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Endocrinology Vol. 144, No. 3 793-801
Copyright © 2003 by The Endocrine Society

ARTICLE

Follicle-Stimulating Hormone and Insulin-Like Growth Factor I Synergistically Induce Up-Regulation of Cartilage Link Protein (Crtl1) via Activation of Phosphatidylinositol-Dependent Kinase/Akt in Rat Granulosa Cells

Guang Wei Sun, Hiroshi Kobayashi, Mika Suzuki, Naohiro Kanayama and Toshihiko Terao

Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan

Address all correspondence and requests for reprints to: Dr. Hiroshi Kobayashi, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu 431-31, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FSH and IGF-I are both important determinants of follicle development and the process of cumulus cell-oocyte complex expansion. FSH stimulates the phosphorylation of Akt by mechanisms involving phosphatidylinositol 3-kinase (PI3-K), a pattern of response mimicking that of IGF-I. Cartilage link protein (Crtl1) is confined to the cartilaginous lineage and is assembled into a macroaggregate complex essential for hyaluronan-rich matrix stabilization. The present studies were performed to determine the actions of FSH and IGF-I on Crtl1 production in rat granulosa cells. Primary cultures of granulosa cells were prepared from 24-d-old rats. After treatments, cell extracts and media were prepared, and the Crtl1 level was determined by immunoblotting analysis using anti-Crtl1 antibodies. Here we showed that 1) treatment with FSH (>=25 ng/ml) or IGF-I (>=25 ng/ml) for 4 h increased Crtl1 production; 2) maximal stimulatory effects of FSH or IGF-I were observed at 100 or 50 ng/ml, respectively; 3) FSH caused a concentration-dependent increase in IGF-I-induced Crtl1 production and vice versa; 4) FSH and IGF-I also up-regulate the expression of Crtl1 mRNA; 5) FSH- and IGF-I-dependent Crtl1 production were abrogated by PI3-K inhibitors (LY294002 and wortmannin), and inhibition of Crtl1 production by p38 mitogen-activated protein kinase inhibitor (SB202190) was partial (~30%), suggesting that PI3-K and, to a lesser extent, p38 mitogen-activated protein kinase are critical for the response. Our study represents the first report that FSH amplifies IGF-I-mediated Crtl1 production, possibly via PI3-K-Akt signaling cascades in rat granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICULAR DEVELOPMENT is dependent on both intraovarian growth regulatory factors, such as IGF-I and estrogen, as well as the pituitary gonadotropins, FSH and LH (1). The mammalian cumulus cells form hyaluronan (HA)-rich matrix on their cell surface during the process of follicle development before ovulation (2). The accumulation of extracellular matrix (ECM) on cumulus cells during the process of ovulation is a highly organized process resulting from the deposition of HA upon the cumulus cell membranes (3). Such a degree of structural organization may result from control by matrix components of cumulus cells under stimulation by gonadotropins (3).

The ECM may comprise proteins, glycoproteins, and proteoglycans (4). Three of these have previously been identified: inter-{alpha}-trypsin inhibitor (I{alpha}I) (5, 6), TNF-stimulated gene 6 (TSG-6) (7, 8, 9), and cartilage link protein (Crtl1) (10, 11), all of which belong to the HA-binding proteins. There are at least seven members of the HA-binding family, which contains aggrecan, versican, neurocan, brevican, Crtl1, CD44, and TSG-6. The G1 domain of aggrecan contains three protein motifs: an Ig fold and two copies of an HA-binding motif, or link module [also referred to as the proteoglycan tandem repeat (PTR)]. The link module is present in tandem in all members of the HA-binding family of proteoglycans and in Crtl1, but it is also present as a single copy in the cell surface HA-binding receptor CD44 and in TSG-6, a secreted matrix protein whose synthesis is induced by inflammatory cytokines. The primary structure of the mature Crtl1 is made up of the Ig fold domain and the PTR domain (12). These repetitive PTR elements are related to those found in the other well characterized members of the aggrecan family (12).

