Protein Tyrosine Phosphatase PTP20 Induces Actin Cytoskeleton Reorganization by Dephosphorylating p190 RhoGAP in Rat Ovarian Granulosa Cells Stimulated with Follicle-Stimulating Hormone

Masayuki Shiota, Tatsuya Tanihiro, Yoshimi Nakagawa, Naohito Aoki, Norio Ishida, Koyomi Miyazaki, Axel Ullrich and Hitoshi Miyazaki

Gene Research Center, University of Tsukuba (M.S., T.T., Y.N., H.M.), Ibaraki 305-8572, Japan; Department of Applied Molecular Biosciences (N.A.), Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; Clock Cell Biology Group (N.I., K.M.), Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8566, Japan; Max-Planck-Institut fur Biochemie (A.U.), D-82152 Martinsried, Germany

Address all correspondence and requests for reprints to: Hitoshi Miyazaki, Ph.D., Gene Research Center, University of Tsukuba, Ibaraki 305-8572, Japan. E-mail: hitomy1{at}sakura.cc.tsukuba.ac.jp.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We identified 25 protein tyrosine phosphatases (PTPs) expressed in rat ovarian granulosa cells. Of these PTPs, the expression levels of at least PTP20, PTP-MEG1, PTP{epsilon}M, and PTP{epsilon}C significantly changed during the estrous cycle. We examined the cellular functions of PTP20 in granulosa cells by expressing the wild type, a catalytically inactive CS mutant in which Cys229 of PTP20 was changed to Ser, or a substrate-trapping DA mutant in which Asp197 was mutated to Ala, using an adenovirus vector. Overexpression of the wild type, but not of the CS mutant, induced retraction of the cell body with the extension of long, dendritic-like processes after stimulation with FSH, a critical factor for the survival and differentiation of these cells. In addition, cell adhesion to the substratum decreased in an FSH-dependent manner. Inhibiting Rho GTPase activity with C3 botulinum toxin caused similar morphological changes. The FSH-enhanced phosphotyrosine (p-Tyr) level of p190 RhoGAP was selectively reduced by the overexpressed wild type, but not by mutated PTP20. Although p190 RhoGAP is tyrosine phosphorylated by c-Src via the tyrosine kinase Pyk2, wild-type PTP20 had little effect on p-Tyr418 of c-Src and no effect on p-Tyr402 of Pyk2, which are required for full c-Src activity and for interacting between Pyk2 and c-Src, respectively. The CS and DA mutants as well as the wild type reduced the formation of p190 RhoGAP-p120 RasGAP complexes. Confocal microscopy analysis revealed that PTP20 intracellularly colocalizes with p190 RhoGAP. These results demonstrate that PTP20 regulates the functions of granulosa cells in an FSH-dependent manner by dephosphorylating p190 RhoGAP and subsequently inducing reorganization of the actin cytoskeleton. Moreover, our data suggest that PTPs play significant roles in controlling the dynamics of ovarian functions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MORE THAN 99% OF all ovarian follicles in mammals undergo a degenerative process called atresia, which is characterized by granulosa cell apoptosis (1). In contrast, only a few follicles mature and reach ovulation under cyclic gonadotropin stimulation. The survival, apoptosis, differentiation, and proliferation of granulosa cells are regulated by a number of endocrine and paracrine factors that are critically involved in determining the fate of follicles. However, the molecular mechanisms underlying the intracellular events induced by these regulatory factors are poorly understood. As in other cells, protein tyrosine kinases (PTKs) play important roles in regulating the intracellular events of granulosa cells after stimulation with various factors (2, 3, 4, 5). Indeed, growth factors including epidermal growth factor and basic fibroblast growth factor, the receptors that possess PTK domains within their molecular structures, inhibit the spontaneous onset of apoptosis in cultured granulosa cells (2, 3). IL-1ß acts through cytoplasmic PTKs called Janus kinases (JAKs), and this cytokine is an effective survival factor for preovulatory follicles in vitro (4). These findings suggest that the protein tyrosine phosphorylation of intracellular signaling molecules by PTKs is significant for regulating the dynamics of follicular atresia, growth, and maturation. Because phosphotyrosine (p-Tyr) levels are regulated by the coordinated activities of PTKs and protein tyrosine phosphatases (PTPs), the roles of PTPs must also be critical for controlling follicular functions. Nevertheless, the roles of PTPs in the ovary have not been described.

The large superfamily of PTPs comprises cytoplasmic and receptor-like forms (6, 7), each of which contains at least one catalytic domain of approximately 250 amino acids characterized by the signature motif [I/V]HCxAGxxR[S/T]. A Cys residue and an Asp residue in the catalytic domain are essential for the activity of PTP. In general, while the functions of many PTKs have been investigated in detail, far less is understood about the physiological roles of PTPs. The present study identified multiple PTPs expressed in granulosa cells using a cloning approach based on the PCR. We found, using a model system of immature female rats primed with gonadotropins, that the expression levels of some of these PTPs, including PTP20 (8) [also called PTP-HSCF (9), FLP1 (10), PTP-K1 (11), or BDP-1 (12)], significantly changed during the estrous cycle. Here we focused on PTP20 and examined the cellular functions of this phosphatase in granulosa cells.

PTP20 is a cytoplasmic enzyme that constitutes a separate subfamily of PTPs with PTP-Pro-, Glu-, Ser-, and Thr-rich [PEST (13), also named p19 (14) or PTP G1 (15)], and PEST domain phosphatase [PEP (16)]. The subfamily is characterized by one catalytic domain at the N terminus, a large middle region that is rich in Pro, Glu, Ser, and Thr residues, and a Pro-rich C-terminal region consisting of 24 amino acids. Proline, Ser, Thr phosphatase interacting protein 1 (PSTPIP1), and the cytoplasmic PTKs, c-Abl and the Src-related kinases, are known substrates for PTP20 (17, 18, 19). PSTPIP1 also interacts with the C-terminal part of the cytosolic PTK c-Abl, serves as a substrate for c-Abl, and can bridge interactions between c-Abl and PTP20 with the dephosphorylation of c-Abl by PTP20 (18). PTP20 associates with the PTK Csk, a potent negative regulator of the Src-family kinases, via the Csk Src homology 2 domain and two putative sites of tyrosine phosphorylation of the phosphatase (19). This association is thought to allow Csk and PTP20 to synergistically inhibit Src-family kinase activity by phosphorylating and dephosphorylating the negative and positive regulatory tyrosine residues, respectively. Unlike the interaction of PTP20 with these proteins, the physiological roles of PTP20 remain unclear. However, these molecules associated with PTP20 have been implicated in the actin cytoskeleton reorganization (17, 20, 21), suggesting that PTP20 may be involved in similar cellular functions.

