Lysophosphatidic Acid Signals through Mitogen-Activated Protein Kinase-Extracellular Signal Regulated Kinase in Ovarian Theca Cells Expressing the LPA1/edg2-Receptor: Involvement of a Nonclassical Pathway?
Lygia T. Budnik,
Bärbel Brunswig-Spickenheier and
Amal K. Mukhopadhyay
Institute for Hormone and Fertility Research, University of Hamburg, D-20251 Hamburg, Germany
Address all correspondence and requests for reprints to: Dr. Lygia T. Budnik, Institute for Hormone and Fertility Research, University of Hamburg, Centrum for Innovative Medicine, Falkenried 88, D-20251 Hamburg, Germany. E-mail: Budnik{at}IHF.de.
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ABSTRACT
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We investigated the mechanism of lysophosphatidic acid (LPA) signaling in ovarian theca cells and observed that stimulation with this bioactive lipid markedly enhanced Thr/Tyr phosphorylation of the MAPK ERK1/2. Activation of ERK was transient, showing a peak at 5 min that declined thereafter, and was not associated with a concomitant nuclear translocation of the enzyme, suggesting that a cytosolic tyrosine phosphatase may be responsible for switching off the signal. Epidermal growth factor (EGF)-induced activation of the enzyme in the same cell system was more rapid (peaking at 1 min), sustainable for at least 60 min, and could be suppressed by prior treatment with either pertussis toxin or a noncompetitive inhibitor of Ras acceptor protein, manumycin A. This functional inhibition of either Gi or Ras failed, however, to affect the LPA-induced ERK-phosphorylation. Surprisingly, functional inhibition of Rho-GTPase, in C3-exotoxin-lipofected cells, markedly reduced LPA-stimulated phosphorylation of ERK, without affecting the EGF-induced stimulation of MAPK. Theca cells labeled with anti-LPA1/edg2-type antibody showed a distinct cell surface labeling, which is reflected in the expression of (LPA1)-type LPA receptors at both mRNA and protein levels. The findings indicate that LPA transiently stimulates MAPK ERK in LPA1/edg2-expressing theca cells and suggest an alternative mechanism regulating the activation of ERK that differs from the canonical EGF-Ras-MAPK kinase pathway.
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INTRODUCTION
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A PRESUMED ROLE of the bioactive lipid lysophosphatidic acid (LPA) in carcinogenesis (1, 2, 3, 4) may be reflected in normal physiological functions related to growth, differentiation, or survival. LPA is found not only in malignant effusions (3, 4) but also in the follicular fluid of healthy mammalian ovaries, implying a local physiological relevance (5, 6). The cyclical demands on the ovary make the ovulatory follicle one of the most extreme tissues in the body, in terms of growth, differentiation and, in the absence of fertilization, regression. The developing follicle, as one of the fastest growing and differentiating tissues in the body, is also known to undergo processes similar to those involved in inflammation and wound healing (7, 8). Interestingly, all such systems are known to involve participation of LPA (9, 10). Therefore, cells, like those from the theca of developing follicles, could serve as highly amenable models for LPA effects and help to identify the mechanisms responsible for LPA signaling under physiological conditions. Primary theca cells are capable of growth and differentiation in culture. In vivo, upon stimulation by the ovulatory-surge gonadotropin, LH, the ovarian granulosa and theca cells differentiate during the late follicular phase to large and small luteal cells, which form the corpus luteum (luteinization). In earlier studies, cell type- and ovarian cycle stage-dependent effects of LPA were observed (6), with radioactively labeled LPA binding to a 38-/40-kDa receptor protein, and LPA producing dramatic changes on the actin cytoskeleton of luteal cells stimulated with LH (11, 12, 13).
LPA is known to evoke hormone and growth factor-like responses through the activation of its cognate G-protein coupled (GPC) receptor in a variety of cell systems (14). Provisionally termed "edg" (endothelial differentiation gene) or "LPL" (lysolipid-like) receptor, the subfamily includes these receptors for LPA (three members) and five for SP1, sphingosine-1-phosphatide (15, 16, 17). Chun et al. (18) have proposed a provisional nomenclature for this receptor family (which has been recently recognized by the pharmacological society, International Union of Pharmacology) in which the edg-2.-4.-7-receptors are designated LPA1/A2/A3 and bind LPA with high affinity (19). LPA receptors are expressed widely (with a few exceptions such as HL60, B103, or yeast cells), but from the eight currently known LPA-receptor genes only the LPA1 receptor shows a high sequence conservation ranging from Xenopus to mammals (19, 20). Physiological events that follow LPA receptor activation may include mitogenesis, cell survival (antiapoptosis), wound healing, immune modulation, cellular differentiation, or carcinogenesis (9, 14, 15, 19). Analysis of LPA receptors by semiquantitative RT-PCR and Western blotting shows that epithelial ovarian cancer cells express LPA2/edg-4-receptor prominently (21). In contrast, the levels of the LPA1/edg2-receptor appear to be higher in nonmalignant epithelium (21). There is no information on the presence of LPA receptor subtypes on hormonally active ovarian cell compartments. The presence of a specific type of LPA receptor on granulosa, theca-, or luteal cells is a necessary prerequisite for the demonstration of a physiological function of LPA in the ovary.
LPA receptors make use of a variety of heterotrimeric G proteins including Gi2, Gq11, G
12, and G
13 (9, 14, 16). It is as yet poorly understood which individual LPA receptor couples to which G protein, and which adapter or exchange factors are involved in transducing the LPA signals. Further downstream, small GTPases like Ras or Rho are activated and may initiate either LPA-mediated mitogenic signaling (via Gi-Ras) or the G
12/13-Rho-dependent regulation of the actin cytoskeleton (14, 15, 16). A variety of cell responses initiated after binding of LPA to its LPA receptor are associated with the activation of the classic MAPKs, ERK1/2, (p44/p42) (22).
The classic serine/threonine kinases ERK1/2 can integrate signals from multiple agonists (23). The growth factor-stimulated ERK cascade is initiated by the MAPK kinase kinase (MAPKKK), Raf-1, also called c-Raf, which is ubiquitous in proliferation pathways (24). c-Raf is usually activated by Ras in a complex upstream process, which is relatively well understood only for epidermal growth factor (EGF) stimulation. The canonical Ras-Raf-MAPK kinase (MEK)-ERK pathway is known to be initiated by tyrosine phosphorylation of several substrates (including EGF-receptor-tyrosine-kinase) with subsequent utilization of adapter proteins such as son of sevenless (SOS) (25, 26, 27). Like other switch kinases, ERKs are themselves regulated by phosphorylation in the absence of a regulatory subunit (28). Two upstream dual specificity enzyme kinases MEK1/MEK2 (MAPKKs) phosphorylate threonine and tyrosine residues of ERK1/2 and initiate an ultrasensitive downstream phosphorylation cascade (25). Phosphorylation of the residues Thr 202/Tyr 204 (for human ERK, or Thr 185/Tyr 187 for bovine ERK) in the conserved ERK sequences Thr/Glu/Tyr is necessary and sufficient for full activation of the enzymes and serves as an indicator of their activation status (28).
