Molecular Human Reproduction, Vol. 8, No. 6, 552-558,
June 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases, Axl and Sky, in human uterine endometrium and ovarian endometriosis
Department of Obstetrics and Gynecology, Gifu University School of Medicine, Tsukasamachi-40, Gifu City, 500-8705, Japan
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We demonstrated the expression of Gas6, the protein product of the growth arrest-specific gene 6 (gas6) and a member of the vitamin K-dependent protein family, and its receptor tyrosine kinases, Axl and Sky, in human uterine and ovarian endometriotic endometria using RT–PCR–Southern blot analysis and immunohistochemistry. Gas6, Axl and Sky mRNA were detected in all samples analysed. There was no significant difference between the levels of Sky mRNA in normal uterine and endometriotic endometria; however, the levels of Gas6 and Axl mRNA in endometriotic endometria were significantly higher than in normal endometria. These mRNA levels showed no significant alteration during the menstrual cycle. In the immunohistochemical study, Gas6, Axl and Sky were found in endometrial glandular cells and stromal cells in all samples analysed. This study demonstrates the coexpression of receptor tyrosine kinases and their ligand, Gas6, in normal uterine and ovarian endometriotic endometria, and the overexpression of Axl and Gas6 in endometriotic endometria. It is suggested that Gas6 and Axl signal transduction is aberrantly stimulated in endometriotic endometria, and is plausibly related to its growth potential.
growth arrest-specific gene 6/ovarian endometriosis/receptor tyrosine kinase/uterine endometrium
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Introduction |
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Gas6, the protein product of growth arrest-specific gene 6 (gas6), is a member of the vitamin K-dependent protein family. Gas6 has 46% amino acid identity to protein S, a serum protein which negatively regulates blood coagulation (Manfioletti et al., 1993
). Similar to protein S, Gas6 is composed of defined structural motifs: a 
-carboxylated amino-terminus (Gla domain), four tandem repeats of epidermal growth factor (EGF)-like domains, and a large carboxy-terminal domain with similarity to the sex hormone-binding globulin (SHBG). Gas6 lacks the consensus domain recognized by thrombin, which is involved in the regulation of the biological activity of protein S (Lundwall et al., 1986; Manfioletti et al., 1993).
Gas6 has been identified as a ligand for the Axl/Sky family of receptor tyrosine kinases including Axl (also called Ufo, Ark and Tyro7), Sky (Rse, Brt, Tif, Dtk, Etk-2 and Tyro3) and Mer (c-Eyk, Nyk and Tyro12) (Godowski et al., 1995; Varnum et al., 1995; Chen et al., 1997). Gas6 binds these receptors with its binding affinities in the order of Axl > Sky > Mer (Nagata et al., 1996), and thereafter induces receptor phosphorylation. These receptors are typified by the cell adhesion molecule-related extracellular ligand-binding domain, composed of two immunoglobulin-like motifs and two fibronectin type III motifs.
Gas6 acts as a growth-potentiating factor for thrombin-induced proliferation of vascular smooth muscle cells (Nakano et al., 1996, 1997). Gas6 is also able to induce cell cycle re-entry and protect serum-starved NIH3T3 cells from cell death by apoptosis (Goruppi et al., 1996, 1999). Recently, Gas6 has been shown to induce ß-catenin stabilization and T-cell factor transcriptional activation in mammary cells (Goruppi et al., 2001). Moreover, in the mouse, Axl, Sky and Mer are all normally expressed in Sertoli cells, whereas Gas6 is produced by Leydig cells before sexual maturity, and by both Leydig and Sertoli cells thereafter, suggesting a relationship with the reproductive system (Lu et al., 1999).