The aims of the present study are to 1) identify PTPs in rat ovarian granulosa cells, 2) find PTPs of which expression levels change during the estrous cycle, and 3) analyze the cellular function of PTP20 in granulosa cells, focusing on a protein in which the p-Tyr content is decreased by PTP20. Specifically, we examined the roles of PTP20 in the presence of FSH because this gonadotropin is essential for granulosa cell survival and differentiation, which are closely related to the determination of follicular fate.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of PTPs Expressed in Rat Ovarian Granulosa Cells
We examined PTPs expressed in rat ovarian granulosa cells using PCR amplification with degenerate primers derived from conserved sequences in the tyrosine phosphatase domains. Template cDNAs were obtained from the granulosa cells of ovaries containing mostly healthy follicles, and from those of ovaries with mostly atretic follicles. Analysis of approximately 400 PCR-amplified cDNA fragments showed 24 different sequences containing the hallmark residues found in all PTPs (Table 1Go). A database comparison indicated that 15 of the isolated PTP fragments were identical to PTPs that have already been cloned in the rat. Another nine sequences exhibited high homology to those of human or mouse PTPs. Therefore, the nine clones are likely to be the rat homologs of these known PTPs. Of 24 different clones, PTP{epsilon} has both cytoplasmic (PTP{epsilon}C) and receptor-type forms (PTP{epsilon}M) that are transcribed from a single gene by the alternative usage of the promoters. Thus we amplified the template cDNAs by PCR using oligonucleotide primers specific for PTP{epsilon}C and PTP{epsilon}M, respectively, and identified the existence of these two forms. We therefore identified 25 PTP clones in total, of which 13 and 12 clones encoded receptor-type and cytosolic-type PTPs, respectively.


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Table 1. PTPs Expressed in Rat Granulosa Cells

 
Changes in PTP Expression Levels during the Estrous Cycle
We examined, using a model system of immature females primed with gonadotropins (see Materials and Methods), the expression levels of the following PTPs among the identified 25 PTPs during the estrous cycle: PTP{epsilon}M, PTP{epsilon}C, PTPS, Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2), PTP-megakaryocyte (MEG)1, PTP20, density-enhanced PTP (DEP)1, and Fas-associated phosphatase (FAP)1. The expression of PTPs was quantified by RT-PCR with a trace amount of [{alpha}-32P]dCTP. The expression levels of PTP{epsilon}M, PTP{epsilon}C, PTP-MEG1, and PTP20, but not the remaining four PTPs, considerably differed depending on each stage (Fig. 1Go). For example, the content of PTP20 mRNA in granulosa cells from healthy follicles (stage 1) was 4-fold higher than that in granulosa cells from atretic follicles (stage 2) and from corpus luteal cells (stage 3). Expression levels of both PTP{epsilon}M and PTP{epsilon}C in luteal cells (stages 3 and 4) were less than 15% of those in granulosa cells from healthy follicles (stage 1). The mRNA content of PTP{epsilon}M slightly increased as atresia progressed, whereas that of PTP{epsilon}C became remarkably reduced (stage 2). The presence of multiple types of PTPs in granulosa cells and the fact that their expression levels change during the estrous cycle suggest that these enzymes play important roles in regulating follicular growth by dephosphorylating their specific substrates. We select PTP20 and examine its cellular function in granulosa cells in subsequent experiments because its expression changes remarkably during the estrous cycle and its biological function is largely unknown.



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Figure 1. PTP mRNA Levels in Granulosa Cells at Each Stage of the Estrous Cycle

Total RNA was prepared from fresh ovarian granulosa cells at each stage of the estrous cycle. The mRNA contents of the indicated PTPs examined by quantitative RT-PCR as described in Materials and Methods were normalized to the amount of ß-actin mRNA. The amount of ß-actin mRNA was simultaneously determined in the same manner as PTP mRNA, to correct for variations in RNA loading and RT-PCR efficiency. The mRNA content is expressed as a percentage of stage 1 (PMSG 2 d). Data for all PTPs except for PTP20 are averages of two independent experiments, each performed in duplicate. Data for PTP20 are expressed as means ± SE of three independent experiments, each determined in triplicate. S1–S4, Stages 1–4.

 
Preparation of Polyclonal Antibodies Against N-Terminal 20 Amino Acids of PTP20
We raised polyclonal antibodies that recognize PTP20 by immunizing rabbits with the N-terminal 20 amino acids. The antibody recognized a 50-kDa protein in lysates prepared from COS-7 cells transfected with recombinant PTP20, but not with the vector pCDNA3, and in lysates from granulosa cells from healthy (stage 1) or atretic (stage 2) follicles (Fig. 2Go). The molecular mass of 50 kDa agrees well with that of PTP20. The relative levels of this band between stages 1 and 2 are consistent with those of PTP20 mRNA (compare Fig. 1Go to Fig. 2Go). These results show that the polyclonal antibodies specifically recognize PTP20.



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Figure 2. Preparation of Polyclonal Antibodies against N-Terminal 20 Amino Acids of PTP20

COS-7 cells were transfected with either the expression vector pcDNA3 possessing PTP20 or vector alone and cultured for 24 h. Granulosa cells were prepared from the ovaries of immature female rats treated with 20 IU PMSG for 2 d (stage 1) and 5 d (stage 2). Lysates of these cells were resolved (30 µg of protein) by 10% SDS-PAGE and immunoblotted with rabbit antiserum (1:2000) against the N-terminal 20 amino acids of PTP20 prepared as described in Materials and Methods. The same blot was stripped and reprobed with an anti-{alpha}-tubulin antibody to demonstrate that comparable amounts of protein were loaded in each lane. Data are representative of two separate experiments yielding similar results. IB, Immunoblotting.

 
Infection of Cultured Granulosa Cells with Adenoviruses Containing ß-Galactosidase
To define the cellular function of PTP20, wild-type and mutated PTP20 cDNAs had to be efficiently introduced into granulosa cells. We therefore constructed recombinant adenoviruses harboring these cDNAs because the transfection efficiency for granulosa cells is extremely low. We created a catalytically inactive mutant of PTP20 in which Cys229 was changed to Ser (PTP20CS) and an HA-tagged PTP20CS (HA-PTP20CS). We also created an HA-tagged substrate-trapping mutant in which Asp197 was mutated to Ala (HA-PTP20DA). The cDNAs for these mutants and the wild type (PTP20WT and HA-PTP20WT) were inserted into adenovirus vectors, and the resultant recombinant viruses (Ad-PTP20WT, Ad-HA-PTP20WT, Ad-PTP20CS, Ad-HA-PTP20CS, and Ad-HA-PTP20DA) were introduced into granulosa cells. We first determined the multiplicity of infection (moi) appropriate for granulosa cells using an adenovirus expressing ß-galactosidase (Ad-ß-gal). Cells were infected with increasing concentrations of Ad-ß-gal (moi = 5, 10, and 20 plaque-forming units/cell), and then the expression of ß-galactosidase was assessed by staining cells with X-gal (Fig. 3Go). More than 80 and 95% of granulosa cells were transduced when infected at moi of 5 and 10 or 20, respectively. Therefore, we subsequently infected these cells at moi = 15.



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Figure 3. Infection Efficiency of Adenovirus Vector in Granulosa Cells

Granulosa cells were infected with adenovirus expressing ß-galactosidase (Ad-ß-gal) at the indicated multiplicity of infection. After a 48-h incubation, the ß-galactosidase activity was detected using X-gal substrate as described in Materials and Methods. Data are representative of two separate experiments yielding similar results.

 
PTP20 Induces Morphological Change and Detachment of Granulosa Cells
We first examined the effect of PTP20 on cell morphology. Granulosa cells were infected with one of Ad-ß-gal (control), Ad-PTP20WT, or Ad-PTP20CS. After serum starvation, the cells were further incubated with or without FSH for 48 h and cell morphology was observed (Fig. 4AGo). Cells overexpressing PTP20WT (panel b) or PTP20CS (panel c) exhibited the same cell morphology as control cells (panel a) in the absence of FSH stimulation. That is, most cells tightly adhered to the dishes while some cells were rounded. In contrast, PTP20WT in the presence of FSH remarkably induced rounded cells accompanied with the extension of long, dendritic-like processes (panel e), similar to the effect of disrupting small Rho GTPase in fibroblasts (22, 23). Neither PTP20CS (panel f) nor ß-galactosidase (control) (panel d) had such effects even in the presence of FSH. Comparable levels of PTP20WT and PTP20CS protein expression were confirmed by immunoblotting cell lysates with an anti-PTP20 antibody (Fig. 4BGo). Since many PTP20WT-overexpressing cells detached from the substratum, we investigated cell viability by trypan blue staining after observing the morphology (Fig. 4CGo). The viability among control, PTP20WT-, and PTP20CS-expressing cells did not significantly differ in the absence of FSH. In contrast, PTP20WT overexpression reduced viability significantly compared with that of control cells and those overexpressing PTP20CS in the presence of FSH.