More recently, GPC receptors have also been implicated as MAP effectors (29) although the biochemical events involved in this process have not been fully elucidated. LPA is also known to stimulate the classical MAPK cascade, involving Gi, tyrosine kinase (s)-Grb2/SOS adapter proteins, and RasGTPase (22). Given the variety of diverse and essential cellular functions regulated by MAPK (22) and the variety of LPA receptor-G protein couplings (14, 15, 19), it is easy to imagine the involvement of other pathways. In fact, indirect pathways to MAPK, involving c-src- or integrin-focal adhesion proteins, have also been implicated in LPA signaling to MAPK (22, 30). We have, therefore, investigated whether LPA stimulates the classical ERK pathway under physiological conditions. We show here that primary theca interna cells express LPA1-type LPA receptors and that stimulation with LPA results in a transient activation of the ERK kinase cascade in these cells. Interestingly, the route of activation leading to LPA-stimulated MAPK appears to differ from the canonical EGF-Ras-MEK-ERK pathway.
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RESULTS
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LPA Induces Transient, MEK1-Dependent Activation of the MAPK ERK1/2 in Theca Cells
To investigate possible LPA effects on ERK activation, we used immunoblot assays with a monoclonal antibody recognizing the fully phosphorylated form of ERK1/2 [P(T,Y)-ERK1/2]. Treatment of theca cell monolayers with 5 µM LPA for 15 min showed activation of MAPK in cytosolic fractions isolated from the LPA-treated cells (Fig. 1A
). Neither the membrane nor nuclear fraction indicated any detectable amounts of P-ERK (data not shown). No modification in the protein level of total ERK kinases was observed after stimulation with LPA (analyzed with antibody recognizing nonphosphorylated p44/p42-ERK) indicating that the increase in ERK phosphorylation was not due to an increase in total ERK protein (shown in lower panels). As an additional control, a mixture consisting of purified nonphosphorylated p42 ERK protein and fully phosphorylated P(T,Y)-ERK2 were used (Fig. 1A
, Con. Prot.). Pretreatment with PD 98059, an inhibitor of the MAPKK, MEK-1 (31), completely abolished LPA-induced phosphorylation of ERK1/2, whereas the holoenzyme (ERK1/2) remained unaffected (Fig. 1A
). Addition of vanadate, as a broad spectrum inhibitor of tyrosine phosphatase, slightly enhanced the basal phosphorylation of ERK, whereas the serine-threonine phosphatase inhibitor, okadaic acid, had no effect. The LPA-induced stimulation of ERK in theca cells was transient, appearing gradually within 15 min, peaking at 5 min and declining thereafter (Fig. 1B
). Four different patterns of activation/deactivation of the enzyme were reported for growth factor-stimulated mammalian cells (23). Alessi et al. (23) could show that, depending on the cell system studied and on growth factor stimulus applied, either vanadate- or okadaic acid-sensitive phosphatases could participate in inactivating the MAPK signal in the cytosol. Alternatively, the enzyme can be translocated and probably deactivated in the nucleus.

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Fig. 1. LPA Induces Transient, MEK1-Dependent Activation of the MAPK ERK1/2 in Theca Cells
A, Theca cells were pretreated in serum-free medium for 90 min and stimulated either with medium only (basal), with okadaic acid (OA, 10 nM), vanadate (Van., 1 mM), LPA (5 µM), or with LPA plus PD 98059 (40 µM) for 15 min. The last lane shows control peptide mixture (nonphosphorylated ERK2 and phosphorylated ERK2), detailed in Materials and Methods. B and C, Cells were treated with either LPA (5 µM), with LPA plus okadaic acid (10 nM), or with LPA plus vanadate (1 mM) for 030 min. Cytosolic fractions were resolved by SDS-PAGE. Immunoblots were performed to assess activation of the MAPK ERK with an antibody recognizing dually phosphorylated P(T,Y)-ERK1/2. All blots were rehybridized with an antibody directed against the nonphosphorylated ERK1/2. Immunocomplexes were detected by chemiluminescence after the incubation with the respective peroxidase-conjugated secondary antibody. Data show representative experiments repeated three times with similar results.
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We found that the inhibition of protein phosphatase 2A with 10 nM okadaic acid had no effect on LPA-induced phosphoactivation of ERK1/2 (Fig. 1B
) whereas, in the presence of 1 mM vanadate (a broad spectrum inhibitor of tyrosine phosphatase), the signal remained unchanged throughout the time period (Fig. 1C
).
Parallel experiments determined that, under the same experimental conditions, no appreciable activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) could be observed (data not shown).
Neither the Phorbol Ester PMA (4ß-Phorbol-12-Myristate-13-Acetate) nor the cAMP Analog 8-Br-cAMP Can Mimic the Effects of LPA on the ERK1/2 Activation
Before the MEK1/2 kinases can initiate the phosphorylation of ERK1/2, the canonical growth factor-stimulated pathway utilizes the activation of Ras-Raf (25). The upstream serine kinase (MAPKKK) c-Raf (also called Raf-1) is subject to activation by another kinase. Although the identity of this kinase is poorly defined at present, several kinases such as protein kinase C or protein kinase A have been reported to play a functional role as modulators of c-Raf (32, 33). In some cell systems, modulatory effects of either the cell-permeable cAMP analog (8-Br-cAMP) or the phorbol ester, PMA have been reported. To check whether 8-Br-cAMP, its activator luteotropin, LH, or PMA can mimic the stimulatory effects of LPA on ERK, we stimulated the cells with either 1 mM 8-Br-cAMP, 100 ng/ml LH, or 10 nM PMA for various time periods. The phosphoactivation of ERK was analyzed as described above. Neither 8-Br-cAMP/LH nor PMA was able to activate ERK under the given conditions (data not shown).
No Nuclear Relocalization of P(T,Y)-ERK1/2 Can Be Observed in LPA-Stimulated Theca Cells
In addition to phosphorylation and dephosphorylation, P(T,Y)-ERK1/2 is known to be rapidly translocated to the nucleus after agonist-mediated activation. Because we could not detect any active ERK proteins in the nuclear fractions using the Western blotting procedure (see above), we performed more detailed analysis at a single cell level. Cells grown on chamber slides were stimulated with LPA for various time periods (using the same experimental conditions as described for Western blotting). Immunohistochemical analysis using phosphospecific-anti-ERK antibodies revealed that there was no appreciable relocalization of activated ERK after stimulation of the theca cells with LPA for 30 min (Fig. 2A
). Even at higher magnification, no nuclear staining could be observed after stimulation with LPA (examined by laser scanning confocal microscopy). To enhance visualization of the nuclei, the cells were additionally counterstained with 4'6'-diamidino-2-phenylindole (DAPI) (shown in control panels). No nuclear localization of the enzyme could be observed when the cells were treated with LPA for various time periods, from 15 min until overnight (data not shown). In contrast, LPA was able to induce 6090% nuclear translocation of activated ERK in control NIH 3T3 cells within 30 min (Fig. 2C
). Because the inhibition of tyrosine phosphorylation with vanadate prevented rapid dephosphorylation (deactivation) of the ERK signal in the cytosolic fractions, cells were additionally treated with vanadate to check whether this inhibitor could also influence the cellular localization of the enzyme. We could observe (Fig. 2B
) that, whereas this phosphatase inhibitor alone had no effect, its presence during the stimulation with LPA led to some (20%) perinuclear and nuclear relocalization of P-ERK (Fig. 2B
, lower part).

