown that in human pregnant myometrium ERK1/2 phosphorylation is one of several signaling events lying downstream of CRH-R1 or -R2 activation (18) in a PKC-dependent manner, with possible contribution from other additional mechanisms, such as activation of ß
-subunits of G-proteins in a Ras-dependent, PKC-independent process. The data presented in this study indicate that UCN II/CRH-R2-induced ERK1/2 phosphorylation also requires upstream activation of PKC, although the inability of bisindolylmaleimide to completely block the UCN II effect might indicate synergistic effects of additional pathways. The human myometrium expresses multiple PKC isoforms, and the identity of the specific PKC isoforms mediating the actions of CRH-R1 and/or -R2 is currently under investigation. In addition, the identity of G-proteins involved is currently unknown; UCN/CRH-R1 actions on myometrial ERK1/2 activity are primarily mediated via Gq-proteins and phospholipase C (PLC) activation (18); therefore, it is possible that UCN II /CRH-R2 effects on ERK1/2 activation and MLC phosphorylation may also involve Gq-protein and PLC activation. Interestingly, in a recent study using various CRH-Rs expressed in the fission yeast Schizosaccharomyces pombe containing yeast-human G
-protein transplants, UCN II failed to activate Gq-proteins (25), whereas it induced activation of Gs-, Gi-, and G16-proteins. Previous studies in a number of G-protein-coupled receptor models have demonstrated G16-protein potential to activate PLC-ß and ERK1/2 (26). Although these data should be interpreted with caution, due to 1) the limitations of the yeast system containing a modified yeast G-protein where only the last five amino acids of the C terminus of the G-protein were replaced with the corresponding residues from the different human G-proteins and 2) the well established tissue-specific CRH-R G-protein coupling (15, 27, 28), it is possible that in cells expressing G16-protein, CRH-R2-induced activation of G16-protein may represent an alternative pathway involved in UCN II-ERK1/2 interactions.
Our studies also revealed the importance of the RhoA/ROK signaling pathway in mediating UCN II effects on MLC phosphorylation, a pathway that plays a crucial role in Ca2+-independent regulation of smooth muscle cell contraction by inhibiting myosin phosphatase activity through myosin binding subunit phosphorylation (29). This is the first demonstration of a functional link between CRH-R2-specific agonists and the myometrial RhoA/ROK signaling pathway. Myometrial RhoA and ROK are up-regulated during human pregnancy (30) and are thought to be responsible for the uterotonin-stimulated (such as by oxytocin) Ca2+ sensitization found in the development of smooth muscle contraction (31, 32). The precise mechanism leading to RhoA translocation and ROK activation is under investigation; however, our preliminary studies suggest that it might require activation of MEK1/ERK1/2 because inhibition of its activating kinase MEK1 (by U0126) effectively blocked RhoA recruitment to the plasma membrane. In contrast, myometrial ERK1/2 activation does not require intact RhoA/ROK activity because inhibition of either RhoA or ROK activity (by exoenzyme C3 or Y27632, respectively) did not affect UCN II-induced ERK1/2 activation. Collectively, these results suggest the presence of a single signaling pathway, rather than two parallel cascades, in human pregnant myometrial cells potentially involving sequential activation by UCN II of PKC, MEK1, ERK1/2-RhoA, and ROK (Fig. 8). This hypothesis is supported by our findings showing that inhibition of either ERK1/2 or RhoA totally abolished UCN II-induced MLC phosphorylation. However, it is possible that intracellular molecules other than the ERK1/2, such as specific G-proteins (33) or the PLC/ PKC pathway (34), are also involved in RhoA or ROK and MLC phosphorylation.
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Acknowledgments |
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Footnotes |
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Abbreviations: CRH-R2, Type-2 CRH receptor; FISH, fluorescent in situ hybridization; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; PKC, protein kinase C; PLC, phospholipase C; ROK, RhoA-associated kinase; UCN, urocortin.
Received September 11, 2003.
Accepted for publication October 21, 2003.