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Figure 4. Effect of Overexpressed Recombinant PTP20 on Cell Morphology

A, Granulosa cells were infected with either Ad-ß-gal (control) (a and d), Ad-PTP20WT (b and e), or Ad-PTP20CS (c and f) and serum starved for 24 h. The cells were further incubated with (d–f) or without (a–c) FSH (50 ng/ml) for 48 h, and then cell morphology was observed by microscopy. Original magnification is x100. Data represent three separate experiments. Results were similar in two other experiments. B, Lysates (30 µg of protein) from cells that were manipulated as in panel A were sequentially immunoblotted with anti-PTP20 (upper panels) and anti-{alpha}-tubulin (lower panels) antibodies to confirm that comparable amounts of recombinant PTP20 were expressed and equivalent amounts of protein were loaded in each lane, respectively. C, After observations shown in panel A, cell viability was assessed by trypan blue staining as described in Materials and Methods. Cell viability is expressed as a percentage of control value obtained from cells expressing ß-galactosidase. Data are means ± SE of three independent experiments. D, Actin fibers of cells manipulated as in panel A were stained with phalloidin and observed by fluorescence microscopy. Original magnification is x200. The same experiments were performed twice. Similar results were obtained between the two experiments. E, Total DNA was extracted from cells undergoing the same manipulations as in panel A, and DNA fragmentation was analyzed as described in Materials and Methods. Representative data are shown from three independent experiments. Similar results were obtained in all experiments. F, Apoptotic death of cells manipulated as in panel A was evaluated after staining with Hoechst 33342 followed by fluorescence microscopy. Original magnification is x100. Data are representative of two independent experiments that yielded similar results. IB, Immunoblotting; WT, wild type.

 
To detect the effect of PTP20 on the actin cytoskeleton, cells cultured as shown in Fig. 4AGo were fixed and stained with phalloidin (Fig. 4DGo). Actin stress fibers were generated in controls and in cells expressing PTP20CS regardless of the presence or absence of FSH (panels a, c, d, f). In contrast, stress fibers appeared in cells expressing PTP20WT without (panel b) but not with (panel e) FSH, suggesting that FSH induces the dissolution of actin stress fibers in cells overexpressing PTP20WT. These results are in good agreement with the data shown in Fig. 4Go, A–C.

To examine whether the FSH-dependent decrease in the viability of cells overexpressing PTP20WT was due to apoptosis, cellular DNA was resolved by electrophoresis to detect fragmentation, a biochemical characteristic of apoptosis. Figure 4EGo shows that although DNA was fragmented in every lane, FSH obviously augmented fragmentation in cells expressing PTP20WT. We considered that some DNA was fragmented due to extraction from the rounded and detached cells observed under all experimental conditions. Apoptosis was confirmed further by chromatin staining with Hoechst 33342 (Fig. 4FGo). Chromatin condensation and nuclear cleavage, which are morphological characteristics of apoptosis, were evident in most cells overexpressing PTP20WT in the presence of FSH (panel e). In contrast, most cells that expressed ß-galactosidase or PTP20CS in the presence and absence of FSH, and PTP20WT in the absence of FSH, assumed the normal shape of viable cells (panels a–d and f). Together, these data suggest that wild-type PTP20 leads to reorganization of the actin cytoskeleton with decreased cell adhesion upon FSH stimulation in a PTP activity-dependent manner and that PTP20 can induce the apoptosis of at least primary cultured granulosa cells, as a result of decreased adhesion.

PTP20 Selectively Reduces the Tyrosine Phosphorylation Level of 190-kDa Protein
To identify a substrate for PTP20, granulosa cells were infected with either Ad-ß-gal (control), Ad-PTP20WT, or Ad-PTP20CS, after which the phosphorylation state of proteins in cell lysates was analyzed by anti-p-Tyr immunoblotting (Fig. 5Go). Infected cells were stimulated with FSH for 1 h. The p-Tyr content of a protein of approximately 190 kDa in cells overexpressing PTP20WT, but not PTP20CS, was significantly lower than that in control cells (top panel). Reprobing of the immunoblot with either anti-{alpha}-tubulin or anti-PTP20 antibodies demonstrated that equivalent amounts of proteins were loaded in each lane (middle panel) and comparable levels of the recombinant PTP20 were expressed (bottom panel). In some experiments, the tyrosine-phosphorylation levels of several other proteins such as about 120-kDa and about 58-kDa proteins were also slightly reduced in lysates of PTP20WT-overexpressing cells. However, these reduced phosphorylation levels were not reproducible, revealing the selectivity of PTP20 for decreasing the p-Tyr content of the 190-kDa protein, even though PTP20 is overexpressed. Therefore, the 190-kDa protein is a candidate substrate for PTP20. Moreover, a tyrosine-phosphorylated band of 50 kDa, the size of which corresponded to PTP20, was detected when PTP20CS, but not PTP20WT, was overexpressed (see asterisk in top panel). This finding suggests that PTP20 undergoes tyrosine-phosphorylation and mediates its own dephosphorylation. This notion is consistent with the findings of several other reports (16, 17, 18).



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Figure 5. Effect of Overexpressed Recombinant PTP20 on Tyrosine Phosphorylation Levels of Proteins

Granulosa cells infected with either Ad-ß-gal (control), Ad-PTP20WT, or Ad-PTP20CS were serum starved for 24 h, incubated with FSH (50 ng/ml) for 1 h, and lysed. Cell lysates (30 µg of protein) were immunoblotted with an anti-p-Tyr antibody (top panel). The same blots were stripped and sequentially reprobed with anti-{alpha}-tubulin (middle panel) and anti-PTP20 (bottom panel) antibodies. Data represent three separate experiments that yielded similar results. IB, Immunoblotting; WT, wild type.

 
FSH Increases p-Tyr Content of the 190-kDa Protein in a Time-Dependent Manner
We tested whether the p-Tyr level of the 190-kDa protein increases in response to FSH stimulation. Uninfected granulosa cells were cultured with FSH for the indicated periods and Tyr-phosphorylated bands were detected (Fig. 6AGo). Figure 6BGo shows quantified results of Fig. 6AGo with respect to the 190-kDa protein. The p-Tyr content of the 190-kDa protein increased within 1 min after the addition of FSH and lasted for at least 60 min. Although the difference was not significant, this increase tended to last for 24 h and then returned to the basal level by 48 h. Thus, tyrosine phosphorylation of the 190-kDa protein may be required for FSH function in granulosa cells.



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Figure 6. Time Course of FSH-Induced Increase in p-Tyr Content of the 190-kDa Protein

A, Granulosa cells were serum-starved for 24 h, stimulated with FSH (50 ng/ml) for the indicated periods, lysed, and immunoblotted (30 µg of protein) against an anti-p-Tyr antibody (upper panel). The same blot was stripped and reprobed with anti-{alpha}-tubulin antibody. Data represent three separate experiments. B, Band intensity of the 190-kDa protein in panel A was quantified using a LAS-1000 imaging analyzer. Values of the 190-kDa band were normalized to amount of {alpha}-tubulin and expressed as a percentage of the value at time 0. Data are means ± SE of three independent experiments. *, P < 0.05 vs. the value at time 0. IB, Immunoblotting.