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Fig. 2. No Nuclear Relocalization of P(T, Y)-ERK1/2 Can Be Observed in LPA-Stimulated Theca Cell
A and B, Theca cells grown on chamber slides were pretreated for 90 min in serum-free medium and stimulated with either 5 µM LPA, with 1 mM vanadate (Van.), or with LPA plus vanadate (LPA +Van.) for 30 min. C, Control NIH 3T3 cells were treated with or without 5 µM LPA as described above for the theca cells. Cells were fixed and immunostained using a monoclonal antibody recognizing P(T,Y)-ERK1/2 followed by Cy3-conjugated antimouse secondary antibody. Each slide was subjected to blue nuclear counterstaining with DAPI and was visualized by laser scanning confocal microscope (magnification shown in A and C, x1600; and in B, x8000). Micrographs were taken with a Leica digital camera. Data show representative experiments repeated three times with similar results.
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EGF Produces More Rapid Activation of P(T,Y)-ERK1/2 than LPA in the Theca Cell
G
q-coupled receptors are known to transactivate EGF receptor tyrosine kinase and stimulate the activatory phosphorylation of ERK utilizing the classic EGF pathway (34). Because LPA could stimulate the MAPK ERK through a pathway using transactivation of the EGF receptor (35, 36), it was of interest for us to check whether EGF itself can stimulate ERK in theca cells. Cells were therefore stimulated with either EGF (10 ng/ml) or LPA (5 µM), and ERK phosphoproteins were analyzed as described above. Figure 3A
shows that EGF induced the activation of ERK in our cell system, peaking at 15 min and declining thereafter more slowly than for LPA (Fig. 3
, A and B). Results from eight independent experiments show, however, that the activation of P(T,Y)-ERK1 in response to both EGF and LPA peaked at 15 min (Fig. 3B
).

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Fig. 3. EGF Mimics the Effects of LPA on the Activation of ERK in Theca Cells
A, Cells were stimulated for 030 min with 10 ng/ml EGF. P(T,Y)-ERK1/2 and control ERK1/2 were analyzed as described in the legend to Fig. 1 . B, Mean data quantitated from eight independent experiments using cells treated with either 5 µM LPA or with 10 ng/ml EGF for 030 min. Plotted are integrated optical density values for the specific P(T,Y)-ERK1/2 bands analyzed with a computer-assisted program (detailed in Materials and Methods). The data show mean values ± SD. C, Cells were pretreated with or without either 10 µM tyrphostin B48 (AG 494) or tyrphostin B44 (AG 527) as described in Materials and Methods, and were stimulated with either 5 µM LPA or with 10 ng/ml EGF for 15 min. Immunoreactive P(T,Y)-ERK1/2 and control ERK1/2 were analyzed as described in the legend to Fig. 1 . D, Cells were stimulated for 15 min without any addition or with either 5 µM LPA or 10 ng/ml EGF. Membrane fractions were resolved by SDS-PAGE. Immunoblots were performed to assess the activation of the EGF receptor using an antibody recognizing the phosphorylated EGF receptor (P-EGF-R). The blot was stripped and rehybridized with anti-ß-actin antibody as control.
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Based on data collected from transfected cell lines, it has been suggested that LPA is able to transactivate the EGF receptor by acting on the EGF receptor tyrosine kinase (35). Pretreatment of theca cells with tyrphostins B48 (AG 494) and B44 (AG 527), known inhibitors of EGF receptor tyrosine phosphorylation (Fig. 3C
), had hardly any effect on LPA-stimulated activation of ERK1/2. When the activated EGF receptor status was evaluated in membranes from theca cells stimulated with either EGF or LPA, we could, however, observe that not only EGF, but also LPA was able to transactivate the EGF receptor (Fig. 3D
) in our cell system. When cells were additionally stimulated with LPA plus EGF, some additive effects were observed (at least for ERK2), especially when immunoblotted with an antibody recognizing ERK phosphorylated on tyrosine (P-Y) only (Fig. 4
).

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Fig. 4. LPA-Induced Phosphorylation of ERK Is Partially Additive in the Presence of EGF
Theca cells were stimulated with either 5 µM LPA or with LPA in combination with 10 ng/ml EGF. To assess activation of the MAPK ERK, the blots were incubated first with an antibody recognizing P(T,Y)-ERK1/2 and were rehybridized thereafter with an antibody recognizing ERK1/2 phosphorylated on tyrosine residues [P-(Y)-ERK1/2] only. As a control, the blots were incubated with an antibody recognizing nonphosphorylated ERK1/2 proteins. Lower plots show quantitated results repeated twice and analyzed as detailed in Materials and Methods.
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Pertussis Toxin (PTX) Inhibits Activating Phosphorylation of ERK Stimulated by EGF, But Not That Induced by LPA
LPA is known to activate Gi proteins that, in turn, can be blocked by PTX catalyzing the ADP ribosylation of G
i (14, 37). Cells were pretreated with PTX (100 ng/ml) for 180 min and were then further stimulated for 15 min with either EGF (10 ng/ml), with LPA (5 µM), or with medium only. The MAPK activity, P(T,Y)-ERK1/2 stimulated after LPA treatment, was not affected by PTX although the toxin also reduced the basal ERK phosphorylation levels. On the other hand, the EGF- induced activation of ERK was fully inhibited by PTX in the same cells (Fig. 5
).

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Fig. 5. The Activating Phosphorylation of ERK1/2 Can Be Inhibited by PTX Only When Induced by EGF, But Not by LPA
Cells pretreated with or without 100 ng/ml PTX were stimulated with either 5 µM LPA or with 10 ng/ml EGF for 15 min. P(T,Y)-ERK1/2 was analyzed as detailed in the legend to Fig. 1 . Control bands show total ERK1/2.
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Inhibition of Ras Function Abolishes the Effects of EGF on ERK but Has No Effect on P(T,Y)-ERK1/2 Phosphorylation Induced by LPA
A common link between EGF- or LPA-induced signaling to the MAPK ERK are the small GTPases from the Ras family (22, 24, 25), which can be blocked by the noncompetitive inhibitor of the Ras-acceptor protein, manumycin A (38). Figure 6A
shows that the inhibition of Ras function abolished the EGF-stimulated activation of ERK but did not influence the activation of ERK induced by LPA. We have then measured active Ras function using the pull-down Ras activity assay in stimulated cell lysates after incubation with a glutathione-S-transferase (GST) fusion protein containing a Ras-binding domain from c-Raf. Figure 6C
shows that whereas either EGF or GTP
S (control) could activate Ras in our cell system, stimulation with LPA did not affect the endogenous Ras activity. Further downstream, an activation of c-Raf in the presence of EGF (10 ng/ml) was observed. At the same time, LPA (5 µM) had hardly any effect on the phosphorylation status of the c-Raf kinase (Fig. 6D
). It would appear that a Gi-mediated pathway connects EGF signaling to Ras and c-Raf, but LPA may probably utilize another pathway in our cell system.