As follicles mature to the preovulatory stage, FSH induces the expression of cumulus expansion-specific genes encoding HA synthase (13) and TSG-6 (7, 8, 9). We had previously demonstrated that Western analyses with an anti-Crtl1 antibody detected a single molecular species of 42 kDa in the granulosa cell extracts and also in follicular fluid (10). Immunocytochemistry with anti-Crtl1 antibody showed localization of Crtl1 exclusively to the ECM and cytoplasm of cumulus cells of the rat (10) and mouse (11) ovary, indicating that Crtl1 is identified as a protein synthesized in the granulosa cells where cumulus cell-oocyte complex (COC) expansion occurs (10, 11). The 42-kDa species was almost the same as or equal in size to a recently reported Crtl1 in chondrosarcoma cells (10). These results allow us to hypothesize that Crtl1 is expressed by granulosa cells and stimulated with gonadotropins; the synthesis of Crtl1 by granulosa cells suggests possible functions as stabilizing HA into the ECM on the cells and that its increased expression just before ovulation supports an autocrine or paracrine role for granulosa cell-derived Crtl1 during follicle development and subsequent ovulation. However, the immunocytochemistry in previous studies (11) is not completely convincing. Crtl1 appears to be made everywhere in the ovary, not just in cumulus cells, and the background appears high. More recently we have studied the regulation and distribution of Crtl1 mRNA (the mRNA sequence is that for the cartilage form) in different cell types during follicle development by semiquantitative RT-PCR analysis (Sun, G. W., and H. Kobayashi, unpublished data). These ongoing experiments showed that granulosa cells, but not thecal-interstitial cells, in culture showed a significant increase in accumulation of Crtl1 mRNA due to treatment with gonadotropin. These results support Crtl1 as one of the ECM glycoproteins that may function as a stabilizer maintaining HA-rich matrexes in cartilage (14) and ovary (10, 11).

IGF-I has been implicated in the chondrocyte differentiation process (15). During differentiation, chondrocytes secrete ECM components characteristic of cartilage, such as type II collagen, HA, aggrecan, and Crtl1, providing an environment that maintains the chondrocyte phenotype (16). The actions of IGF-I on target cells are mediated by the type I receptor, which causes activation of PI3-K, leading to activation of serine-threonine kinase, Akt kinase, or protein kinase B (PKB) (17, 18), a downstream target of PI3-K. Recent publication demonstrated that PKB (Akt) is constitutively expressed, but rapidly phosphorylated, in granulosa cells by exposure to FSH or IGF-I (19). IGF-I via PI3-K phosphorylates PKB in granulosa cells (20, 21). FSH via PI3-K and its downstream target, PDK1, phosphorylates PKB in a manner that mimics and enhances IGF-I-induced phosphorylation and activation of PKB. IGF-I is also secreted by granulosa cells (22). The level of IGF-I in human follicular fluid is approximately 200 ng/ml (23). IGF-I may act in an autocrine mode to stimulate granulosa cell replication and promote granulosa cell differentiation and survival. It has been concluded that the PI3-K/Akt signaling serves as a functional pathway in the ovary (20, 21).

The effects of FSH on granulosa cell function may be enhanced at least in part by IGF-I (20, 21). It has been established that FSH appears to activate both A kinase- and PI3-K-dependent pathways in granulosa cells (19), that FSH impacts the IGF-I pathway via stimulation of the PI3-K cascade, leading to phosphorylation of PKB/Akt (19), that IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity (24), that ovarian IGF-I expression serves to enhance granulosa cell FSH responsiveness by augmenting FSH receptor expression (25), and that in the late stages of folliculogenesis the decrease in IGF-binding proteins (IGF-BPs) participates in the increase in IGF bioavailability, leading to a further amplification of FSH action (26).

The present study was carried out to investigate the effects of FSH and IGF-I on the up-regulation of Crtl1 production via Akt kinase signaling in rat granulosa cells and to determine whether FSH influences the effects of IGF-I on this signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ovine FSH (1 ng = ~1 mU), forskolin, 8-bromo-cAMP (8-Br-cAMP), and the PI3-K inhibitor LY294002 were purchased from Sigma-Aldrich (St. Louis, MO). Crtl1 was purified from bovine nasal cartilage. The generation of anti-Crtl1 antibodies was carried out as described previously (27). These antibodies are highly specific for Crtl1 at the dilution used in this work. Anti-phospho-Akt (no. 9271L) and anti-Akt (no. 9272) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Media and cell culture reagents and materials were purchased from Life Technologies, Inc. (Grand Island, NY), Sigma-Aldrich, and Corning, Inc. (Corning, NY). Wortmannin, protein kinase A (PKA) inhibitor H89, MAPK kinase-1 inhibitor PD98059, p38MAPK inhibitor SB202190, and Streptomyces hyaluronidase were obtained from Calbiochem (San Diego, CA). TRIzol reagent was obtained from Life Technologies, Inc. Electrophoresis and molecular biology grade reagents were purchased from Sigma-Aldrich and Bio-Rad Laboratories, Inc. (Richmond, CA).

Animals
Female Sprague Dawley rats (21 d old) were purchased from SLC Laboratories (Hamamatsu, Japan) and maintained under standard conditions. The animal protocols were approved by the Hamamatsu University animal care and use committee.