 
FSH Stimulation Enhances Dephosphorylation of the 190-kDa Protein by PTP20
Because the PTP20-induced morphological change was dependent on FSH stimulation and FSH increased the p-Tyr level of the 190-kDa protein, we predicted that the ability of PTP20 to dephosphorylate the 190-kDa protein would be enhanced upon FSH stimulation. Thus, this ability of PTP20 was compared in the presence and absence of FSH. Cells infected with Ad-ß-gal or Ad-PTP20WT were serum starved for 24 h and cultured with and without FSH. Thereafter, cell lysates were immunoblotted using an anti-p-Tyr antibody, which showed that FSH increased the phosphorylation level of the 190-kDa protein (Fig. 7AGo, compare lane 1 to 3). This level was reduced by the forced expression of PTP20 regardless of the presence or absence of FSH (Fig. 7AGo, compare lane 1 to 2 and lane 3 to 4, respectively). However, the difference in the phosphorylation level between cells expressing ß-galactosidase and PTP20 was more remarkable in the presence than in the absence of FSH. Figure 7BGo shows the quantified results of Fig. 7AGo. These findings suggest that overexpressed PTP20 has PTP activity without FSH stimulation and that this phosphatase activity or the interaction of PTP20 with the p190-kDa protein is enhanced in an FSH-dependent manner.



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Figure 7. Different Levels of PTP20-Induced Dephosphorylation of the 190-kDa Protein with and Without FSH Stimulation

A, Granulosa cells infected with either Ad-ß-gal (control) or Ad-PTP20WT were serum-starved for 24 h and incubated with or without FSH (50 ng/ml) for 1 h. Thereafter, lysates (30 µg of protein) were immunoblotted with an anti-p-Tyr antibody (upper panel). The same membrane was stripped and reblotted with anti-{alpha}-tubulin antibody (lower panel). Data represent three independent experiments. B, Intensity of 190-kDa protein band in panel A was quantified using the LAS-1000 imaging analyzer. Values of the 190-kDa band were normalized to the amount of {alpha}-tubulin and expressed as a percentage of the value in ß-galactosidase-expressing cells in the absence of FSH. Data are means ± SE of three independent experiments. *, P < 0.05. IB, Immunoblotting; WT, wild type.

 
The 190-kDa Protein Is p190 RhoGAP
We predicted that this 190-kDa protein would be p190 RhoGAP, a GTPase-activating protein (GAP) of the Rho family, based on the following two reasons. First, the molecular mass was identical to that of p190 RhoGAP, which is tyrosine phosphorylated at several sites by the Src kinase family (24, 25, 26, 27, 28, 29). Second, p190 RhoGAP is implicated in regulating reorganization of the actin cytoskeleton (26, 28, 29, 30, 31). The overexpression of wild-type PTP20 induced a rounded cell phenotype accompanied by dendritic-like processes similar to that seen when Rho function is disrupted (Fig. 4Go), suggesting that PTP20 is involved in reorganization of the actin cytoskeleton. To confirm our prediction, lysates from granulosa cells infected with Ad-ß-gal, Ad-PTP20WT, or Ad-PTP20CS with subsequent FSH stimulation for 1 h were immunoprecipitated with an anti-p190 RhoGAP antibody followed by anti-p-Tyr immunoblotting (Fig. 8AGo). The p-Tyr state of the immunoprecipitated p190 RhoGAP protein was significantly reduced by the overexpression of PTP20WT, but not of PTP20CS, compared with the control (upper panel). The same results were obtained when infected cells were incubated with FSH for 6 or 24 h (data not shown). This dephosphorylation profile and extent were almost identical to those in total cell lysates (compare Fig. 8Go to Fig. 5Go). Reprobing of the immunoblot membrane with an anti-p190 RhoGAP antibody indicated that equivalent amounts of anti-p190 RhoGAP was loaded in each lane (lower panel). These data supported our notion that the 190-kDa protein is p190 RhoGAP and that most, if not all, of the tyrosine-phosphorylated 190-kDa band in lysates reflects the tyrosine-phosphorylated p190 RhoGAP protein.



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Figure 8. Identification of 190-kDa Protein as p190 RhoGAP and Its Association with PTP20

A, Granulosa cells infected with Ad-ß-gal (control), Ad-PTP20WT, or Ad-PTP20CS were serum-starved for 24 h and stimulated with FSH (50 ng/ml) for 1 h before lysis. Cell lysates (500 µg of protein) were immunoprecipitated with an anti-p190 RhoGAP antibody followed by immunoblotting with an anti-p-Tyr antibody (upper panel). The same membrane was reprobed with an anti-p190 RhoGAP antibody to demonstrate that equivalent amounts of p190 RhoGAP were immunoprecipitated from each cell lysate (lower panel). Data represent three separate experiments. Results from two other experiments were similar. B, Lysates (500 µg of protein) from cells undergoing the same manipulations as in panel A were immunoprecipitated with an anti-HA antibody, and then immunoblotted against anti-p190 RhoGAP antibody (upper panel). The same blot was stripped and reprobed with an anti-HA antibody to confirm that comparable amounts of PTP20 were immunoprecipitated from each cell lysate (lower panel). Data represent five separate experiments yielding similar results. IB, Immunoblotting; IP, Immunoprecipitation.

 
We examined the association of PTP20 with p190 RhoGAP by immunoprecipitating the PTP20 protein, followed by immunoblotting the precipitate with an anti-p190RhoGAP antibody. Since the anti-PTP20 antibodies were not ideal for immunoprecipitation, we expressed HA-tagged PTP20WT (HA-PTP20WT) or HA-tagged PTP20CS (HA-PTP20CS). The resultant cells were stimulated with FSH for 1 h (Fig. 8BGo). Whereas there was no detectable band of p190 RhoGAP in control cells, p190 RhoGAP coimmunoprecipitated with HA-PTP20WT as well as with HA-PTP20CS (upper panel). However, the efficiency with which the CS mutant coimmunoprecipitated with p190 RhoGAP was much higher than that of the wild type, suggesting that wild-type PTP20 can transiently associate with p190 RhoGAP and that PTP20CS functions as a substrate-trapping mutant.

PTP20 Directly Dephosphorylates p190 RhoGAP
The tyrosine kinase Pyk2 forms a multiple complex with p190 RhoGAP, p120 RasGAP, ErbB-2, and c-Src upon heregulin stimulation and plays an essential role in mediating the tyrosine phosphorylation of p190 RhoGAP by c-Src (26). A mutation of Pyk2 on Tyr402, a c-Src-binding site, abolishes the phosphorylation of p190 RhoGAP. Therefore, there is the possibility that PTP20 might indirectly decrease the phosphorylation level of p190 RhoGAP by dephosphorylating p-Tyr402 of Pyk2 and/or by dephosphorylating p-Tyr418 of c-Src, which is essential for its full activity. To exclude this possibility, we stimulated infected granulosa cells with FSH for 1 h and sequentially immunoblotted cell lysates with anti-p-Tyr (top panel), antiphospho-c-Src (p-Tyr418) (second panel), and antiphospho-Pyk2 (p-Tyr402) (third panel) antibodies (Fig. 9AGo). The results showed that the overexpression of HA-PTP20WT and HA-PTP20CS had no effect on the phosphorylation states of Tyr402 of Pyk2. In contrast, the overexpression of these proteins slightly, but significantly, reduced the phosphorylation states of Tyr418 in c-Src. However, this reduced level was much smaller than the PTP20WT-dependent reduced level of the phosphorylation content in p190 RhoGAP (compare p panel to second panel). Thus, PTP20 decreases the p190 RhoGAP phosphorylation level by a mechanism other than mediating the dephosphorylation of Pyk2 and c-Src.