To fulfil the function of implantation and development of the fertilized ovum, human uterine endometrium undergoes considerable morphological changes, involving the proliferation and differentiation of both epithelial and stromal cells during the menstrual cycle (Johannisson et al., 1982). Various growth factors are involved in its proliferation. Human endometrial stromal cell growth and differentiation are regulated by EGF, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) (Irwin et al., 1991). Expression of human endometrial EGF, transforming growth factor 
and EGF receptor vary with the stage of the menstrual cycle (Imai et al., 1995). Endometriosis is the presence of functioning endometrial glands and stroma outside the uterine cavity. In contrast with eutopic endometrium, morphological changes during the secretory phase are absent in ectopic endometrium. Different mechanisms of proliferation control have been noted between eutopic and ectopic endometria (Nisolle et al., 1997).
The aim of the present study was to test for the presence of Gas6 and its receptors, Axl and Sky, in human uterine and ovarian endometriotic endometria, to evaluate their plausible distinct proliferation mechanisms.
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Materials and methods |
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Patients
The specimens of uterine endometria and endometriotic tissues of the inner lining of the endometriomas were obtained by hysterectomy from 36 patients (aged 35–46 years; 11 proliferative phase and 25 secretory phase of the menstrual cycle) with a regular menstrual cycle and who had uterine leiomyoma or cervical cancer at an early stage and from 32 patients (aged 30–42 years; 13 proliferative phase and 19 secretory phase of the menstrual cycle) with ovarian endometriosis, respectively, at the Department of Obstetrics and Gynecology, Gifu University School of Medicine from October 1998 to September 2001. The patients had not received any previous hormone therapy or chemotherapy. All subjects gave prior, written, informed consent, and this study was approved by the Research Committee of Gifu University School of Medicine and its Institutional Review Board. A part of each specimen was submitted for histological diagnosis and endometrial dating (Noyes et al., 1950
), and the remainder was immediately frozen in liquid nitrogen and later prepared for the subsequent experiments. Endometrial dating of the eutopic endometria in each case of ovarian endometriosis was performed on endometrial biopsy samples taken before surgery.
Preparation of internal standard recombinant RNA (rRNA)
Following the procedures described in our previous study (Misao et al., 1998), the synthesis of internal standard rRNA was performed. DNA construction of the internal standard was synthesized by PCR from the BamH/EcoRI fragment of v-erbB (Clontech Laboratories, Palo Alto, CA, USA) with sets of two oligonucleotide primers containing gas6, Axl or Sky specific primer sequences and the T7 promoter (Janssen et al., 1991; Manfioletti et al., 1993; Vanden Heuvel et al., 1993; Ohashi et al., 1994).
Competitive RT–PCR
Total RNA was isolated from the cells by the acid guanidinium thiocyanate–phenol–chloroform extraction method (Chomczynski and Sacchi, 1987). Its concentration was determined by UV absorption at 260 and 280 nm. To obtain a standard curve each time, a series of diluted recombinant RNA (1 to 103 fmol) and the total RNA (3 µg), were reverse-transcribed with Moloney murine leukaemia virus reverse transcriptase (MMLV-RT, 200 IU; Gibco BRL, Gaithersburg, MD, USA) in 50 mmol/l Tris–HCl, pH 8.3, 75 mmol/l KCl, 3 mmol/l MgCl2, 40 IU of RNAsin (Toyobo, Osaka, Japan), 10 mmol/l dithiothreitol, 0.5 mmol/l deoxyribonucleoside triphosphates (dNTP), and 30 pmol 3' end specific primers (gas6-3', Axl-3' and Sky-3') at 37°C for 1 h. The reaction was incubated for 5 min at 95°C to inactivate MMLV-RT.