Cell culture
Intact immature (21 d of age) rats were primed with estradiol (1.5 mg/0.2 ml propylene glycol) for 3 d. Granulosa cells were harvested by needle puncture, pooled, and plated according to routine procedures (19). All incubations and cell cultures of granulosa cells were performed at a density of 1 x 106 cells in 3 ml serum-free medium (DMEM/Ham’s F-12 containing 100 U/ml penicillin and 100 µg/ml streptomycin) in multiwell (35-mm) dishes that were serum coated and were maintained at 37 C in a humidified atmosphere of 5% CO2. Only primary cultures were used for this study. Rat granulosa cells were cultured for 16 h in serum-free medium, washed, and left untreated or were treated with FSH, IGF-I, a combination of both, and other inhibitors as indicated in the figure legends. In some experiments cells were pretreated with vehicle or inhibitors for 30 min (H89) or 1 h (other inhibitors) before the addition of FSH and/or IGF-I.

High levels of HA synthesized by granulosa cells were organized into an ECM in the presence of serum (28). Maximum retention of HA in the cumulus matrix, and hence complete COC expansion, occur when 1% fetal bovine serum is continuously present during the first 18 h of culture. Regardless of the culture time, HA synthesized when serum was absent was primarily in the medium, whereas HA synthesized when serum was present was primarily in the cell matrix. When granulosa cells were cultured in the absence of serum, Crtl1 was considered not to be organized in the cell ECM, but to be released into the medium. Therefore, media and cell lysates were separately prepared and analyzed for Crtl1 level.

Cell extracts
Cells were rinsed in PBS and then homogenized in 8 M urea/50 mM sodium acetate at pH 5.8, supplemented with 50 U/ml Streptomyces hyaluronidase, 0.1% Triton X-100, 0.2 mM 4-(2-aminoethyl)-benzenesulfonylfluoride HCl (Calbiochem), 1 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin (Roche Molecular Biochemicals, Indianapolis, IN), 1 mM benzamidine, and 1 mM phenylmethylsulfonylfluoride (Sigma-Aldrich) as protease inhibitors (29). Protein was isolated from granulosa cells by homogenization in extraction buffer, followed by centrifugation (1 min in a microfuge) to isolate soluble protein. One milliliter of each cell medium incubated with 50 U/ml Streptomyces hyaluronidase (to obtain free Crtl1) was desalted on a PD-10 column (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) and concentrated. Crtl1 is insensitive to hyaluronidase (10). The amount of protein in each fraction was quantified in a Bradford assay (Bio-Rad Laboratories, Inc.) using BSA as a standard.

Total cell extracts (for Akt) were prepared according to the method described by Ginty et al. (30) by adding to each well hot (100 C) Tris buffer containing 10% sodium dodecyl sulfate and ß-mercaptoethanol.

Immunoblot analysis for Crtl1 and phosphorylation of Akt
Cell extract and medium samples (50–100 µg total cell protein or 15 µl of 10x concentrated medium), solubilized in sodium dodecyl sulfate sample buffer and boiled for 5 min, were electrophoresed on a discontinuous 12% acrylamide gel under nonreducing and reducing conditions. The proteins were transferred onto a polyvinylidene difluoride (Bio-Rad Laboratories, Inc.) membrane. The membranes were blocked in PBS with 2% BSA for 30 min and then probed with anti-Crtl1 antibody (diluted 1:5,000) for 2 h. In a parallel experiment the blots were incubated with specific antibodies that cross-react with total proteins or phosphorylated forms of Akt (1:1,500) protein for 2 h at room temperature. After washing, the blots were incubated with appropriate antirabbit IgG coupled to horseradish peroxidase (dilution 1:10,000) for 1 h. The blots were then developed in an enhanced chemiluminescence detection system (Amersham Japan, Tokyo, Japan) for 30 sec, exposed to Polaroid film (Kodak, Tokyo, Japan), and visualized. The relative intensities of specific protein bands were determined by densitometric scanning of images using a Power Macintosh 7600/200-assissted FAS-II and Electronic UV transilluminator (Toyobo Co. Ltd., Tokyo, Japan). The ratios of intensities of phosphorylated proteins and total proteins were calculated to determine the extent of activation of specific kinases.

RNA extraction and Northern blot analysis
Total RNA from various treatments was extracted using TRIzol reagent, and 30 µg of each were subjected to electrophoresis on a 1% (wt/vol) agarose/formaldehyde gel and transferred to GeneScreen membranes. The probe for rat Crtl1 is the EcoRI insert of 820 bp (31). The cDNA probe for human glyceraldehyde-3-phosphate dehydrogenase was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). Northern blot analyses were performed as previously described (32).