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Figure 9. Direct Dephosphorylation of p190 RhoGAP by PTP20

A, Granulosa cells infected with either Ad-ß-gal (control), Ad-HA-PTP20WT, or Ad-HA-PTP20CS were serum starved for 24 h and stimulated with FSH (50 ng/ml) for 1 h. Cell lysates (30 µg of protein) were sequentially immunoblotted against anti-p-Tyr (first panel), antiphospho-c-Src (p-Tyr418) (second panel), and antiphospho-Pyk2 (p-Tyr402) (third panel) antibodies. The same membrane was stripped and further reblotted with anti-PTP20 (fourth panel) and anti-{alpha}-tubulin (fifth panel) antibodies. Data are representative of three separate experiments that yielded similar results. B, Granulosa cells were infected with Ad-ß-gal (control), Ad-HA-PTP20WT, Ad-HA-PTP20CS, or Ad-HA-PTP20DA, serum-starved for 24 h, and stimulated with FSH (50 ng/ml) for 1 h. Cell lysates (500 µg of protein) were immunoprecipitated with an anti-p120 RasGAP antibody, and then immunoblotted against anti-p190 RhoGAP antibody (upper panel). The same blot was reprobed with anti-p120 RasGAP antibody to confirm that equivalent amounts of p120 RasGAP were immunoprecipitated from each cell lysate (lower panel). IB, Immunoblotting; WT, wild type.

 
To further examine whether the interaction between PTP20 and p190 RhoGAP is direct or not, we investigated the effect of overexpressed HA-PTP20WT, HA-PTP20CS, or HA-PTP20DA (substrate-trapping mutant) on the formation of p120 RasGAP-p190 RhoGAP complexes. Tyrosine1105 on p190 RhoGAP is involved in p-Tyr-dependent complex formation between these two GAP proteins, although a p-Tyr-independent mechanism also mediates this process (25). Thus, we speculated that if PTP20 directly recognizes p-Tyr1105 on p190 RhoGAP, not only PTP20WT but also the substrate-trapping mutants, PTP20CS and PTP20DA, should prevent the formation of these complexes. The immunoprecipitated p120 RasGAP protein was immunoblotted against an anti-p190 RhoGAP antibody. As we expected, control lysates contained considerably more stable complexes of the two GAP proteins than lysates from cells overexpressing PTP20WT, PTP20CS, or PTP20DA (Fig. 9BGo). Therefore, although we cannot completely exclude the possibility that PTP20 reduces the phosphorylation level of p190 RhoGAP by an indirect mechanism, it is very likely that PTP20 directly acts on phosphorylated p190 RhoGAP.

PTP20 Colocalizes with p190 RhoGAP
We further analyzed the direct association of PTP20 with p190 RhoGAP using confocal microscopy. Since p190 RhoGAP preferentially associates with the CS mutant compared to the wild type of PTP20, we expressed HA-PTP20CS in granulosa cells and tested whether the phosphatase colocalizes with p190 RhoGAP. Figure 10Go shows a typical staining profile. Most of the p190 RhoGAP protein was localized to the cytoplasm, particularly in the perinuclear region, and some were localized at the peripheral region (panels A and D). The PTP20 mutant was widely distributed throughout the cell with staining at the peripheral and perinuclear regions (panels B and E). Also, some mutated PTP20 proteins appeared to be associated with cytoskeletal actin. Panels C and F show that the main site of colocalization of the two proteins was the perinuclear region and sometimes, the peripheral region. These data further confirm the notion that p190 RhoGAP is directly dephosphorylated by PTP20.



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Figure 10. Colocalization of PTP20 with p190 RhoGAP

Granulosa cells seeded on glass coverslips placed in eight-well plates were infected with Ad-HA-PTP20CS at moi = 15 and incubated with FSH (50 ng/ml) for 8 h. Thereafter, cells were fixed, permeabilized, and double-stained with rat anti-HA and mouse anti-p190 RhoGAP antibodies followed by Alexa-568-conjugated (red) antirat (B and E) and Alexa-488-conjugated (green) antimouse antibodies (A and D). It should be noted that cells not expressing recombinant PTP20 were not stained with an anti-HA antibody, suggesting the specific staining of HA-PTP20CS. Merged images (overlay) of the same fields are presented on the right (C and F). Infection efficiency was about 50% at moi = 15 when granulosa cells were seeded on glass coverslips placed in eight-well plates whereas more than 95% of these cells cultured in six-well or 10-cm diameter tissue plates were transduced at moi = 15.

 
ADP Ribosylation of Rho with C3 Botulinum Toxin Exhibits Morphological Change Similar to That Induced by PTP20
The overexpression of PTP20 induced retraction of the granulosa cell body with long, dendritic-like processes. This phenotype was similar to that generated when Rho function is disrupted by ADP ribosylation in fibroblasts. To test whether inhibiting Rho functions really is associated with this phenotype in granulosa cells, we incubated the cells with or without C3 botulinum toxin (4 µg/ml) in the presence of FSH for 48 h and stained actin fibers with phalloidin (Fig. 11Go). This was the same length of time as PTP20-overexpressing cells were observed. The phalloidin-staining profile and morphology of the treated cells was very similar to those of cells overexpressing wild-type PTP20 (compare Fig. 11Go with Fig. 4Go, A and D), suggesting that PTP20 reduces Rho functions via the dephosphorylation of p190 RhoGAP.



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Figure 11. Effect of C3 Botulinum Toxin on Cell Morphology

Uninfected granulosa cells were serum starved for 24 h and incubated with (C and D) or without (A and B) C3 botulinum toxin (4 µg/ml) in the presence of FSH (50 ng/ml) for 48 h. Thereafter, cells were fixed, stained with Alexa Fluor 488 phalloidin and examined by fluorescence microscopy. Original magnifications are x100 (A and C) and 200 (B and D).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present study showed the function of PTP20 in ovarian granulosa cells. Our results suggest that a principal cellular function of PTP20 is to dephosphorylate p190 RhoGAP, a negative regulator of Rho GTPase, in these cells. This causes reorganization of the actin cytoskeleton with decreased focal adhesion probably by reducing Rho function. To date, only SHP-1, SHP-2, and FAP-1 among PTPs have been investigated with respect to whether or not their expression levels change in granulosa cells in response to several stimuli (32, 33). However, the physiological roles of these PTPs have not been further analyzed. Of the three PTPs, only the SHP-1 mRNA level is influenced by hormones (32). We demonstrated, using a model system of immature female rats primed with gonadotropins, that the expression of at least PTP20, PTP{epsilon}M, PTP{epsilon}C, and PTP-MEG1 clearly changes during the estrous cycle. Together with the finding that at least 25 PTPs are expressed in granulosa cells, these data suggest that the physiological roles of PTPs as well as PTKs should be defined to understand the molecular mechanisms regulating the dynamics of ovarian functions.

