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Mechanisms of Hormone Action |
Stimulation of Bovine Luteal Cells1
Departments of Biochemistry/Biophysics and Animal Sciences,3 Oregon State University, Corvallis, Oregon 97331
Laboratory of Molecular Pharmacology, Biosignal Research Center,4 Kobe University, Kobe 657-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF2 treatment and 2) evaluate PGF2-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF2 (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 µM). Treatment of cells expressing wild-type MARCKS-GFP with PGF2 and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF2 treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.
corpus luteum, mechanisms of hormone action, oxytocin, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2) results in phosphorylation and translocation of MARCKS, events closely correlated with exocytosis of luteal oxytocin [9].
Although biochemical approaches have provided evidence for an association of actin disassembly with exocytosis of oxytocin in the bovine CL, there is a need to examine further the subcellular changes in actin cytoskeletal filaments and vesicle transport that characterize the exocytotic process in this endocrine gland. This can be accomplished through stimulation of large luteal cells containing transfected green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) constructs [10] and concomitant immunocytochemical tracking of oxytocin granules within large luteal cells. The present study was conducted to 1) examine cytologically the translocation of MARCKS-GFP in luteal cells in response to PGF2 stimulation and 2) evaluate the dynamics of oxytocin exocytosis that are concomitant with changes in the actin cytoskeleton as a consequence of MARCKS translocation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Four beef heifers were checked twice daily for behavioral estrus (Day 0), using a vasectomized bull. The CL was collected on Day 8 of the cycle per vaginum under lidocaine (2%)-induced caudal epidural anesthesia [11]. All animal experimental procedures were reviewed and performed in accordance with the institutional Animal Care and Use Committee guidelines at Oregon State University. Upon removal, the CL was transported to the laboratory in cold (4°C) Ham F-12 medium (Life Technologies, Rockville, MD) supplemented with 44 mM NaHCO3, 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin. The CL was cut in half, weighed, and minced into pieces approximately 2 mm3. Cell dispersion was achieved by incubating the minced tissue in 20 ml of F-12 medium with collagenase (3000 U/g of tissue; Worthington Biochemical Corp., Lakewood, NJ) for 2 h at 37°C. Dispersed cells were rinsed three times with F-12 medium. After centrifugation (1000 x g) cells were resuspended in 1 ml and counted with a Coulter counter (Beckman Coulter, Brea, CA). Cells were maintained in 6 ml of F-12 medium in a humidified atmosphere containing 5% CO2 at 37°C. The mean percentage of viable cells was 79% ± 2.3% as determined by the trypan blue stain exclusion method [12].
Luteal cells were spread onto round coverslips (8 mm in diameter; Fisher Scientific, Pittsburgh, PA) placed into wells of a Falcon 24-well plate (Becton Dickinson, Lincoln Park, NJ) containing 1 ml F-12 medium and cultured for at least 18 h. Prior to treatments, culture medium was replaced with normal Hepes buffer (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM Hepes, and 10 mM glucose, pH 7.3).
Expression of Wild-Type and Mutant MARCKS-GFP in Luteal Cells
One wild-type (wt) and two mutant (m3 and G2A) MARCKS-GFP cDNA constructs utilized by Ohmori et al. [10] were provided by N. Saito (Kobe University, Kobe, Japan). In m3, the serine residues were replaced with alanine in the phosphorylation site domain by site-directed mutagenesis so that mutant MARCKS-GFP cannot be phosphorylated by PKC. The N-terminal glycine was replaced with alanine in G2A resulting in a nonmyristoylated molecule, thus disrupting its membrane binding ability [10]. MARCKS and GFP were conjugated at the C-terminus of the MARCKS protein. Upon receipt, pBluescript II KS(-) subcloned MARCKS-GFP cDNAs were transfected into Escherichia coli colonies, which were then spread onto a plate containing ampicillin and then incubated at 37°C. After 24 h, a single colony was isolated and amplified in ampicillin containing LB medium. When bacterial growth reached a stable phase, the presence of correct size cDNAs was confirmed by enzymatic digestion with the restriction enzyme EcoRI. After visualization of correct size cDNA fragments on 1% agarose gel, remaining plasmids were isolated and purified using the Plasmid Maxi Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.
Amplified and purified wt MARCKS-GFP construct and its mutants were evaluated for functional integrity by transfecting Chinese hamster ovary (CHO-K1) cells (Center of Cell Research, Oregon State University, Corvallis, OR). These cells were transfected with the cDNAs and treated with PGF2 and 12-O-tetradecanoylphorbol-13-acetate (TPA) 24 h after detection of the fluorescence. The results (data not shown) were strikingly similar to those described by Ohmori et al. [10].