Statistical analysis
All experiments were performed at least twice using different cell preparations. The data are presented for representative experiments. Differences between groups were analyzed for statistical significance using ANOVA (StatView 5.0 software, Abacus Concepts, Inc., Berkley, CA). P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Crtl1 production induced by FSH or IGF-I in rat granulosa cells
Rat granulosa cells were cultured for 16 h in serum-free medium, washed, and left untreated or treated with FSH, IGF-I, or a combination of both. Cells and media were separately collected, and aliquots of cell extracts and media were analyzed by SDS-PAGE under nonreducing and reducing conditions, followed by immunoblotting. The levels of Crtl1 expression in cell extracts and media were quantified densitometrically and analyzed statistically. Figure 1AGo shows that treatment of rat granulosa cells with IGF-I (6.3–100 ng/ml) for 8 h markedly up-regulated Crtl1 production, as evidenced by immunoblotting. We detected immunoreactive Crtl1 corresponding to a 42-kDa form of cartilage link protein in cell extracts and media under nonreducing and reducing conditions. A signal for the 48-kDa band was detected in media. The effect of IGF-I on Crtl1 up-regulation was dose dependent over the dose range of 12.5–50 ng/ml. Compared with controls, 50 ng/ml IGF-I caused a significant (3.6-fold; P < 0.05; n = 3) stimulation of Crtl1 production (Fig. 1AGo). Figure 1BGo shows a time course of up-regulation of Crtl1 in cell extracts and media; the effect was evident from 4 h and peaked at 8 h. No additional Crtl1 was accumulated between 8 and 12 h. Therefore, immunoblotting experiments revealed that IGF-I could up-regulate Crtl1 production in a time- and dose-dependent manner.



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  		Figure 1. FSH or IGF-I induces up-regulation of Crtl1 production in rat granulosa cells in a dose- and time-dependent manner. Serum-starved rat granulosa cells were treated with IGF-I (A and B), FSH (C–E), forskolin (E), 8-Br-cAMP (E), or a combination of IGF-I and FSH (F). The cells were lysed by the addition of extraction buffer. Fifty micrograms (for IGF-I) and 100 µg total protein (for FSH) were subjected to 12% SDS-PAGE and analyzed with Western blot using anti-Crtl1 antibody under nonreducing (NR) and reducing (R) conditions. Crtl1 contents in the cell extracts and media were determined by immunoblotting and were analyzed densitometrically. Cells were treated with IGF-I from 6.3–100 ng/ml (A) or with FSH from 12.5–200 ng/ml (C) for 8 h, respectively. Cells were treated with IGF-I (50 ng/ml; B) or FSH (100 ng/ml; D) from 1–12 h as indicated. The 48-kDa (arrow) and 42-kDa (arrowhead) protein bands, respectively, were recognized in solubilized preparations. The control nonimmune rabbit IgG did not recognize these bands (data not shown). The data illustrated are representative of at least three independent experiments, and the mean density ± SD of the sums of these bands in cell extract plus medium samples in each lane are presented in the graph. Circles with different letters indicate that group means are significantly different at P < 0.05.

 
In a parallel experiment to check the effect of FSH on Crtl1 production, rat granulosa cells were treated with increasing concentrations of FSH (12.5–200 ng/ml; Fig. 1CGo). The cells and media were collected, and the levels of Crtl1 were separately quantified densitometrically and analyzed statistically. Thin, but specific, bands (48 and 42 kDa) were detected in the untreated cells. Compared with controls, FSH caused a significant (3.1-fold; P < 0.05; n = 3) stimulation of Crtl1 production when the cells were treated with FSH at a concentration of 100 ng/ml for an 8-h incubation (Fig. 1DGo). We repeated some experiments using forskolin (10 µM) and 8-Br-cAMP (1 mM), which mimic the action of FSH by bypassing FSH receptor and G proteins, to document the specificity of the FSH effect (Fig. 1EGo). Forskolin is a direct activator of adenylate cyclase, and 8-Br-cAMP is a stable analog of cAMP. As expected, forskolin and 8-Br-cAMP also significantly enhanced Crtl1 levels, indicating that the action of FSH was mimicked in vitro by forskolin and 8-Br-cAMP.

As shown in Fig. 1FGo, both FSH (100 ng/ml) and IGF-I (50 ng/ml) caused a significant stimulation (3.8- and 5.5-fold, respectively) of Crtl1 production (P < 0.05; n = 3) in rat granulosa cells, and the effects of the two agonists were synergistic (~18-fold; P < 0.05; n = 3).