The protein p190 RhoGAP contains an N-terminal GTP-binding domain, a middle domain that mediates interaction with p120 RasGAP, and a C-terminal GAP domain with specificity for the Rho family. Whereas p190 RhoGAP is tyrosine phosphorylated at sites including Tyr1105 by the Src-family kinases (25, 26, 27, 28, 29), whether p190 RhoGAP is positively or negatively regulated by tyrosine phosphorylation remains controversial. In our study, PTP20 appears to reduce the Rho activity by dephosphorylating and hence activating p190 RhoGAP. This is because granulosa cells overexpressing wild-type PTP20 exhibited a morphological phenotype similar to that seen when Rho function was disrupted with C3 botulinum toxin (Figs. 4Go and 11Go). The c-Src phosphorylation of tyrosine residues in the GTP-binding domain of p190 RhoGAP results in the loss of its GTP-binding activity, thereby disrupting the GAP activity in vivo (27, 30). Therefore, tyrosine-phosphorylation of this domain is predicted to negatively regulate p190 RhoGAP. Thus, one possible mechanism by which PTP20 reduced the Rho function is that PTP20 dephosphorylates p-Tyr residues located in the GTP-binding domain in addition to Tyr1105 in the middle domain of p190 RhoGAP, which would activate the GAP protein. The idea that PTP20 decreases the Rho function by activating p190 RhoGAP is in good agreement with a report indicating that stable overexpression of PTP20 in PC12 cells accelerated neurite formation after exposure to nerve growth factor (8), because the inhibition of Rho activity in neural cells promotes extensive neurite outgrowth.

Some reports suggest that tyrosine phosphorylation by the Src family kinases leads to p190 RhoGAP activation (25, 34, 35, 36, 37, 38). For example, the interaction between p190 RhoGAP and p120 RasGAP that is enhanced by the phosphorylation of p190 RhoGAP on Tyr1105 by Src-family kinases correlates with actin stress fiber dissolution upon epidermal growth factor stimulation (25, 38), a phenotype that is usually observed when the p190 RhoGAP activity increases concomitantly with a decrease in Rho activity. As described above, our findings suggest that PTP20 dephosphorylates the Tyr1105 of p190 RhoGAP probably with a subsequent decrease in p190 RhoGAP activity. Therefore, p190RhoGAP activation induced by c-Src via Tyr1105 phosphorylation is inconsistent with our present findings. A p-Tyr-independent mechanism is also known for the formation of p190 RhoGAP-p120 RasGAP complexes. Thus, dephosphorylation of the GTP-binding domain by PTP20 may be more critical for regulating p190 RhoGAP function than dephosphorylation of Tyr1105 in FSH-stimulated granulosa cells.

Our data suggest that PTPs are important in p190 RhoGAP regulation. The focus to date has been directed toward the tyrosine phosphorylation of p190 RhoGAP. Consequently, little is known about how p190 RhoGAP is dephosphorylated. Two studies describe the interaction of RhoGAPs with PTPs (39, 40). Overexpressed low molecular weight PTP (LMW-PTP) tyrosine dephosphorylates p190 RhoGAP after platelet-derived growth factor stimulation in NIH3T3 cells, which potentiates platelet-derived growth factor-induced cell adhesion, spreading, and migration (40). Thus, the authors concluded that LMW-PTP regulates reorganization of the actin cytoskeleton by dephosphorylating and hence inhibiting p190 RhoGAP activity. A novel RhoGAP of 150 kDa has been cloned as a protein that interacts with the PTP FAP-1 (39). However, tyrosine phosphorylation of 150-kDa RhoGAP was undetectable even in the presence of the PTP inhibitor vanadate, indicating that this RhoGAP is not a substrate for FAP-1. Therefore, our finding that p190 RhoGAP is dephosphorylated and probably activated by PTP20 represents a novel mechanism for regulating p190 RhoGAP activity.

Our data suggest that PTP20 undergoes autodephosphorylation and/or dephosphorylation by other cellular PTPs (Fig. 5Go). Wild-type and/or catalytically inactive PTP20 are phosphorylated by c-Abl and Src-related kinases (17, 18, 19). The biological significance of tyrosine phosphorylation of PTP20 by c-Abl is unclear. However, Src-related kinase-dependent phosphorylation of PTP20 is critical for PTP20 to dephosphorylate Src-related kinases themselves via Csk. PTP20 may undergo tyrosine phosphorylation in an FSH-dependent manner and recruit p190 RhoGAP via p-Tyr residue(s). Alternatively, whereas PTP20 has basal phosphatase activity without undergoing tyrosine phosphorylation, such activity might be enhanced by phosphorylation like that of LMW-PTP and SHP-2 (41, 42). Our finding that the ability of PTP20 to reduce the p-Tyr level of p190 RhoGAP is augmented by FSH agrees with both of these notions (Fig. 7Go).

We did not find that PTP20 reduces the p-Tyr content of p190 RhoGAP in vitro (data not shown). Nevertheless, we consider that p190 RhoGAP is a substrate for this phosphatase in granulosa cells. PSTPIP1, c-Abl, and the Src-related kinases are known substrates for PTP20 (18, 19, 20). However, we could not detect the tyrosine phosphorylation of c-Abl even in immunoprecipitated c-Abl protein (data not shown), indicating that c-Abl is not a substrate for PTP20 in granulosa cells. PSTPIP1 associates with PTP20 via the C-terminal 24 amino acids of the phosphatase. A PTP20 mutant lacking the C-terminal 24 amino acids and the wild type similarly reduced the p-Tyr content of p190 RhoGAP (data not shown). Phosphorylated Tyr418 of c-Src, an essential site for its full activity, was little affected by PTP20 (Fig. 9Go). Moreover, p-Tyr402 on Pyk2, which is required for Pyk2 binding to c-Src and for the phosphorylation of p190 RhoGAP by c-Src, was not affected by PTP20 (Fig. 9Go). These data indicate that Pyk2 and c-Src as well as PSTPIP1 are not responsible for the PTP20-dependent p190 RhoGAP dephosphorylation as substrates of PTP20 in granulosa cells. In contrast, several types of recombinant PTP20 prevented the interaction of p190 RhoGAP with p120 RasGAP that is enhanced by Tyr1105 phosphorylation in p190 RhoGAP, showing that PTP20 directly binds and dephosphorylates p190 RhoGAP. It is unclear why PTP20 did not reduce the p-Tyr content of p190 RhoGAP when glutathione-S-transferase-PTP20 was added to lysates from granulosa cells. One explanation is that a mediator may be necessary for the direct binding of PTP20 to p190 RhoGAP, and such complexes consisting of three proteins including the mediator cannot be formed under our in vitro experimental conditions.

Fibronectin is a major secretion product of cultured granulosa cells (43) and enhances their substratum attachment, spreading, survival, and growth (44, 45). Therefore, interaction between these cells and the extracellular matrix is highly significant for controlling follicular growth. However, our knowledge about this issue is very limited. Indeed, although Rho plays critical roles in the process of cell-matrix interaction, Rho functions or molecules regulating the Rho activity in granulosa cells have not been described. One role of PTP20 in the follicles may be to promote granulosa cell proliferation by inducing reorganization of the actin cytoskeleton in response to gonadotropin and/or other humoral factors. This idea is consistent with the fact that far more PTP20 is expressed in healthy than in atretic follicles. However, the opposite function of PTP20 is also conceivable. The detachment of granulosa cells overexpressing wild-type PTP20 from the substratum could suggest that cell proliferation is negatively controlled by PTP20 to avoid excess proliferation because the number of these cells noticeably increases within a short period during the estrous cycle. It is unlikely that PTP20 is critically involved in the progression of apoptosis as a negative regulator for FSH because little PTP20 is expressed in atretic follicles.