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Fig. 6. Inhibition of the Ras Function Abolishes the Effects of EGF on ERK, but Has No Effect on p44/42 Phosphorylation Induced by LPA
A and B, Cells pretreated with or without 2 µg/ml manumycin A (Manu.) were stimulated with either 10 ng/ml EGF or with 5 µM LPA for 15 min. P(T,Y)-ERK1/2 was analyzed as described in the legend to Fig. 1 . B, Quantitated data from three independent experiments (mean ± SD) analyzed statistically as detailed in Materials and Methods. ***, P < 0.001; **, P < 0.01; NS, not significant (P > 0.05). C, To measure Ras activity, cells were stimulated with or without LPA (5 µM) or EGF (10 ng/ml) for 5 min, washed, pelleted, and lysed. Cell lysates were used to affinity purify the activated Ras on SwellGel-immobilized glutathione discs adding GST-fusion protein containing Ras-binding domain of c-Raf (GST-Raf1-RBD) as detailed in Materials and Methods. To ensure that the pull-down procedure is working properly, control membranes were treated in vitro for 30 min with either 0.1 mM GTP S or 1 mM GDP. The pulled-down active Ras was detected by Western blot analysis using anti-Ras antibody. D, Membranes were isolated from theca cells treated as above (in the presence or absence of either LPA or EGF). Phospho-specific c-Raf antibody was used to detect the activation of c-Raf using Western blot analysis.
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Functional Inhibition of Rho GTPase Affects the LPA-Stimulated Activation of ERK, but Has No Effect on P(T,Y)-ERK Phosphorylation Induced by EGF
To examine further whether other members of the Ras superfamily, e.g. RhoGTPases, transmitted the activatory signal from the LPA receptor to ERK, we lipofected theca cells with C3-exotoxin and stimulated them with either LPA or EGF. C3-toxin from Clostridium botulinum ADP-ribosylates RhoA and thereby impairs its function (39). Theca cells grown on chamber slides show actin reorganization upon C3-toxin infection, indicating that Rho function was indeed attenuated in our cell system (Fig. 7A
). Treatment with C3-exotoxin had no effect on the EGF-induced activation of the enzyme (Fig. 7B
). Surprisingly, the inhibition of RhoGTPase function resulted in an attenuation of the LPA-stimulated phosphorylation of ERK1/2. Control experiments showed that Lipofectamine alone had no effect on LPA-stimulated activation of ERK (Fig. 7B
, inset). We have carefully analyzed the effects of C3-exotoxin in five independent experiments (Fig. 7C
) and have demonstrated a consistent inhibition of LPA- induced activation of ERK1 and ERK2 by 4080%. Additionally, when the active Rho function was assessed in cell lysates from LPA- and EGF-stimulated theca cells, we could observe that LPA was able to activate endogenous RhoGTPase, whereas EGF was ineffective (Fig. 7D
).

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Fig. 7. Functional Inhibition of the Rho GTPase Effects LPA-Stimulated Activation of ERK, But Has No Effect on the Phosphorylation of ERK Induced by EGF
AC, Cells were infected with C3 exotoxin from botulinum toxin (C3) using Lipofectamine (Lipo)-based infiltration system (described in Materials and Methods) and stimulated with either LPA (5 µM) or EGF (10 ng/ml) for 15 min. A, Immunohistochemical analysis using antiactin antibody to ensure changes in morphological appearance after infection with C3-exotoxin. B, P(T,Y)-ERK1/2 was analyzed as detailed in the legend to Fig. 1 . The inset shows control cells treated with Lipofectamine alone. C, Mean data (±SD) quantitated from five independent experiments analyzed statistically as described in the legend to Fig. 4 . ***, P < 0.001; **, P < 0.01. D, To measure the activity of Rho the cells were stimulated with or without LPA (5 µM) or EGF (10 ng/ml) for 5 min, washed, pelleted, and lysed as detailed in Materials and Methods. To specifically isolate active Rho-GTP, the assay is using GST-fusion protein containing the Rho-binding domain of Rhotekin. The active Rho was detected by Western blot analysis using anti-Rho antibody. To ensure that the pull-down procedure is working properly, control membranes were treated in vitro for 30 min with either 0.1 mM GTP S or 1 mM GDP (as shown).
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Why is LPA not using the Gi-Ras pathway? One possibility is that the theca cells express a different subtype of the LPA receptors (see below); the other is that LPA perhaps couples to the ERK pathway through a different set of effector molecules such as MEK kinase 1 (MEKK1) (40). Theca cell membranes express high amounts of 194- to 196-kDa MEKK1 (Fig. 8
), which is a dual activity kinase able to signal between growth factor receptors and ERK in other cell systems (41). Although it is difficult to show endogenous signaling between MEKK1 and MEK, the appearance of various immunoactive fragments of this MEKK1 may provide some information on presumed function of the kinase. After stimulation with LPA, the full-length MEKK1 protein (
196 kDa) vanished from the cell membrane and an approximately 78-kDa fragment was seen. This MEKK1 fragment has been suggested in the literature as a putative proline-rich pleckstrin homology-containing fragment associated with the regulation of actin stress fibers and focal adhesion (see below). A prominent 50-kDa fragment inducible upon cell stimulation (even under basal conditions) implicates putative ubiquitin ligase (E3 Ub ligase) fragment (42). Putative proapoptotic fragments (
90100 kDa) were not observed (Fig. 8
, first four lanes) even when the incubation was continued for up to 120 min (data not shown). However, when the cells were incubated in the absence of serum for longer time periods (240 min) an approximately 90-kDa fragment was observed (Fig. 8
, last three lanes). Interestingly, the incubation with LPA, and to some extent with EGF, appeared to prevent the formation of this proapoptotic fragment. The importance of the various immunoreactive fragments remains to be elucidated because some MEKK1 functions may not be related to MAPK activity (43).

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Fig. 8. Endogenous Expression of MEKK1 Protein in Theca Cells
Cells were stimulated for various time periods (0, 15, 240 min, as shown) with or without either 5 µM LPA or 10 ng/ml EGF. Particulate fractions were immunoblotted with an antibody recognizing 194200 kDa MEKK1 protein and various immunoreactive MEKK1 fragments (90100, 78, and 50 kDa).
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Theca Cells Express Mainly mRNA for LPA1-Type LPA Receptor, but Neither LPA2-, nor LPA3- Are Endogenously Expressed
Because different LPA receptor isoforms may couple to a different set of G proteins, it was necessary to determine which of the known edg/LPA receptors are present in the theca cell. Edg/LPA receptors are widely expressed in various tissues and cell lines (44). Using RT-PCR, we found that the LPA1/edg2 receptor mRNA is present in all hormonally active ovarian compartments, especially in theca and luteal cells (Fig. 9A
) and to a lesser extent in granulosa cells. Sequence analysis of the PCR products revealed 99.6% identity with the known edg-2 sequence from bovine brain. As a negative control, HL-60 cells and ovaries from patients suffering from ovarian carcinoma (h.Ov.Car) with no or extremely low amounts of endogenous LPA1 receptors were used. As a positive control, primate ovary (from marmoset monkeys, Marm.Ov.) with high amounts of LPA1-type was used. We were unable to detect any signal for either LPA2- or LPA3-type LPA receptor in theca cells, although the positive controls (whole testis and prostate) showed strong signals (Fig. 9A
, right panels).