The sequences of primers used to amplify the gas6, Axl and Sky genes were as follows: 5'-TGCTGTCATGAAAATCGCGG-3' (gas6-5'; 1328–1347), 5'-CATGTAGTCCAGGCTGTAGA-3' (gas6-3'; 1594–1613), 5'-GGTGGCTGTGAAGACGATGA-3' (Axl-5'; 1820–1839), 5'-CTCAGATACTCCATGCCACT-3' (Axl-3'; 2103–2122), 5'-CACTGAGCTGGCTGACTAAGCCCC-3' (Sky-5'; 2719–2742) and 5'-AATGCATGCACTTAAGCAGCAGGG-3' (Sky-3'; 3039–3062), all synthesized by Rikaken Co. Ltd (Nagoya, Japan). PCR with reverse-transcribed RNAs as templates (1 µl) and 5 pmol of each specific primer was carried out using a DNA Thermal Cycler (Perkin–Elmer Cetus, Norwalk, CT, USA) with 0.5 IU of Amplitaq DNA polymerase (Perkin–Elmer Cetus) in a buffer containing 50 mmol/l KCl, 10 mmol/l Tris–HCl, pH 8.3, 1.5 mmol/l MgCl2, and 0.2 mmol/l dNTP. Amplification was performed for 38 cycles at 94°C for 45 s for denaturing, 55°C for 45 s for annealing, and 72°C for 90 s for extension. To assess the quality of the RNA samples, all samples were confirmed by RT–PCR analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression using specific primers (GAPDH-5': 71–96, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; GAPDH-3': 1030–1053, 5'-CATGTGGGCCATGAGGTCCACCAC-3').
Southern blot analysis
Amplified PCR products were applied to a 1.2% agarose gel for electrophoresis performed at 100 V, and capillary-transferred to a nylon membrane (Immobilon-S; Millipore, Burlington, MA, USA) for 20 h using 10xstandard sodium citrate solution (SSC; 1.5 mol/l NaCl, 0.15 mol/l sodium citrate, pH 7.0). After blotting, the membrane was dried at 75°C and then cross-linked by UV irradiation (33 000 µJ/cm2 at 254 nm). The membrane was prehybridized in hybridization buffer (1 mol/l NaCl, 50 mmol/l Tris–HCl, pH 7.6, 1% sodium dodecyl sulphate) at 42°C for 2 h, and then in the same solution with the biotinylated gas6, Axl or Sky gene-specific oligonucleotide probe (gas6 probe: 5'-TCGTCTGGATGGCTGCATG-3'; Axl probe: 5'-TGTCTGTTTCCAGGGTTCTG-3'; Sky probe: 5'-TTGATGGAAGTGGGCCAGTC-3') and a biotinylated internal standard gene-specific oligonucleotide probe (5'-TGTTATACAGGGAGTGAAA-3') specific genes, or hybridized with biotinylated gas6-, Axl- or Sky-5' (10 pmol/µl; Ritaken Co. Ltd) to detect their precise intensities at 42°C for 24 h (Figure 1). The membrane was washed with 2xSSC for 15 min at room temperature, then with 2xSSC for 15 min at 42°C, and finally with 0.5xSSC for 15 min at 42°C. The detection reaction for hybridized biotin was performed using a Plex Chemiluminescent Kit (New England BioLabs, Beverly, MA, USA). Kodak XAR-5 film (Eastman Kodak, Rochester, NY, USA) was exposed to the membrane for 15 min. The strength of the recorded signal on film was analysed densitometrically, using Bio Image (Millipore).