Expression of Crtl1 mRNA by FSH or IGF-I
We next examined whether FSH or IGF-I can induce the expression of Crtl1 gene. Rat granulosa cells were first exposed over time to FSH or IGF-I. RNA was isolated and probed with Crtl1 cDNA. Figure 2Go shows the results from blots probed for Crtl1 mRNA. As expected, Crtl1 mRNA is increased by approximately 3- and 10-fold, respectively, after exposure to 100 ng/ml FSH or 50 ng/ml IGF-I within 4 h and persists for at least 12 h (Fig. 2AGo). No additional Crtl1 mRNA is accumulated between 4 and 12 h. This would be more consistent with the decrease in protein beyond the 8 h point (Fig. 1Go). Induction of Crtl1 mRNA by FSH or IGF-I was dose dependent, presenting at concentrations as low as 25 ng/ml FSH or 12.5 ng/ml IGF-I (Fig. 2BGo). The effects of the two agonists were synergistic (~23-fold; n = 4; Fig. 2CGo).



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  		Figure 2. FSH or IGF-I induces Crtl1 mRNA expression in a time- and concentration-dependent manner. Serum-starved rat granulosa cells were incubated with FSH, IGF-I, or a combination of both as described in Fig. 1Go. A, Cells were incubated for different periods of time (1, 4, or 12 h) in the presence of FSH (100 ng/ml) or IGF-I (50 ng/ml). B, Cells were incubated for 4 h in the presence of various concentrations of FSH or IGF-I. C, Cells were incubated for 4 h with a combination of both FSH and IGF-I. RNA was extracted with time and subjected to Northern analysis using Crtl1 cDNA. Crtl1 mRNA induction was apparent within 4 h. Crtl1 mRNA induction was apparent at 25 ng/ml FSH or 12.5 ng/ml IGF-I. Data are representative of three separate experiments. The PCR products are shown in the upper panel, and the ratios of PCR products (target/internal control) are graphed in the lower panel. Bars with different letters indicate that group means are significantly different at P < 0.05.

 
FSH and IGF-I induce Crtl1 production via phosphorylation of Akt in rat granulosa cells
First, we investigated the ability of FSH and IGF-I to induce phosphorylation of Akt in rat granulosa cells. The cell lysate from rat granulosa cells treated with FSH or IGF-I was subjected to immunoblotting with respective antibodies. In unstimulated cells, the expression of phosphorylated Akt protein was weak, whereas IGF-I significantly raised (5.7-fold at 50 ng/ml; P < 0.05; n = 3) the levels of the phosphorylated form of this protein (Fig. 3AGo). FSH also caused a stimulation of Akt phosphorylation (2.4-fold at 100 ng/ml; P < 0.05; n = 3; Fig. 3BGo). Like FSH, forskolin (10 µM) and 8-Br-cAMP (1 mM) also enhanced phosphorylation of Akt (Fig. 3BGo), indicating that the action of FSH was mimicked in vitro by forskolin and 8-Br-cAMP.



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  		Figure 3. Effects of IGF-I or FSH on phosphorylation of Akt protein. Serum-starved cells were left untreated or were treated with either IGF-I (A, C, and E) or FSH (B, D, and F). Further, cells were preincubated with or without LY294002 (2 and 10 µM), wortmannin (0.02 and 0.1 µM), PD98059 (10 µM), H89 (10 µM), or SB202190 (2 µM) and either left untreated or treated with IGF-I (50 ng/ml) or FSH (100 ng/ml) for 30 min. Cell lysates were immunoblotted using activation-specific polyclonal antibody to Akt (i.e. anti-phospho-Akt) or anti-total Akt antibody. No other protein bands except those shown were observed. Data are representative of three separate experiments. The ratios of phospho-Akt/total Akt are graphed in the lower panel. Bars with different letters indicate that group means are significantly different at P < 0.05.

 
The effects of FSH and IGF-I on phosphorylation of Akt were dependent on concentrations of FSH and IGF-I. Maximal induction of Akt phosphorylation was observed with 100 ng/ml FSH or 50 ng/ml IGF-I in three separate experiments. Exposure of the cells to IGF-I (Fig. 3CGo) or FSH (Fig. 3DGo) caused a time-dependent increase in phosphorylation of the Akt protein. We found that phosphorylation was most pronounced at 30 min after FSH and IGF-I stimulation and then returned to the uninduced state after 12 h (data not shown). The relationship between FSH/IGF-I stimulation and Akt phosphorylation was further determined by application of the specific PI3-K inhibitors (LY294002 and wortmannin; Fig. 3Go, E and F). Based on densitometric scanning, both PI3-K inhibitors dose-dependently inhibited FSH- and IGF-I-induced Akt phosphorylation (~40% at 2 µM and ~90% at 10 µM LY294002). Inhibitors such as H89 (10 µM), PD98059 (10 µM), and SB202190 (2 µM) inhibited PKA, MAPK, and p38MAPK activation, respectively. In a separate experiment, we confirmed that inhibitors of MAPK, PKA, and p38MAPK failed to inhibit IGF-I or FSH stimulation of phosphorylated Akt levels. FSH-dependent phosphorylation of Akt was not affected by preincubation with PD98059 and H89, whereas SB202190 inhibited FSH-mediated phosphorylation of Akt, by about 30% at 2 µM. Immunoblotting of cell extracts with antibodies against Akt indicated the presence of total immunoreactive proteins. FSH and IGF-I did not elevate total antigen levels of Akt.