In conclusion, we identified multiple PTPs that are expressed in rat ovarian granulosa cells. The expression levels of some of them, including PTP20, significantly changed during the estrous cycle. Granulosa cells overexpressing PTP20 showed a phenotype of rounded cells with long, dendritic-like processes in an FSH-dependent manner by dephosphorylating p190 RhoGAP and hence probably decreasing Rho activity, with subsequent actin cytoskeleton reorganization. Our results suggest that PTPs play significant roles in controlling the dynamics of ovarian functions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Ovine FSH (NIDDK-oFSH-20) was provided by the National Institutes of Health (Bethesda, MD). The biological potency of this compound was 175-fold greater than that of NIH-FSH-S1. We converted the weight of NIDDK-oFSH-20 into that of NIDDK-oFSH-15, the potency of which is 20-fold greater than that of NIH-FSH-S1. We and others have widely applied NIDDK-oFSH-15 in many studies of the ovary (46, 47). Gonadotropin derived from pregnant mares serum (PMSG), human chorionic gonadotropin, fetal bovine serum (FBS), type I deoxyribonuclease, L-glutamine, penicillin, and streptomycin were purchased from Sigma Modified McCoy’s 5A medium was obtained from Life Technologies, Inc. (Gaithersburg, MD). The total RNA preparation reagent ISOGEN and the poly(A)+ RNA purification reagent Oligotex were purchased from Nippongene (Osaka, Japan) and Roche (Indianapolis, IN), respectively. The first-strand cDNA synthesis kit and [{alpha}-32P]dCTP were from Amersham Pharmacia Biotech (Arlington Heights, IL). AmpliTaq Gold DNA polymerase was obtained from PE Applied Biosystems (Foster City, CA). Hoechst 33342 and Alexa Fluor 488 phalloidin were from Wako (Osaka, Japan) and Molecular Probes, Inc. (Eugene, OR), respectively. Antibodies were obtained from the following sources: anti-HA antibody for immunoblotting, Roche Molecular Biochemicals; anti-HA for immunoprecipitation, p120 RasGAP, c-Abl, and Pyk2 (p-Tyr402) antibodies, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-p190 RhoGAP antibody, Transduction Laboratories, Inc. (Lexington, KY); anti-p-Tyr antibody (4G10), Upstate Biotechnology, Inc. (Lake Placid, NY); anti-{alpha}-tubulin antibody, Oncogene Science, Inc. (Manhasset, NY); and antiphospho-c-Src antibody (p-Tyr418), BioSource International (Camarillo, CA).

Model System of the Estrous Cycle
Immature female Wistar-Imamichi rats (3-wk-old) obtained from the Imamichi Institute for Animal Reproduction (Ibaraki, Japan) were housed in air-conditioned quarters with a 12-h light, 12-h dark cycle. To construct a model of the estrous cycle, the animals (21-d-old) were injected sc with 20 IU of PMSG for 2 (stage 1) or 5 d (stage 2) (46, 47). The ovary contains mostly healthy preovulatory follicles at stage 1 and mostly atretic follicles at stage 2. After an injection with 20 IU of PMSG for 2 d, some rats were further administered with 40 IU of human chorionic gonadotropin for 7 d (stage 3) or 10 d (stage 4) (48, 49, 50). The ovary contains a number of corpora lutea at stage 3 and several degenerating corpora lutea at stage 4. Gonadotropin-primed rats for RNA extraction and untreated female rats (26-d-old) for primary culture of granulosa cells were anesthetized with ether and decapitated, and ovaries were removed. The animals were maintained and manipulated in accordance with the Guidelines for Animal Experiments of Tsukuba University.

Preparation of Granulosa and Luteal Cells and Culture of Granulosa Cells
Granulosa cells were prepared as described (47). Luteal cells were prepared according to the method of Nelson et al. (50). Granulosa cells prepared from untreated female rats (26-d-old) were seeded into six-well (5 x 105 cells per well) or 10-cm diameter tissue plates (5 x 106 cells per plate) and cultured in McCoy’s 5A medium supplemented with 25 mM HEPES, 26 mM NaHCO3, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 IU/ml penicillin, and 1% FBS at 37 C under a humidified atmosphere of 5% CO2-95% room air for 24 h. The cells were then serum starved for 24 h and further incubated with or without FSH.

Construction of Recombinant Adenoviruses and Infection
Recombinant adenoviruses were constructed according to the manufacturer’s protocol (Takara, Tokyo, Japan). In brief, cDNAs encoding a wild type and mutants of PTP20 (see Results) were inserted into the Swa I site of the cosmid shuttle vector pAxCAwt, the expression of which is controlled by the chick ß-actin promoter, a cytomegalovirus enhancer, and the poly (A) adenylation site of bovine GH. These adenoviral shuttle vectors together with the Ad5 genomic DNA Ad5-dlX were transfected into human embryonic kidney 293 cells, which provide the E1A gene product necessary for viral replication. The resultant recombinant viruses were propagated in 293 cells, titered, and stored at -80 C. Granulosa cells were infected as follows. Cells were cultured with 1% FBS for 24 h. The medium was then removed and the cells were exposed to recombinant adenoviruses in McCoy’s 5A medium at 37 C for 1 h with occasional rocking. Thereafter, the viruses were removed and cells were cultured without serum for 24 h before the addition of FSH. The adenovirus vector containing ß-galactosidase (Ad-ß-gal) was used as a control. The expression of the P450 aromatase gene was induced in response to FSH under these experimental conditions, which is a typical feature of granulosa cells (data not shown).

Identification of PTPs using PCR Amplification
Total RNA was isolated using ISOGEN (Nippongene), according to specifications provided by the manufacturer, from the granulosa cells of rats treated with PMSG for 2 (stage 1) or 5 d (stage 2). First-strand cDNAs were synthesized using random primers from poly(A)+ RNA (200 ng) as the template. The resultant cDNA mixtures were amplified by PCR to isolate cDNAs for PTPs. Degenerate oligonucleotide primers were designed based on highly conserved amino acid sequences within the catalytic domain of PTPs, FWXMXW, WPDXG, and HCSAG. One sense primer corresponding to FWXMXW and four and six antisense primers corresponding to WPDXG and HCSAG, respectively, were synthesized. The PCR products were size fractionated on 5% acrylamide gels, and amplified fragments of 200–480 bp were excised, subcloned into pBluescript II KS+, and sequenced.