Dispersed luteal cells (106 cells/dish) from each of four heifers were cultured in glass-bottom dishes (MatTek, Ashland, MA) for 24 h before transfection. Cells were transfected with plasmids including wt MARCKS-GFP and its mutant cDNAs by lipofection using Trans IT-LT1 (Mirus, Madison, WI). A ratio of 1 µg cDNA/6 µl reagent was used (total of 2 µg cDNA/dish). Supplementation of F-12 medium with 10% fetal bovine serum resulted in a higher transfection efficiency with primary luteal cells (23%–35%). The fluorescence of MARCKS-GFP was detectable 15 h after transfection. Luteal cells were incubated 48 h after transfection until treatments were imposed. Duplicate dishes containing live luteal cells (79%) transfected with one of the three cDNAs (wt, m3, G2A) were assigned randomly for the following treatments: ethanol (control, 10 µl), PGF2 (56 nM), and TPA (1 µM). Numbers of large luteal cells (mean ± SEM: wt, 14.7 ± 1.85; m3, 12.4 ± 1.9; G2A, 13.2 ± 2.1) were recorded before the treatment (0 min) and 2.5 and 5 min after the treatments in 1-sec exposures using a Axiovert S100 TV (Zeiss, Thornwood, NY) fluorescent microscope with attached camera and MetaMorph 4.6 software (Universal Imaging, Downingtown, PA).
Immunostaining of Luteal Cells Expressing MARCKS-GFP
Luteal cells were cultured on round coverslips and transfected with one of the cDNAs as described above. After observing the fluorescence of MARCKS-GFP with the Axiovert S100 TV fluorescent microscope on Day 3 of the culture, culture medium was replaced with Hepes buffer. Luteal cells expressing three different MARCKS-GFP constructs were treated in duplicate with ethanol (control, 10 µl) and PGF2 (56 nM) for 5 min. Luteal cells were then fixed with 4% paraformaldehyde in 0.1 M PBS for 30 min. Cells were then rinsed three times with 0.1 M PBS and treated with 0.3% Triton X-100 in PBS and 10% normal goat serum for 20 min. Next, cells were incubated with rabbit anti-oxytocin polyclonal antibody (1:5000; Chemicon International, Temecula, CA) for 60 min with 0.03% Triton X-100 and 10% normal goat serum at room temperature. The antibody-oxytocin complex was detected by exposing cells to rhodamine-labeled goat anti-rabbit IgG (Alexa Fluor 546; Molecular Probes, Eugene, OR) for 30 min at room temperature. For staining actin filaments, cells were subsequently rinsed with PBS and incubated with Alexa Fluor 350 phalloidin (absorption 346, emission 442, 300 U/dish; Molecular Probes) for 30 min at room temperature. After three washes with PBS, coverslips carrying cells were removed to air dry and then were mounted on glass slides using Prolong Antifade (Molecular Probes). Cells were examined using a fluorescent microscope, and images were recorded as described in the previous section. From each of four heifers, a total of 20 large luteal cells per treatment (vehicle or PGF2) containing either transfected wt, m3, or G2A MARCKS-GFP were examined microscopically.
Statistical Analysis
Data on percentages of large luteal cells transfected with wt MARCKS-GFP exhibiting translocation of the protein in response to PGF2 and TPA were analyzed by one-way ANOVA. Differences among means were tested for significance with the least significant difference test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The translocation of wt and mutant MARCKS-GFP was visualized by monitoring the changes in GFP fluorescence. Apparently, all three types of the plasmids were intensely expressed by the large luteal cells, as determined by the abundant GFP signal (Fig. 1). Treatment of cells expressing transfected wt and mutant MARCKS-GFP with ethanol (vehicle control) produced no changes in the distribution of the GFP-attached MARCKS protein (Fig. 1, A, D, and G; wt, m3, and G2A, respectively). Treatment of cells expressing wt MARCKS-GFP with PGF2 and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min (Fig. 1, B and C, respectively). The majority of the wt MARCKS-GFP protein translocated from the periphery of the cells and accumulated in the paranuclear region by 5 min. The mean percentage of large luteal cells in which translocation of wt MARCKS-GFP occurred in response to stimulation with PGF2 or TPA was greater than that of controls (P < 0.001; Fig. 2). Phosphorylation mutant (m3) MARCKS-GFP expression was detected solely on the plasma membrane, and neither PGF2 nor TPA stimulation induced translocation of this mutated MARCKS protein from the membrane to the cytoplasm at either time period studied (Fig. 1, E and F). In transfected cells containing the myristoylation site-mutated MARCKS-GFP, the protein product remained exclusively in the cytoplasmic compartment, and treatments with PGF2 and TPA failed to effect a change in its subcellular localization after either 2.5 or 5 min (Fig. 1, H and I).