Second, we investigated directly the role of Akt signaling cascades in mediating FSH- and IGF-I-induced up-regulation of Crtl1 production. In some experiments cultures were treated with test drugs. After treatments, media and cell lysates were separately prepared, and the expression of Crtl1 at mRNA and protein levels was determined by Western and Northern blot analyses, respectively. When cells were preincubated with LY294002 or wortmannin, IGF-I-induced up-regulation of Crtl1 secretion (Fig. 4AGo) was significantly reduced in media. FSH-induced up-regulation of Crtl1 secretion was affected by preincubation with these inhibitors without any detectable cytotoxicity (Fig. 4BGo). In addition, these PI3-K inhibitors selectively inhibited IGF-I- and FSH-induced Crtl1 expression in cell extracts (data not shown). Furthermore, FSH- or IGF-I-stimulated expression of Crtl1 mRNA was abrogated in cells pretreated with LY294002 or wortmannin (Fig. 4Go, C and D). SB202190 did block FSH-mediated Crtl1 up-regulation at the mRNA (Fig. 4DGo) and protein (Fig. 4BGo) levels by about 30%, whereas H89 and PD98059 had no effect on Crtl1 up-regulation. Unlike FSH, SB202190 (2 µM) did not block IGF-I-mediated Crtl1 expression (Fig. 4Go, C and D). Although it has been reported that IGF-I rapidly increased the phosphorylation of ERK1/2 (21), PD98059 did not block IGF-I-mediated phosphorylation of Akt (Fig. 3EGo) or up-regulation of Crtl1 mRNA (Fig. 4CGo) and protein (Fig. 4AGo). In addition, inhibition of PKA and MAPK by H89 (10 µM) and PD98059 (10 µM), respectively, did not reduce Akt phosphorylation by IGF-I (Fig. 3EGo) or Crtl1 mRNA and protein levels (Fig. 4Go, A and C). These results suggest that both FSH and IGF-I specifically stimulate Crtl1 expression via phosphorylation of Akt protein, that PI3-K is a possible candidate involved in FSH- and IGF-I-mediated expression of Crtl1 at the gene and protein levels, and that there is a partial involvement of the p38MAPK route in the effects of FSH on Crtl1 up-regulation in granulosa cells.



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  		Figure 4. Akt mediates FSH/IGF-I-induced up-regulation of Crtl1 production. Serum-starved rat granulosa cells were preincubated with or without the PI3K inhibitors LY294002 (10 µM) and wortmannin (0.1 µM), the MAPK inhibitor PD98059 (10 µM), the PKA inhibitor H89 (10 µM), or the p38MAPK inhibitor SB202190 (2 µM) and were either left untreated or treated with FSH (100 ng/ml) or IGF-I (50 ng/ml) for different periods of time [8 h for Western blotting (A and B) and 4 h for Northern blotting (C and D)]. A and B, Media were immunoblotted using anti-Crtl1 antibody as described in Fig. 1Go. The 48- and 42-kDa bands were observed. C and D, RNA was extracted and subjected to Northern analysis using a Crtl1 cDNA. Data are representative of three separate experiments. Bars with different letters indicate that group means are significantly different at P < 0.05.

 
Synergistic effects of FSH and IGF-I on Akt signaling and up-regulation of Crtl1 production
Figures 1FGo and 2CGo showed that FSH and IGF-I have been shown to act synergistically to regulate Crtl1 expression at the mRNA and protein levels. We examined more precisely whether FSH and IGF-I synergistically up-regulate Crtl1 production via Akt phosphorylation in rat granulosa cells. Cells were cultured overnight and then stimulated for 8 h (for protein expression), 4 h (for Crtl1 mRNA expression), and 30 min (for Akt phosphorylation) with IGF-I, FSH, or their combination. As shown in Fig. 5Go, low concentrations of either IGF-I (6.3 ng/ml) or FSH (12.5 ng/ml) alone did not cause significant accumulation of Crtl1 at the protein (Fig. 5AGo) and mRNA (Fig. 5BGo) levels, but their cotreatment produced significant expression (3.6-fold for protein level and 11-fold for mRNA level; P < 0.05; n = 4). Although Akt was not phosphorylated by either IGF-I (6.3 ng/ml) or FSH (12.5 ng/ml), and Akt phosphorylation was enhanced (8.9-fold) by costimulation with IGF-I and FSH (Fig. 5CGo), suggesting that FSH and IGF-I did phosphorylate Akt synergistically. Crtl1 production induced by these agonists was blocked by cotreatment of cells with the PI3-K inhibitor, LY294002. Further, FSH plus IGF-I-induced Crtl1 mRNA and protein up-regulation was blocked by approximately 30% by SB202190 (Fig. 5Go, A and B). Therefore, the p38MAPK route is also partially involved in Crtl1 up-regulation in response to FSH plus IGF-I. These results suggest that FSH and IGF-I synergistically activated the Akt signaling pathway, which results in marked expression of Crtl1 at the mRNA and protein levels, and that there is a partial involvement of the p38MAPK pathway in the synergistic action of FSH and IGF-I in granulosa cells.