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Immunohistochemical staining
Immunohistochemical staining was conducted using formalin-fixed, paraffin-embedded sections of tissues by the avidin–biotin–peroxidase complex (ABC) method (Hsu et al., 1981
). Sections (4 µm) of formalin-fixed, paraffin-embedded tissues were cut with microtome and dried overnight at 37°C on a silanized-slide (Dako, Carpinteria, CA, USA). Samples were deparaffinized in xylene at room temperature for 80 min and washed with a graded ethanol/water mixture and then with distilled water. The samples were soaked in a citrate buffer and then microwaved at 100°C for 10 min (Shi et al., 1991). Before addition of the primary antibodies, endogenous peroxidase activity was blocked by incubation in methanol containing 1% H2O2 for 20 min, followed by 60 min incubation with normal donkey serum to reduce background staining. The primary antibodies, goat anti-human Gas6, Axl and Sky antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were then incubated at 4°C for 8 h, followed by incubation with the biotinylated secondary antibodies (donkey anti-goat IgG, Santa Cruz Biotechnology) for 30 min and ABC complex for 30 min. The primary and secondary antibodies were used at 1:80 and 1:100 dilutions respectively. The peroxidase binding sites were demonstrated by the diaminobenzidine method. A phosphate-buffered solution instead of the primary antibody was used in the protocols for negative controls. The results of immunohistochemical staining for Gas6, Axl and Sky were semiquantitatively evaluated as previously described (McCarty et al., 1985). Each stained section was given a histochemical score (histoscore) calculated by the formula: 
(i+1)xPi, in which i = nuclear staining intensity (range; 1–4; 0 indicates no staining) and Pi = percentage of stained nuclei of endometrial epithelial and stromal cells (McCarty et al., 1985).
Statistics
Statistical analysis was performed with Student's t-test for two comparisons. Correlation coefficients were determined by Pearson's correlation coefficient. Differences were considered statistically significant at P < 0.05. Data were expressed as mean ± SD.
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In the competitive RT–PCR–Southern blot analysis for Gas6, Axl or Sky mRNA, two predicted PCR products were detected without non-specific products in all samples analysed. The level of Gas6, Axl or Sky mRNA was determined using a standard curve of a serial dilution of rcRNA by competitive RT–PCR–Southern blot analysis, as detailed in our previous study (Misao et al., 1998
) (Figure 2). We performed total RNA isolation and competitive RT–PCR–Southern blot analysis in three different parts of each individual sample. Gas6, Axl and Sky mRNA levels in normal endometria and in ovarian endometriotic endometria showed no significant alteration (P > 0.05) during the menstrual cycle (normal endometria; r = 0.115, 0.036, ovarian endometriotic endometria; r = 0.011, 0.022, 0.009 and 0.007 respectively). While there was no significant difference between the levels of Sky mRNA in normal uterine endometria (2.7 ± 1.2 fmol/µg total RNA) and ovarian endometriotic endometria (2.4 ± 0.6 fmol/µg total RNA), the levels of Gas6 and Axl mRNA in the endometriotic endometria (33.4 ± 12.4 and 33.8 ± 14.8 fmol/µg total RNA respectively) were significantly higher (both P < 0.01) than those in the normal endometria (16.6 ± 8.5 and 10.7 ± 3.4 fmol/µg total RNA respectively) (Figure 3). The level of Sky mRNA was 10-fold lower than the levels of Gas6 and Axl mRNA in both tissues.
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In the immunohistochemical study, positive staining of Gas6, Axl and Sky was found dominantly in endometrial glandular cells and in some stromal cells in all normal uterine and ovarian endometriotic endometria analysed (Figure 4
). The histoscores of immunohistochemical staining for Gas6 and Axl in ovarian endometriotic endometria (275.34 ± 61.68 and 361.72 ± 72.20 respectively) were significantly higher (P < 0.05) than those in the normal endometria (212.78 ± 53.91 and 279.56 ± 61.65 respectively) (Figure 5), while there was no significant difference between Sky histoscores in normal endometria (149.00 ± 46.95) and in endometriotic endometria (153.63 ± 45.96).
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Discussion |
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The biological activities of Gas6, namely the potentiation of cell proliferation and prevention of cell death, are very likely mediated by receptor tyrosine kinases. The C-terminal D domain, the SHBG-like domain, is necessary for receptor activation since chimeric proteins which contain this domain are able to induce tyrosine phosphorylation, while proteins which lack this region fail to do so (Mark et al., 1996
). SHBG binds sex steroid hormones such as dihydrotestosterone, testosterone and 17ß-estradiol with high affinity (Mercier-Bodard et al., 1970). Serum SHBG level increases after the pre-ovulatory peak of estradiol in the normal menstrual cycle (Plymate et al., 1985), and endometrial SHBG mRNA synthesis is regulated by estrogen and progesterone (Misao et al., 1994). Therefore, whether Gas6 with its SHBG-like domain binds to steroid hormones and whether Gas6 synthesis is hormone-dependent or not are intriguing questions. However, Gas6 mRNA levels in normal endometria showed no significant alteration during the menstrual cycle in the present study. Therefore, Gas6 might maintain continuous basic proliferation activity during the menstrual cycle in normal endometria.