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Fig. 9. Theca Cells Express Mainly LPA1/edg2-Type Receptor at the mRNA Level (A), as LPA-Receptor Protein (B), and on the Surface of Growing Cells (D)
A, cDNAs from bovine theca (Tk), granulosa (GC), luteal cells (LC), and from various control tissues [primate ovary (Marm. Ov.); HL-60 cells; bovine ovary from the luteal phase (bov.luteal); bovine theca (bov. Tk); human ovarian carcinoma (h.Ov.Car1, h.Ov.Car2); whole rat testis and whole prostate (as shown) were used as a template for PCR amplification of LPA receptors and GAPDH as a housekeeping gene. Primers designed on the basis of known sequences were commercially synthesized. PCR conditions are described in Materials and Methods. The arrows show the position of specific fragments. Last lanes (each) show 100-bp marker, respectively. B, Membrane fractions from two different theca cell preparations (Tk-1, Tk-2) and from HL-60 cells (negative control) were separated on SDS-PAGE, blotted to polyvinylidine difluoride membranes, and incubated with a polyclonal antibody recognizing edg-2 type LPA1 receptor. Chemiluminescence analysis was performed as detailed in Materials and Methods. Lower control bands show ß-actin staining in rehybridized blot. C, Theca cells grown on chamber slides were fixed and immunohistochemical analysis performed using an anti-LPA1/edg2-receptor antibody as detailed in Materials and Methods. D, Growing cells were stained for the antigens present at the cell membrane (live cell staining) using anti-LPA1/edg2-antibody and Cy-3-conjugated secondary antibody at 4 C (to prevent the antigen internalization and to stabilize the membrane) as detailed in Materials and Methods. Further analysis was performed as above.
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Endogenous Expression of LPA1-Type LPA-Receptor Protein in Theca Cell Membranes and on the Cell Surface
Using an antibody recognizing the edg2/LPA1-subtype LPA receptor, we could observe a signal at 39 kDa and a tiny additional band at 48 kDa (Fig. 9B
) in Western blots of theca cell membranes. HL60 cells (which have no endogenous LPA receptor) were used as a negative control and showed no specific immune reaction. Theca cells grown on chamber slides (Fig. 9C
, upper panel) show an immune reaction at the cell membrane, but also in areas associated with extracellular matrix (Fig. 9C
). Staining performed on growing cell cultures (live cell staining, Fig. 9D
) showed that the immunohistochemical signal was more clearly and distinctly associated with the cell membrane.
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DISCUSSION
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We have shown here that LPA stimulates the activating phosphorylation of ERK in ovarian theca cells expressing the LPA1/edg2 receptor. The stimulation of ERK in theca cells is also achieved by EGF, which can be blocked using PTX. The stimulation of ERK initiated by LPA, however, appears to follow a different pattern of activation. Not only is this stimulation independent of PTX pretreatment, but it is not abolished after treatment with the farnesyltransferase inhibitor, manumycin A, which in contrast ablates the EGF signal in the same experiment. Interestingly, silencing the RhoGTPase, in C3-exotoxin-lipofected cells, has no effect on EGF-stimulated ERK, while, at the same time, an obliteration of the LPA response is observed. Whereas LPA-induced ERK phosphorylation gradually declines after 15 min, there is no significant translocation of the active enzyme to the nucleus.
Activation of the ERK-MAPK pathway is known to result from stimulation either of a growth factor tyrosine kinase receptor (e.g. EGF-R) or a GPCR (25, 26, 29). When LPA signals as a growth factor, it is known to stimulate the Gi-Ras-Raf-MEK pathway in several cell lines (19). When we compared the kinetics of ERK activation using either EGF, LPA, or a combination of both, we were able to observe an additive effect, at least with respect to the ERK2 phosphorylation. On the other hand, the effect of LPA is retarded in comparison with the effect of EGF. Using an EGF receptor-specific tyrosine kinase inhibitor, Cunnick et al. (45) were able to inhibit LPA-stimulated MAPK kinase and the phosphorylation of the EGF receptor both in HeLa cells and NIH 3T3 cells. In contrast, we could not observe any significant effect of the EGF receptor-specific tyrphostins B48 or B44, whereas both EGF and LPA induced phosphoactivation of the EGF receptor. Although it is currently considered that LPA signals by transactivation of the EGF receptor (36), there is, on the contrary, also evidence that there may not be any strict correlation between the transactivation of the EGF receptor and the activation of ERK by LPA under physiological conditions (22). The complex process, however, could also involve additional kinases (such as c-src) or a metalloprotease (46, 47).
So far, most data indicate that LPA acts as a classical growth factor utilizing mainly the Ras-Raf-ERK pathway, although in a few cases, the involvement of members of the RhoGTPase family has also been implicated (48, 49). From a mechanistic point of view, the Rho pathway has been considered as separate from the ERK pathway (22). Even so, cross-cascade effects between the Rho family members and the MAPK pathway have been suggested (50), indicating either a ligation of integrin or an interaction involving focal adhesion kinase (29, 51). On the other hand, Andreev et al. (52) showed that neither src nor Pyk2 is required for LPA-stimulated MAPK. Results published recently (53) provide evidence for a direct link between G
12/13 and the activation of p115RhoGEF. Concomitantly, the p115RhoGEFs have also been shown (54) to directly couple to the MEKK1 pathway. MEKK1 (55) could serve as a link between the LPA-induced G
12/13 response, RhoGTPase, and ERK. The hypothesis is shown in Fig. 10
. Is this link possible for theca cells? LPA is activating RhoGTPase in our cell system, MEKK1 (known to stimulate ERK) is abundantly present in theca cell membranes, and endogenous activation of MEKK1 may occur. Because there is also no activation of SAPK/JNK under the given experimental conditions, it is tempting to speculate that LPA may stimulate ERK via a pathway involving MEKK1. Athough this hypothesis is attractive, additional biochemical evidence is still required. First, it would be difficult to differentiate between the effects of other Raf- and MEK-kinases under physiological conditions. Second, MEKK1 is known to synergize with Raf and bind to c-Raf in various cell systems (56, 57). Additionally, recent work has revealed that MEKK1 may also have additional broader functions, which appear to be independent of MAPK activation (43). Also broader-spectrum functions, associated with the ability of MEKK1 to interact with actin fibers and integrin-mediated cell adhesion contacts, should not be forgotten. On the other hand, recently published data (53, 54) do provide evidence for a direct link between G
12/13 and Rho, and between Rho and MEKK1 (54), thus supporting our hypothesis. Future studies should throw more light onto this pathway and determine whether the interaction between the LPA-receptor and Rho has any functional consequences for the theca cell.

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Fig. 10. Model Mechanisms for LPA- and EGF-Induced Activation of MAPK-ERK in the Theca Cell
Oversimplified scheme showing our current working hypothesis.