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Correlation of Phosphorylation and Translocation of MARCKS with the Changes in Cross-Linked Actin Filaments and Oxytocin Localization
To understand the functional role of phosphorylation and translocation of MARCKS protein, along with MARCKS-GFP expression, cells were fixed and stained sequentially for oxytocin and filamentous actin with fluorescent probes of different emission-excitation wavelengths. Images from three different channels were combined to superimpose the three subcellular constituents of the observed cell; actin cytoskeleton (blue), oxytocin granules (orange-red), and MARCKS-GFP (green). Treatment of control cells with vehicle (ethanol) had no significant effect on translocation of wt MARCKS-GFP, movement of vesicles containing oxytocin, or changes in actin filament composition (Fig. 3A). Wild-type MARCKS-GFP was associated with the cell membrane, whereas oxytocin staining was localized in the paranuclear area (Fig. 3A). The actin filaments (Fig. 3A, arrows) appeared intact, similar to those of neighboring fibroblasts. PGF2 treatment resulted in increased translocation of MARCKS from the plasma membrane to the cytoplasm and induced movement of oxytocin from the paranuclear area to the cell periphery and probably secretion (Fig. 3B). The actin filaments appeared shorter; an indicator of possible disintegration (Fig. 3B, arrows). Neither ethanol (control; data not shown) nor PGF2 stimulation provoked any changes in MARCKS translocation, oxytocin granule mobilization, and composition of actin filaments in cells transfected with the phosphorylation mutant MARCKS-GFP (m3) (Fig. 3C). Similarly, in cells expressing the myristoylation mutant MARCKS-GFP, treatment with ethanol (data not shown) or PGF2 failed to cause any change in the location of the protein, which remained uniformly distributed throughout the cytoplasm (Fig. 3D). In these latter cell types, oxytocin was localized to the paranuclear area and actin filaments showed no change in integrity or structure.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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has been shown to stimulate secretion of oxytocin from bovine luteal cells both in vivo [11, 13] and in vitro [14]. However, whether uterine PGF2 induces secretion of luteal oxytocin to promote luteolysis in the cow as apparently occurs in the ewe [15] is controversial. Although there is some evidence that episodic luteal secretion of oxytocin might contribute to luteal regression in the cow [16], more recent evidence suggests otherwise [17]. Nevertheless, the present study was conducted to determine, using MARCKS-GFP transfected primary luteal cell cultures, whether MARCKS protein plays any role in PGF2-induced secretion of luteal oxytocin. Our data demonstrate that phosphorylation of the plasma membrane-associated MARCKS protein is essential for exocytosis of oxytocin by the bovine large luteal cell. Mutations that prevent phosphorylation or myristoylation of MARCKS protein preclude initial intracellular mobilization of oxytocin granules characteristic of exocytosis of this nanopeptide. MARCKS protein contains three highly conserved regions [18]. One of these regions is an eight-residue domain in the amino terminal region of the protein that has unknown function (MARCKS homology 2 domain). Another region is the myristoylation site that directs the cotranslational addition of the 14-carbon myristate moiety. The third domain is a 25-amino acid basic effector domain that contains PKC phosphorylation sites. This region, known as the phosphorylation site domain, contains 12 or 13 positively charged Lys/Arg residues [6] and interacts electrostatically with the membrane. Myristoylation and phosphorylation site domains provide hydrophobic and electrostatic interactions of MARCKS with the plasma membrane, respectively [6]. Phosphorylation of MARCKS by PKC disrupts its cross-linking and anchoring capacities and causes its translocation from membrane to cytoplasm [6]. Phosphorylation-dependent translocation of MARCKS is a pivotal event associated with the disassembly of actin cortex in a variety of secretory cells [7, 8, 19].
Translocation of MARCKS has been visualized using GFP-tagged MARCKS cDNA constructs in living CHO-K1 cells in a study elegantly conducted by Ohmori et al. [10]. Wild-type MARCKS-GFP translocated rapidly from membrane to cytoplasm in response to TPA treatment. However, a MARCKS-GFP construct containing the mutated phosphorylation site domain remained associated with membrane and actin cortex after treatment with TPA [10]. In experiment 1 of the present study, bovine luteal cells were transfected successfully with wt or mutant MARCKS-GFP constructs to observe cytological changes in response to treatment with PGF2. Cells expressing either type of MARCKS-GFP plasmid appeared solely green under the fluorescence microscope. Based on morphology, only large luteal cells were observed and recorded. The percentage of large luteal cells exhibiting translocation of MARCKS protein did not differ among those responding to PGF2 and TPA (P > 0.05; Fig. 2). However, the effects of PGF2 stimulation, as observed by the decrease in fluorescence intensity over time, appeared more profound than those of TPA stimulation (Fig. 1, B and C). We speculate that unlike TPA stimulation that activates PKC because of its structural resemblance to diacylglycerol (DAG), PGF2 in addition to activating PKC also increases intracellular free Ca2+ via formation of inositol 1,4,5-trisphosphate (IP3), which activates Ca2+-dependent F-actin-severing enzymes scinderin and gelsolin [20]. Treatment of chromaffin cells with phorbol esters causes only a partial disruption of the cortical actin cytoskeleton [21].