Quantification of PTP mRNA by RT-PCR
Total RNA (2 µg) was reverse transcribed with random primers, and the resultant cDNA mixtures were amplified by PCR with a trace amount of [{alpha}-32P]dCTP to selectively detect PTP{epsilon}M, PTP{epsilon}C, PTPS, SHP-2, PTP-MEG1, PTP20, DEP-1, and FAP-1. Denaturation, annealing, and polymerase reactions proceeded at 94 C for 1 min, at temperatures described below for 1 min, and at 72 C for 1.5 min, respectively. After 24 cycles of amplification, the incubation was continued at 72 C for an additional 8.5 min to complete polymerization. Oligonucleotide primers and annealing temperatures were: PTP{epsilon}M (62 C), 5'-cttgcagcctacttcttcagg-3' (sense primer) and 5'-ttgaactcctctcggaaccg-3' (antisense primer); PTP{epsilon}C (57 C), 5'-aaacagttcagataccgctgg-3' (sense primer) and 5'-ttgaactcctctcggaaccg-3' (antisense primer); PTPS (59 C), 5'-atacctgtctcgttctgatgg-3' (sense primer) and 5'-taggtgtctgtcaatcttggc-3' (antisense primer); SHP-2 (57 C), 5'-caagtgcaacaattcaaaacc-3' (sense primer) and 5'-ttctctctgtgtttccctgg-3' (antisense primer); PTP-MEG1 (59 C), 5'-ggatgctaccaagttacctgc-3' (sense primer) and 5'-cggtacacagagtaacttacgt-3' (antisense primer); PTP20 (59 C), 5'-actgggtggcgttctcagg-3' (sense primer) and 5'-cgtggccctttaggtcttcc-3' (antisense primer); DEP-1 (59 C), 5'-acatcagaagttgttcttccgg-3' (sense primer) and 5'-acatgctcactcgctcgagg-3' (antisense primer); FAP-1 (57 C), 5'-caggaattcgtgtacattgcc-3' (sense primer) and 5'-tagcagaagacatactgaccc-3' (antisense primer). The PCR products were size fractionated on 5% acrylamide gels. Amplified DNA fragments were detected on dried gels using a BAS-2000 imaging analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). The resultant values were normalized to a specific amount of ß-actin mRNA, which was also simultaneously determined under the same conditions as PTP mRNA, to correct for variations in RNA loading and RT-PCR efficiency.

ß-Galactosidase Assay
Cells treated with recombinant adenovirus containing the ß-galactosidase gene (Ad-ß-gal) were fixed with 0.5% glutaraldehyde and stained in 100 mM sodium phosphate (pH 7.5), containing 0.05% X-gal, 1 mM MgCl2, 10 mM KCl, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, and 0.1% Triton X-100 at 37 C for 2 h.

Preparation of Antibodies Against PTP20
The N-terminal peptide of PTP20 (MSRQSDLVRSFLEQQEARDH), to which a cysteine residue was added to the C terminus, was coupled to keyhole limpet hemocyanin. Rabbits were immunized sc at intervals of 2 wk with this antigen mixed with complete Freund’s adjuvant for the primary immunization and incomplete adjuvant for the next three injections.

Assessment of Cell Viability
After detached cells in culture were collected, adsorbed cells were harvested by trypsinization. Thereafter, all cells, including detached cells, were counted by trypan blue staining. Cell viability was calculated and expressed as a percentage of the control values obtained in control cells expressing ß-galactosidase.

DNA Fragmentation Analysis
To detect apoptotic DNA fragmentation, total DNA was extracted from cultured cells and quantified by reading the absorbance at 260 nm. DNA samples (2 µg) were resolved through 2% (wt/vol) agarose gels, stained with SYBR Green I nucleic acid gel stain (Wako, Osaka, Japan), and visualized under UV light using LAS1000 film (Fuji Photo Film Co., Ltd.).

Hoechst 33342 Staining
Detached and adsorbed cells were washed with PBS and fixed in 4% (wt/vol) paraformaldehyde at 4 C overnight. Thereafter, the cells were washed with PBS, stained with 1.6 µM Hoechst 33342 for 10 min, resuspended, and seeded on glass slides for observation by fluorescence microscopy.

Phalloidin Staining
Cells cultured on micro coverglass (Matsunami, Japan) in 12-well dishes were fixed in 4% (wt/vol) paraformaldehyde at room temperature for 30 min, washed with PBS three times, and permeabilized with 0.05% (vol/vol) Triton X-100 in PBS for 15 min. After three PBS washes, the cells were incubated in PBS containing 5% (wt/vol) BSA at 4 C overnight, and then stained with 5 U/ml of Alexa Fluor 488 phalloidin in PBS containing 1% (wt/vol) BSA at room temperature for 20 min. Thereafter, cells were washed with PBS three times and observed by fluorescence microscopy.

Cell Lysis, Immunoblotting, and Immunoprecipitation
To immunoprecipitate p190 RhoGAP and c-Abl, cells were washed with PBS and lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EGTA, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10% glycerol, 5 mM sodium pyrophosphate, 100 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml antipain. HA-tagged PTP20 was immunoprecipitated in the same lysis buffer except that 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, and 0.5% sodium deoxycholate were replaced with 0.5% Nonidet P-40. Lysates were prepared for immunoblotting as described for immunoprecipitation except that cells were harvested in lysis buffer consisting of 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 5 mM sodium pyrophosphate, 100 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml antipain. Lysates were clarified by centrifugation, the supernatant was recovered, and protein concentrations were assayed using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL).

For immunoprecipitation, cell lysates (1 mg of protein) clarified with protein A-Sepharose or protein G-Sepharose beads (5 mg) for 2 h at 4 C were mixed with primary antibodies at 4 C for 2 h. Protein A-Sepharose or protein G-Sepharose beads (3 mg) were then added and the mixture was gently rocked overnight at 4 C. The immunoprecipitates were washed three times with ice-cold buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 10% glycerol, resolved by SDS-PAGE, and immunoblotted. Lysates for immunoblotting (30 µg of protein) were separated on 10% SDS-polyacrylamide gels under reducing conditions, followed by electrophoretic transfer to polyvinylidine difluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA). After blocking by a 1-h incubation with 5% BSA or 5% skim milk in Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with the relevant primary antibodies in the same buffer for 1 h. The membranes were washed in Tris-buffered saline containing 0.1% Tween 20 three times and further incubated with horseradish peroxidase-linked second antibodies (1:1000). After three more washes, proteins were visualized using an enhanced chemiluminescence kit (Roche). All immunoblotting procedures proceeded at room temperature. Bound antibodies were stripped for subsequent analysis by boiling for 5 min.

Confocal Microscopy Analysis
Granulosa cells seeded on glass coverslips placed in eight-well plates in McCoy’s 5A medium were incubated with 1% FBS for 24 h, and then infected with recombinant adenovirus encoding HA-PTP20CS at moi = 15 after an 8-h incubation with 50 ng/ml of FSH. Cells were fixed in 0.1 M phosphate buffer (pH 7.5), containing 4% paraformaldehyde and 3% sucrose for 30 min, and permeabilized with 0.1% Triton X-100 in 10 mM phosphate buffer (pH 7.3), containing 0.5 M NaCl and 0.1% Tween 20. After blocking with 0.8% BSA and 5% goat serum at 4 C for 1 h, cells were incubated with rat anti-HA monoclonal antibody 3F10 and mouse anti-p190 RhoGAP monoclonal antibody at 4 C overnight. Thereafter, cells were washed with PBS three times and incubated with Alexa-568-conjugated antirat IgG and Alexa-488-conjugated antimouse IgG at room temperature for 1 h. Coverslips were mounted on microscope slides and observed using a laser-scanning confocal microscope (Leica Corp., Deerfield, IL).


    ACKNOWLEDGMENTS
 
We thank Ms. Norma Foster for help in preparing the manuscript and Miss Chisa Shinotsuka and Miss Naoko Fukushima for technical assistance.


    FOOTNOTES
 
This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (11460134).

Abbreviations: FAP, Fas-associated phosphatase; FBS, fetal bovine serum; GAP, GTPase-activating protein; HA, hemagglutinin; LMW-PTP, low molecular weight PTP; MEG, megakaryocyte; moi, multiplicity of infection; PMSG, pregnant mare’s serum gonadotropin; PSTPIP, Pro, Ser, Thr phosphatase interacting protein; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; p-Tyr, phosphotyrosine; SHP, Src homology 2-containing protein tyrosine phosphatase.

Received for publication May 22, 2002. Accepted for publication December 26, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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