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Depending on the physiological functions of LPA in a given cell, various effector proteins could transduce diverse signals merging at ERK. Alessi et al. (23) have revealed four different patterns of activation/inactivation of MAPKK and MAPK. One pattern is exhibited by platelet-derived growth factor (PDGF)-stimulated endothelial cell line, PAE, which has a similar ERK-activation status as in LPA-stimulated theca cells, with transient activation (declining within 30 min) of the enzyme and no nuclear relocalization. Another pattern is shown by PDGF-stimulated Swiss 3T3 cells, with sustained activation of ERK and its nuclear translocation. It is suggested that cells showing rapid, transient activation of ERK are likely to be deactivated in the cytosol by a protein tyrosine phosphatase (23). This may also be the case for the theca cell, whereas the deactivation of ERK could be prevented in vanadate-treated cells.
Kranenburg and Moolenaar (22) have carefully reviewed the literature on LPA-induced MAPK signaling and have suggested that the early transient phase of MAPK activation is probably fundamental to housekeeping functions, such as rapid stimulation of cellular metabolism, survival, and early gene transcription. Not only are the normal physiological functions of LPA just beginning to be appreciated, there is still only limited information on ERK itself in the normal cycling ovary (6, 10, 22). In a few cases, elevated MAPK is observed in response to either gonadotropins or mitogenic growth factors in ovarian cells. In porcine granulosa cells, the ERK enzyme is shown to be activated by EGF (65), whereas in porcine theca cells a transient activation of ERK is observed after stimulation with PDGF (66). Although in porcine theca cells the Ras-Raf-MEK-ERK pathway is implicated in PDGF-stimulated theca cell growth and inhibition of LH-stimulated progesterone production (60), the activation of ERK by gonadotropins in rat granulosa cells was associated with increased steroidogenesis (59). We have so far been unable to observe any correlation between LPA activation and steroid production, but rather an impact on cell survival (data not shown).
Fundamental data on LPA receptor genes, and the molecular pharmacology of LPA receptors, have been based mainly on nonovarian cells and tissues (15, 18, 20). Chun and colleagues (18, 20, 44) have estimated the size of mouse and human LPA1-3 receptors to be between 38.9 and 41.2 kDa. In our earlier studies we have observed specific binding sites for LPA using [3H]-LPA-cross-linking techniques and were able to demonstrate that LPA binds specifically to a 38- to 40-kDa protein in luteal cell membranes (11). In the present study, we show that the LPA1/edg-2 receptor antibody recognizes an approximately 39-kDa protein apparently localized to the plasma membrane in growing theca cell cultures. Using PCR-based analysis we can attribute this binding to the presence of an LPA1/edg2 type of receptor.
In summary, the MAPK ERK is one of the key enzymes associated with various physiological functions (22). Using cultured primary theca cells, we have been able to show that both LPA and EGF stimulate an activating phosphorylation of the enzyme. Theca cells express a classical LPA1/edg2-type LPA receptor, which appears to couple preferentially to a different set of effector proteins from the classical Gi-Ras-ERK-pathway taken by EGF in the same cell system. Until now, it was presumed that the route deviations adopted by LPA, in some cell systems, are determined by the different set of G proteins available in any given cell type. This is clearly not the case in the theca cell. The activation of ERK by LPA ignores the available Gi-Ras pathway used by EGF and, instead, prefers to mediate stimulation by the Rho-dependent pathway. Whether this nonclassical pathway is restricted to ovarian theca cells remains to be seen.
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MATERIALS AND METHODS
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The sources of various chemicals were as follows: 1-oleoyl-glycero-3-phosphate (18:1 LPA), pertussis toxin (islet activating protein from Bordella pertussis), protease inhibitors, and 4'6'-diamidino-2-phenylindole were from Sigma-Aldrich (Deisenhofen, Germany). Clostridium botulinum C3-exotoxin was from Biomol (Hamburg, Germany). Manumycin A (Streptomyces parvulus), phorbol ester (4ß-phorbol-12-myristate-13-acetate), PD 98059 (2'-amino-3'methoxyflavone), okadaic acid, sodium-ortho-vanadate, and tyrphostins were provided by Calbiochem (Bad Soden, Germany). EGF was from Roche (Mannheim, Germany) and 8-Br-cAMP was from BIOLOG (Bremen, Germany). Nonphosphorylated and fully phosphorylated ERK2 (control proteins) were from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-based Western-detection system was from Pierce (Oud Beijerland, The Netherlands). Bovine dermal CSB Collagen was from Cellon (Strassen, Luxemburg). Bovine LH was a gift from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Bethesda, MD). Centricon filters were from Amicon-Millipore (Eschborn, Germany). RNA purification kit was from Peqlab (Erlangen, Germany), and SuperScript II reverse transcriptase, 100-bp size ladder standard, low-DNA-mass ladder standard, and Lipofectamine reagents were from Invitrogen (Karlsruhe, Germany; and GIBCOBRL, Eggenstein, Germany). dNTP and oligo(dT)1218 were from Amersham Pharmacia Biotech (Freiburg, Germany); BioTherm polymerase was from GeneCraft (Münster, Germany); and QiaQuick gel extraction kit was from QIAGEN (Hilden, Germany). Abi Prism dye terminator sequencing-ready reaction kit was provided by Perkin Elmer Corp. (Branchburg, NJ), and oligonucleotides were from MWG Biotech (Ebersberg, Germany). Pull-down Ras- and Rho-activity assays were from Pierce (PERBIO, Bonn, Germany). All other reagents were obtained from commercial sources and were of the highest purity grade.
Antibodies
The antibody against fully phosphorylated P(T,Y)-ERK1/2 was from New England BioLabs (Cell Signaling Technology). This monoclonal antibody detects T202/Y204 of human ERK, T197/Y199 of rat ERK, and T185/Y187 of bovine ERK1/2. Polyclonal antiphosphotyrosine, P(Y)-ERK1/2 antibody, the antibody against nonphosphorylated CT-ERK1/2, and the polyclonal LPA1/edg2 antibody (recognizing the p45 edg2 receptor in rat- and p3840 LPA receptor in bovine tissues) were from Upstate, Biomol. Antibodies directed against active c-Raf, activated phospho-SAPK/JNK were from Cell Signaling Technology (New England BioLabs, Frankfurt, Germany) and against activated EGF-receptor from Calbiochem. The antibody against MEK1-kinase was from Santa Cruz Biotechnology (Heidelberg, Germany) and those against
-, and ß-actin were from Sigma. All affinity-purified peroxidase-conjugated secondary antibodies used for Western analysis as well as the fluorochrome-Cy3-conjugated secondary antibodies used in immunohistochemistry were from Jackson Immunochemicals (Dianova, Hamburg, Germany).
Methods
Theca Cell Isolation and Culture Procedures.
Detailed methods for isolation and purification of bovine theca cells have been published elsewhere (13, 61). Briefly, the theca interna layer was peeled away from the theca externa and surrounding stromal tissue of large healthy antral follicles before digestion in Mosconas balanced salt solution containing 0.5% collagenase, 0.1% hyaluronidase, 0.1% pronase, 0.1% BSA, and 0.1 mg/ml DNase. The crude cell suspension was layered over a stable Percoll density gradient. Purified cells were resuspended in medium 1 (DMEM/HAMs F-12 medium, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin), plated on 0.3% Cellon collagen, cultured in medium 1 containing 1.5% heat-inactivated fetal calf serum for 48 h, and maintained further in serum-free medium 1 plus 1 µg/ml BSA and 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite (ITS medium) until d 6.