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  		Figure 5. Synergistic effects of IGF-I and FSH on the activation of Akt and up-regulation of Crtl1 expression. The cells were treated with IGF-I (6.3 ng/ml) alone, FSH (12.5 ng/ml) alone, or IGF-I (6.3 ng/ml) plus FSH (12.5 ng/ml) for 8 h in the presence or absence of specific inhibitors. After incubation, cell extracts were prepared to determine Crtl1 expression at protein (A) and mRNA (B) levels. Crtl1 production was assayed and expressed as the relative increase compared with the level observed in control cells. C, After 30 min, the cells were lysed, and phosphorylated and total Akt were assayed and expressed as the fold increase compared with the level observed in control cells. Data are representative of two separate experiments. Bars with different letters indicate that group means are significantly different at P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apart from the characterization of Crtl1 in cartilage (12), the functional importance in the ovary has not yet been elucidated. In the present study we focused our attention on one of the COC expansion-specific proteins, Crtl1. We describe the regulated expression of the proteoglycan Crtl1 mRNA and protein in cultured rat granulosa cells in response to FSH and IGF-I. Here we show 1) that both FSH and IGF-I can increase Crtl1 mRNA and protein; 2) that they act synergistically, i.e. the combination of FSH and IGF-I appears to be more effective than either alone, suggesting that more than one pathway may be involved; and 3) that the effects of FSH and IGF-I are mediated primarily by activation of the PI3-K pathway, as inhibitors of PI3-K (wortmannin and LY294002) block the phosphorylation of Akt, a downstream target of PI3-K, and abrogate the expression of Crtl1. Inhibitors of the PKA and MAPK do not inhibit the induction of Crtl1 expression by FSH and IGF-I. The p38MAPK inhibitor SB202190 partially blocked FSH-mediated (but not IGF-I-mediated) Crtl1 expression, suggesting that FSH also operates partially through p38MAPK signaling pathway. Therefore, these studies establish a signal transduction cascade or cross-talk emanating from IGF-I and FSH to Crtl1. Our study represents the first report that IGF-I and FSH synergistically stimulate Crtl1 up-regulation at the mRNA and protein levels mainly through PI3-K-Akt-dependent activation mechanism.

Our results on the effects of FSH on IGF-I-induced Akt phosphorylation in rat granulosa cells confirm the findings of earlier studies (33), in which they reported that IGF-I rapidly and potently activates the PI3-K-Akt signaling pathway in rat Sertoli cells and that the endocrine factor FSH dramatically enhances IGF-I-dependent Akt phosphorylation. The inhibition of synergistic effects of FSH and IGF-I on Akt phosphorylation by the PI3-K inhibitor (LY294002) in rat Sertoli cells (33) and in rat granulosa cells (19) indicates that FSH enhances the activity of PI3-K. They also reported that as the stimulatory effect of FSH was completely blocked by IGF-BP3, it is logical to assume that the FSH effects on PI3-K are dependent on IGF-I signaling. Another possibility is that FSH may regulate the activity of small G proteins that could possibly alter the activity of p21ras, which, in turn, could up-regulate the catalytic activity of PI3-K (34, 35). However, this possibility may not be supported by the fact that the inhibitor of MAPK did not block Crtl1 production in rat ovary.

It has been established that granulosa cell stimulation by IGF-I in the presence of FSH activates several signaling pathways (36). FSH effects may involve increased secretion of endogenous IGF-I or inhibition of IGF-BPs (37). IGF-I showed positive interaction with FSH in granulosa cells on meiotic maturation and synergistically enhanced DNA synthesis, protein synthesis, and steroidogenesis (38). They reported that these synergistic effects are mainly caused by the increase in IGF-I receptors in granulosa cells caused by FSH (38). Alternatively, a recent study of IGF-I knockout mice demonstrated that IGF-I up-regulates FSH receptor gene expression. Therefore, we speculate that ovarian IGF-I/FSH may serve to up-regulate the expression of Crtl1 mRNA and protein, possibly through enhancement of granulosa cell IGF-I/FSH responsiveness by augmenting each receptor’s expression.