Experiment 2 was conducted to ascertain whether translocation of MARCKS protein was associated with obvious structural changes in the actin cortex and mobilization of oxytocin granules. Unlike experiment 1, where observations were made on living cells, for experiment 2 cells were fixed with 4% paraformaldehyde. Thus, cellular micrographs may appear slightly different in Figures 1 and 3. Within 5 min of PGF2 stimulation, release-ready oxytocin vesicles were scattered in the cytoplasm, usually in close proximity to the plasma membrane, and relatively fewer vesicles were detected in the paranuclear region (Fig. 3B). However, oxytocin immunoreactivity was particularly intense in the paranuclear region, and only limited movement of vesicles containing oxytocin was observed in PGF2-treated cells expressing either type of mutant MARCKS-GFP construct. In these cells, the actin cytoskeleton appeared to be intact and consisted of long filamentous actin (Fig. 3, C and D). Because the actin filaments remained intact, one would expect to observe accumulation of oxytocin granules in PGF2-stimulated cells just beneath the actin cortex. However, as visible in Figure 3C, the anticipated mobilization of vesicles containing oxytocin never occurred. These findings suggest that interrupting the functionality of MARCKS protein (inhibition of phosphorylation) not only fails to affect the integrity of the actin filaments but also suppresses oxytocin granule mobilization. Lack of oxytocin exocytosis in stimulated cells suggests that MARCKS protein is somehow involved in promoting downstream signal transduction. This hypothesis is supported by the findings of Rauch et al. [22], who suggested that MARCKS sequesters phosphatidylinositol-4,5-bisphosphate (PIP2) while in the unphosphorylated state, with phosphorylation of MARCKS causing the release of PIP2 on the plasma membrane. Thus, in the case of the phosphorylation mutant of MARCKS there would be little or no PIP2 to generate DAG and IP3 in response to PGF2. Hence, no downstream signal would be generated. Trifaro et al. [23] suggested that exocytosis of secretory granules coincides with disassembly of actin filaments. Although various proteins bind and cross-link actin filaments and alter cytoskeletal dynamics, MARCKS possesses demonstratable properties that are correlated with the exocytotic process [10, 24].
The results of the present research confirm the existence of MARCKS protein in bovine large luteal cells and demonstrate that phosphorylation of the protein is essential for initiation of the exocytotic process. It appears from these observations that phosphorylation of MARCKS protein is involved with the generation of a signal propagated via the interior cytoskeletal matrix to ensure mobilization of secretory vesicles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Fredrick Stormshak, Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331. FAX: 541 737 4174; fred.stormshak{at}orst.edu
Received: 27 March 2003.
First decision: 18 April 2003.
Accepted: 18 August 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activation. Mol Brain Res 1999 64:236-245[Medline]-induced oxytocin secretion from the bovine corpus luteum. Endocrinology 1994 134:78-83-induced release of oxytocin from bovine corpora lutea in vitro. Biol Reprod 1987 37:550-555[Abstract]This article has been cited by other articles:
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  K. Satoh, M. Matsuki-Fukushima, B. Qi, M.-Y. Guo, T. Narita, J. Fujita-Yoshigaki, and H. Sugiya Phosphorylation of myristoylated alanine-rich C kinase substrate is involved in the cAMP-dependent amylase release in parotid acinar cells Am J Physiol Gastrointest Liver Physiol, June 1, 2009; 296(6): G1382 - G1390. [Abstract] [Full Text] [PDF]
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 J. Li, K. L. O'Connor, G. H. Greeley Jr., P. J. Blackshear, C. M. Townsend Jr., and B. M. Evers Myristoylated Alanine-rich C Kinase Substrate-mediated Neurotensin Release via Protein Kinase C-{delta} Downstream of the Rho/ROK Pathway J. Biol. Chem., March 4, 2005; 280(9): 8351 - 8357. [Abstract] [Full Text] [PDF]
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 C. Ehre, A. H. Rossi, L. H. Abdullah, K. De Pestel, S. Hill, J. C. Olsen, and C. W. Davis Barrier role of actin filaments in regulated mucin secretion from airway goblet cells Am J Physiol Cell Physiol, January 1, 2005; 288(1): C46 - C56. [Abstract] [Full Text] [PDF]
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 M. Shibaya, K. M. Deptula, A. Korzekwa, K. Okuda, and D. J. Skarzynski Involvement of the Cytoskeleton in Oxytocin Secretion by Cultured Bovine Luteal Cells Biol Reprod, January 1, 2005; 72(1): 200 - 205. [Abstract] [Full Text] [PDF]
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