Cell Treatments.
Cell monolayers were synchronized by incubation with medium 1 containing 5 µg/ml fatty acid-free BSA for 90120 min and overlayed with fresh medium 1 containing either of the following agonists: 25 µM LPA (1-oleoyl-sn-glycero-3-phosphate), 10 nM phorbol ester, PMA (4ß phorbol-12-myristate-13-acetate), 10 ng/ml epidermal growth factor (EGF), 1 mM 8-Br-cAMP, or 100 ng/ml bovine LH (as indicated). Cells were stimulated for 15 min or for varying time periods as indicated. The various pharmacological inhibitors were added directly to the cell culture medium during the preincubation period. Because LPA might be sensitive to air and light, stock solutions were made up in methanol and, shortly before stimulation, aliquots were diluted in albumin containing serum-free medium.
C3-exotoxin (C3-toxin from Clostridium botulinum) was introduced into the cells using the Lipofectamine-based infiltration system, as described by others (30). Briefly, cells were grown until d 6. To generate liposomes, a complex of Lipofectamine (176 µg/ml) and C3-exotoxin (100 µg/ml) was formed by gently mixing the components and incubating them at room temperature for 20 min, according to the procedure described by the manufacturer. Control cells were incubated with the same concentrations of Lipofectamine without C3-exotoxin. While the complex was forming, the cells were rinsed with transfection medium (medium 1 without albumin and antibacterial agents). The liposome complex was diluted in transfection medium and was used to overlay the rinsed, adherent cells. Cells were incubated with the complex for 6 h, medium was replaced with fresh ITS- medium also containing C3-exotoxin (100 ng/ml), and the cells were allowed to recover overnight (1618 h). On the following day, cells were stimulated as usual.
Culture of NIH3T3 Cells.
Cells were cultured in DMEM containing 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin containing 10% heat-inactivated fetal calf serum until the cells reached 80100% confluence and were maintained in serum-free medium 1 containing 5 µg/ml BSA for 90 min before stimulation with LPA.
Culture of HL 60 Cells.
Cells were cultured in DMEM containing 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin containing 10% heat-inactivated fetal calf serum until the cells reached 80100% confluence.
Subcellular fractionation is described in detail elsewhere (13). Cells were harvested by gently scraping with a rubber policeman into ice-cold isotonic sucrose buffer A containing 10 mM Tris-HCl (pH 7.4) and 0.25 M sucrose plus 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml amastatin, and 1 mM phenylmethylsulfonic acid (PI) before gentle homogenization. The resulting homogenate was centrifuged first at 100 x g for 15 min to yield pellet 1 and supernatant 1. Pellet 1 was resuspended in lysis buffer (B) containing 0.5% Nonidet P-40, 10 mM Tris-HCl (pH 7.4) plus protease inhibitors, PI. From this suspension, the nuclei were pelleted at 200 x g (pellet 2). Supernatant 1 was recentrifuged at 100,000 x g for 60 min to yield a particulate (nonnuclear) membrane fraction and the supernatant (cytosolic fraction). Subfractions were filtered through Centricon Ultrafilters with 10 k-cutoff, and protein content was determined according to Bradford (62).
Immunoblot Analysis of P-ERK1/2.
Equal amounts of proteins were separated by SDS-PAGE (80 µg), transferred to polyvinylidine difluoride membranes, and stained with Ponceau S. Blots were blocked with I-Block (Serva, Heidelberg, Germany) and incubated overnight at 4 C with affinity-purified monoclonal antibodies [P(T,Y)-ERK1/2] described above, recognizing the fully phosphorylated (T/Y) form of p44/42 ERK1/2 (1:1000). After incubation with the peroxidase- conjugated and affinity-purified rabbit antimouse secondary antibody (1:3000), immune complexes were detected using a luminol-based chemiluminescence system. Additionally, control blots were run with nonimmune serum and the secondary antibody. Immunoanalysis with control antibodies [i.e. P(Y)-ERK1/2, nonphosphorylated total ERK1/2] was performed in a similar way as described above with the appropriate species-specific (affinity-purified) secondary antibodies (all from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
Endogenous Expression of LPA1/edg2 Receptors.
To investigate the expression of edg 2-type receptors, particulate fractions from theca cells (or other control cells) were subjected to Western blot analysis using antibodies recognizing the LPA1 receptor. As control, blots were rehybridized with antibodies directed against ß-actin. Immunoblotting and chemiluminescence techniques were performed as described above.
RT-PCR Analysis of LPA-Receptor Subtypes.
Total RNA was isolated from ovarian cells by the modified method of Chomczynski and Sacchi (63) using the PeqGold RNA purification kit. The resulting RNA was dissolved in TE [10 mM Tris/HCl (pH 7.4), 1 mM EDTA] and the concentration determined by measuring the spectrophotometric absorbance at 260 nm. To estimate RNA integrity, an aliquot was electrophoretically separated on 1.5% agarose gel and visualized by staining with ethidium bromide. Following the manufacturers instructions, first-strand cDNA was synthesized from 3 µg total cDNA using SuperScript II reverse transcriptase, 0.5 mM of each deoxyribonucleoside triphosphate, and 500 ng oligo(dT)12-18 as primer. After completion of the reaction, 80 µl of water were added to yield a final volume of 100 µl. Three microliters of cDNA from theca, granulose, and luteal cells and controls were used as template for PCR amplification of transcripts for LPA receptors, and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a marker housekeeping gene in ovarian cells (64, 65). In a total volume of 50 µl, the PCR mixture contained 0.5 U BioTherm polymerase, 5 µl 10x BioTherm buffer, 1.5% Ficoll 400, 100 pmol of each primer, and 0.2 mM dNTP. For GAPDH, only 1 µl total RNA and 50 pmol of each primer were used in the PCR reaction. Primers were designed on the basis of known sequences obtained from the NCBI data bank and were commercially synthesized. The following primers (66) were employed for the PCR: GAPDH: sense, 5'-GTCTTCACCACCATGGAG-3'; antisense, 5'-GTCATGGATGACCTTGGC-3' (yielding a fragment with an expected size of 198 bp); edg-2(LPA1): sense, 5'-ATCTTTGGCTATGTTCGCCA-3'; antisense, 5'-TTGCTGTGAACTCCAGCCA-3' (yielding a fragment with an expected size of 394 bp); edg-4(LPA2): sense, 5'-TGGCCTACCTCTTCCTCATGTTCCA-3'; antisense, 5'-GGGTCCAGCACACCACAAAT GCC-3' (yielding a fragment with an expected size of 516 bp); edg-7 (LPA3): sense, 5'-AGTGTCACTATGACAAGC-3'; antisense, 5'-GAGATGTTGCAGAGGC-3' (yielding a fragment with an expected size of 513 bp). The primers chosen recognize edg-type receptors from human, marmoset, bovine, and rat. PCR conditions were as follows: after a 2-min denaturation step at 95 C, samples were subjected to 30 cycles at 95 C for 1 min, 48 C [edg-7 (LPA3)], 59 C [edg-4(LPA2)], or 53 C for 1 min [edg-2 (LPA1) and GAPDH, respectively] and 72 C for 1 min, followed by an additional elongation step at 72 C for 10 min. As negative control, cDNA was replaced by water in the reaction mixture. The PCR products were separated on a 1.5% agarose gel and the fragment lengths were determined by comparison with a 100-bp size ladder standard. In some experiments the number of PCR cycles was changed to analyze their influence on the intensity of the amplification products. To verify the identity of GAPDH and edg-2 (LPA1), bands were cut from the gel and isolated with a QiaQuick gel extraction kit. The purified DNA fragments were dissolved in TE and quantified spectrophotometrically and by comparison to a low-DNA-mass ladder standard. A 100-ng aliquot of the respective PCR products was amplified using ABI Prism Dye Terminator Sequencing Ready Reaction Kit according to the manufacturers manual and sequenced on an ABI Prism 377 sequencer. The sequences of the PCR fragments showed 99.6% identity to the published bovine edg2- sequences (67).