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References |
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-induced activation of RhoA in airway smooth muscle cells: role in the Ca2+ sensitization of myosin light chain20 phosphorylation. Mol Pharmacol 63:714–721 and R2ß CRH receptor subtypes and stimulation of Gq-proteins. Mol Endocrinol 14:2076–209112, G13, and Gq induce Rho-dependent neurite retraction through different signaling pathways. J Biol Chem 273:28700–28707This article has been cited by other articles:
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  J Lartey and A Lopez Bernal RHO protein regulation of contraction in the human uterus Reproduction, September 1, 2009; 138(3): 407 - 424. [Abstract] [Full Text] [PDF]
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 M. Torricelli, L. Galleri, C. Voltolini, G. Biliotti, P. Florio, M. De Bonis, and F. Petraglia Changes of Placental Kiss-1 mRNA Expression and Maternal/Cord Kisspeptin Levels at Preterm Delivery Reproductive Sciences, October 1, 2008; 15(8): 779 - 784. [Abstract] [PDF]
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 I. Garcia-Verdugo, Z. Tanfin, E. Dallot, M.-J. Leroy, and M. Breuiller-Fouche Surfactant Protein A Signaling Pathways in Human Uterine Smooth Muscle Cells Biol Reprod, August 1, 2008; 79(2): 348 - 355. [Abstract] [Full Text] [PDF]
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 D. Markovic, A. Punn, H. Lehnert, and D. K. Grammatopoulos Intracellular Mechanisms Regulating Corticotropin-Releasing Hormone Receptor-2{beta} Endocytosis and Interaction with Extracellularly Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling Cascades Mol. Endocrinol., March 1, 2008; 22(3): 689 - 706. [Abstract] [Full Text] [PDF]
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Dan Jin, Ping He, Xingji You, Xiaoyan Zhu, Ling Dai, Qian He, Chunmin Liu, Ning Hui, Jinyan Sha, and Xin Ni Expression of Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 in Human Pregnant Myometrium Reproductive Sciences, September 1, 2007; 14(6): 568 - 577. [Abstract] [PDF]
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 D. Markovic, M. Vatish, M. Gu, D. Slater, R. Newton, H. Lehnert, and D. K. Grammatopoulos The Onset of Labor Alters Corticotropin-Releasing Hormone Type 1 Receptor Variant Expression in Human Myometrium: Putative Role of Interleukin-1{beta} Endocrinology, July 1, 2007; 148(7): 3205 - 3213. [Abstract] [Full Text] [PDF]
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S. V. Wu, P.-q. Yuan, L. Wang, Y. L. Peng, C.-Y. Chen, and Y. Tache Identification and Characterization of Multiple Corticotropin-Releasing Factor Type 2 Receptor Isoforms in the Rat Esophagus Endocrinology, April 1, 2007; 148(4): 1675 - 1687. [Abstract] [Full Text] [PDF]
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I. Garcia-Verdugo, D. Leiber, P. Robin, E. Billon-Denis, R. Chaby, and Z. Tanfin Direct Interaction of Surfactant Protein A with Myometrial Binding Sites: Signaling and Modulation by Bacterial Lipopolysaccharide Biol Reprod, April 1, 2007; 76(4): 681 - 691. [Abstract] [Full Text] [PDF]
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A. Punn, M. A. Levine, and D. K. Grammatopoulos Identification of Signaling Molecules Mediating Corticotropin-Releasing Hormone-R1{alpha}-Mitogen-Activated Protein Kinase (MAPK) Interactions: The Critical Role of Phosphatidylinositol 3-Kinase in Regulating ERK1/2 But Not p38 MAPK Activation Mol. Endocrinol., December 1, 2006; 20(12): 3179 - 3195. [Abstract] [Full Text] [PDF]
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 A. Ball, J. W. Wang, S. Wong, B. Zielnik, J. Mitchell, N. Wang, M. B. Stemerman, and B. F. Mitchell Phorbol ester treatment of human myometrial cells suppresses expression of oxytocin receptor through a mechanism that does not involve activator protein-1 Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E922 - E928. [Abstract] [Full Text] [PDF]
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J. R. Lindsay and L. K. Nieman The Hypothalamic-Pituitary-Adrenal Axis in Pregnancy: Challenges in Disease Detection and Treatment Endocr. Rev., October 1, 2005; 26(6): 775 - 799. [Abstract] [Full Text] [PDF]
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 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]
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K. L. Parham, S. Zervou, E. Karteris, R. D. Catalano, R. W. Old, and E. W. Hillhouse Promoter Analysis of Human Corticotropin-Releasing Factor (CRF) Type 1 Receptor and Regulation by CRF and Urocortin Endocrinology, August 1, 2004; 145(8): 3971 - 3983. [Abstract] [Full Text] [PDF]
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