Some matrix proteins and proteoglycans have already been identified in the ECM of cumulus cells. Hyaluronan was the first component described in the matrix of the cumulus cells (2), which is also found in other cartilaginous tissues, such as bones and cartilage. The presence of I{alpha}I (5, 6), TSG-6 (7, 8, 9), and Crtl1 (10, 11) has been reported in cumulus cells during follicle development. Our previous data demonstrated that the specificity of Crtl1 as a cumulus matrix protein was established by Western blot, immunocytochemical data, and in vitro culture experiments (10, 11). It is likely that granulosa-lutein cells, but not thecal-interstitial cells, are involved in gonadotropin-stimulated expression of this matrix protein (10, 11). As gonadotropin could stimulate Crtl1 expression at the mRNA and protein levels, a functional role for Crtl1 in COC expansion is implied. Consistent with this idea, the present studies are enhanced by the provision of a further functional experiment showing that exogenous applied Crtl1 plays a role in the ovary, in COC expansion in particular. Our most recent publication (39) demonstrated that the addition of purified Crtl1 to the medium containing I{alpha}I and FSH resulted in significantly higher expansion levels than those observed in response to I{alpha}I alone, although Crtl1 alone had no or very little effect by itself. Therefore, in vitro Crtl1 might serve to enhance the COC expansion, possibly by stabilizing the HA-I{alpha}I (or heavy chains of I{alpha}I) complex on the surrounding cumulus cell matrexes, suggesting that Crtl1 is one of the possible candidates that may affect the process of COC expansion.

We showed that the Crtl1 species present in the cell lysates and media of cultured rat granulosa cells were 48 and 42 kDa. The 48-kDa species is found in media, whereas the 42-kDa species is found in cell lysates and media. This pattern is similar to that observed in human cartilage, in which it has been shown that there are three different molecular mass Crtl1 species (48, 44, and 41 kDa), that the 48- and 44-kDa molecules differ from one another in glycosylation in a domain close to the NH2 terminus of the protein, and the 41-kDa molecule differs from the 48- and 44-kDa molecules in that it lacks a peptide of 16 amino acids, which bears these two potential glycosylation sites, close to the NH2 terminus (12). It is therefore possible that the 48-kDa species is described as the fully glycosylated mature form of Crtl1 and that the 42-kDa species reported in this work is either a partially glycosylated form of Crtl1 (44 kDa) or a partially degraded fully glycosylated form of Crtl1 (41 kDa). It is likely that the fully glycosylated mature form of Crtl1 is immediately released into medium in the absence of serum.

In conclusion, the activation of PI3-K induced by IGF-I involves a relay of phosphorylation of Akt enhancing the FSH-dependent signaling pathway, and FSH and IGF-I synergistically stimulate the PI3-K-Akt signaling cascade, which results in up-regulation of Crtl1 production in rat granulosa cells. p38MAPK is also partially involved in the FSH-mediated signaling cascade. This is the first report that investigates the linkage between PI3-K-Akt signaling and the regulation of Crtl1 expression in response to FSH and IGF-I in primary rat granulosa cells. Dominant negative studies would be required to confirm that Akt mediates the effects of FSH and IGF-I. Further, we found in a separate experiment (39) that the addition of exogenous Crtl1 altered the morphology of the COC and enhanced expansion. We speculate that the Crtl1 expression induced by granulosa cells in response to FSH and IGF-I may enhance Crtl1-dependent, HA-rich matrix stabilization on cumulus cells and play an important functional role in COC expansion during follicle development. Additional experiments are needed to determine whether specific inhibitors, including PI3-K, A kinase, MAPK, and p38MAPK, block FSH/IGF-I-mediated COC expansion.


   Acknowledgments
 
The authors thank Drs. S. Miyauchi and M. Ikeda (Seikagaku Corp. Kogyo Co. Ltd., Tokyo, Japan); Drs. H. Morishita, K. Kato, and H. Sato (BioResearch Institute, Mochida Pharmaceutical Co., Tokyo, Japan); and Drs. Y. Tanaka and T. Kondo (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan) for their continuous and generous support of our work.


   Footnotes
 
This work was supported in part by a grant from the Yamanouchi Foundation for Research on Metabolic Disorders.

Abbreviations: 8-Br-cAMP, 8-Bromo-cAMP; COC, cumulus cell-oocyte complex; Crtl1, cartilage link protein; ECM, extracellular matrix; HA, hyaluronan; I{alpha}I, inter-{alpha}-trypsin inhibitor; IGF-BP, IGF-binding protein; PI3-K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; PTR, proteoglycan tandem repeat; TSG-6, TNF-stimulated gene 6.

Received August 27, 2002.

Accepted for publication November 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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