Immunohistochemistry Using Immunofluorescence and Confocal Microscopy.
Cells were grown either on collagen-coated eight-chamber glass slides (LAB-TEK, Nunc, Rochester, NY) or on extra-thin glass chambers (for confocal analysis). After stimulation, cells were washed with PBS and fixed with 3% paraformaldehyde before the nonspecific binding sites were blocked by treatment with 5% nonimmune serum (species of the second antibody). Cells were permeabilized with 0.5% Triton X-100 for 4 min, incubated with the primary antibody (anti-P-ERK, antiactin, or anti-edg2, as indicated), and stained with Cy3-conjugated secondary antibody. The conjugate was washed several times and chamber slides were kept in the dark at 4 C before viewing with a Nikon fluorescence microscope (Nikon, Düsseldorf, Germany) or Leica laser scanning confocal microscope (Leica, Inc., Bensheim, Germany). To counterstain the cell nuclei, DAPI was applied after the secondary antibody. Control analysis was performed for each single slide using different Nikon filter blocks with the excitation wavelength 330380 nm for DAPI staining and 450490 nm for Cy3-immunofluorescence (using FITC filter). Micrographs were taken with a Leica digital camera.
Staining on Growing Cells for LPA1/edg2-Type LPA Receptor (Live-Cell Staining for Surface Antigens Using Immunofluorescence Analysis).
Cells, grown and stimulated on chamber slides, were washed twice with PBS, precooled on ice (to stabilize cell membranes and to prevent antigen internalization) for 20 min in medium with 10% serum (species of the second antibody), and washed again, after which nonspecific binding was blocked by incubation with 10% nonimmune serum for 45 min at 4 C. Chamber slides were incubated further for 60 min with primary antibody, washed again, stained for 60 min with Cy3-conjugated secondary antibody, washed, and then fixed with 3% paraformaldehyde. Further analysis with fixed cells was performed as described above.
Ras and Rho Activity Assays (Pull-Down Assays).
Theca cells were synchronized for 90 min as usual, stimulated with either LPA (5 µM) or EGF (10 ng/ml) for 5 min, washed with cold Tris-buffered saline buffer, pelleted, and lysed [lysis buffer: 25 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM dithiothreitol, 5% glycerol]. Cell lysates were used to affinity purify the activated Rho on SwellGel-immobilized gluthathione discs. To specifically isolate active Rho-GTP, the assay uses a GST-fusion protein containing the Rho-binding domain of Rhotekin. To ensure that the pull-down procedure was working properly, control membranes were treated in vitro for 30 min with either 0.1 mM GTP
S or 1 mM GDP (and were treated further as above). The pulled-down active Rho was detected by Western blot analysis using an anti-Rho antibody provided with the assay.
The Ras activation assay is based on the same pull-down principle described for Rho, using the ability of Ras to bind the Ras-binding domain of c-Raf. Briefly, the cells were stimulated and lysed as above. After incubation with a GST-fusion protein containing the Ras-binding domain of c-Raf, affinity-purified activated Ras was detected by Western blot using an anti-Ras antibody (provided with the assay). Control membranes were treated in vitro as above, either with or without either GTP
S or GDP.
Data Analyses.
Each experiment was performed three to eight times using different cell cultures. All blots and films were scanned, and specific bands were analyzed densitometrically using the NIH Scion Image Program (Scion Corp., Frederick, MD). Immunoblots were quantitated with the computer-assisted analysis program using threshold and density-slicing modes to provide area and density measurements of the gray-scale-immunoreactive images. Integrated optical density bands were analyzed statistically as described below. The data are presented as mean values (±SD) from separate experiments (n = 38). Each representative data set show results from the same individual representative experiment (repeated at least three times with similar results). Changes in the intracellular protein relocalization (if any) were monitored using digitalized epifluorescence or confocal scanning microscopy and were calculated as a percentage of cells showing redistribution of immunofluorescence stain vs. the total number of cells (DAPI staining) in 500 µm2 areas. Statistical analysis of the data presented was performed using a program from GraphPad Software, Inc. (San Diego, CA). First tests were performed using standard t test, and further analysis was carried out using repeated measures one-way ANOVA and Dunnetts posttest. P values greater than 0.05 were considered as not significant (NS), values 0.010.05 as significant, 0.0010.01 as very significant, and less than 0.001 as extremely significant.
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ACKNOWLEDGMENTS
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We acknowledge the contribution of the following scientists in collecting tissue samples and/or providing various cDNA samples from reproductive tissues used in the LPA-receptor studies: Dr. N. Schlabritz-Loutsevitch (Department of Obstetrics and Gynecology, New York University Medical School, Southwest Foundation for Biomedical Research, San Antonio, TX); Dr. Andrej Obuchovich (Department of Obstetics and Gynecolgy, Hospital N8, Minsk, Belarus); Dr. R. Zarreh and Dr. D. Müller (Institute for Hormone and Fertility Research at the University of Hamburg, Hamburg, Germany); and Dr. A. Einspanier (German Primate Center, Göttingen, Germany). We also thank Professor F. A. Leidenberger for excellent facilities and encouragement throughout the work; Professor R. Ivell and Dr. K. Willey for critical reading of the manuscript; Dr. C. Osterhoff for her help with the confocal facilities; Ms. U. Steuber for technical assistance; and NIDDK for supplying us with the bovine LH standard preparations.
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FOOTNOTES
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Abbreviations: DAPI, 4'6'-Diamidino-2-phenylindole; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPC, G protein coupled; GST, glutathione-S-transferase; JNK, c-Jun N-terminal kinase; LPA, lysophosphatidic acid; PDGF, platelet-derived growth factor; PMA, 4ß-phorbol-12-myristate-13-acetate; MEK, MAPK kinase; MEKK1, MEK kinase 1; PTX, pertussis toxin; SAPK, stress-activated protein kinase.
Received for publication November 8, 2002.
Accepted for publication April 24, 2003.
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