Recently, an alternative-spliced Gas6 variant has been characterized as containing an additional 43 amino acids between the fourth EGF-like repeat and the SHBG-like domain. Expression of this Gas6 splicing variant has been investigated in various human tissues and found to have a distribution pattern similar to that of wild-type Gas6 (Goruppi et al., 1997; Marcandalli et al., 1997). Gas6 may, therefore, exist in both intact and cleaved forms, which may possess distinct properties.
Gas6 is widely expressed in many tissues and quite abundantly in the lung, intestine and endothelium, while it is almost undetectable in the liver, where protein S can be detected at high levels (Marcandalli et al., 1997; Prieto et al., 1999). Axl and Mer are widely expressed, but the highest levels of Axl mRNA are found in the ovarian follicles, haematopoietic system, skeletal muscle, heart and testis, and the highest levels of Mer mRNA are found in ovary, prostate, lung and kidney (Graham et al., 1994; Mark et al., 1994; Neubauer et al., 1994). While Sky is expressed preferentially in the central nervous system, it is expressed at lower levels in a number of tissues including the kidney, ovary, testis and in a variety of haematopoietic cell lines (Lai et al., 1994; Mark et al., 1994). These different patterns of expression suggest the presence of a distinct role for each receptor. In this study, these ligand and receptor mRNA and proteins were detected in normal uterine and ovarian endometriotic endometria. Gas6 seems to act on receptor tyrosine kinase signal transduction in these tissues in an autocrine fashion.
The dissociation constants of the binding of Gas6 to Axl, Sky and Mer are 0.4, 2.7 and 29 nmol/l respectively (Nagata et al., 1996). Mer may have an original ligand other than Gas6. In the present study, the level of Sky mRNA was 10-fold lower than Axl mRNA in both tissues. Moreover, the intensity of Sky staining in the immunohistochemical study was weaker than that of Axl in all samples analysed. Taking into account the dissociation constants, the effect of Gas6 on normal uterine and ovarian endometriotic endometria appears to be exerted mainly via Axl.
Since Axl/Sky family receptors have cell-transforming activity through overexpression, they are thought to be involved in tumour progression and in normal cell proliferation, but little is known of the physiological functions of these receptors. Axl and Sky are not expressed in primary colon cancer, but are expressed in normal colon mucosa and hepatic metastasis (Chen et al., 1999). Axl is not expressed in normal thyroid tissues, but is faintly expressed in human adenomatous goitre and adenomas, and its expression is detected in ~90% of human thyroid carcinomas (Ito et al., 1999). Axl gene expression is observed in cutaneous malignant melanoma (Bittner et al., 2000). The biological significance of the coexpression of Gas6 and receptor tyrosine kinases in normal uterine and ovarian endometriotic endometria still remains to be clarified, but might be related to the proliferation of uterine endometrium and the development and growth of ovarian endometriosis. Previous studies comparing eutopic and ectopic endometria have shown different mechanisms of proliferation control (Schweppe et al., 1984; Vierikko et al., 1985; Nisolle et al., 1997). In the current study, the levels of Gas6 and Axl mRNA in endometriotic endometria were significantly higher than those in normal endometria. This evidence is plausibly related to aberrantly stimulated growth of ovarian endometriosis.
In conclusion, the present study is the first to demonstrate the coexpression of Gas6 and receptor tyrosine kinases, Axl and Sky, and the high expression of Gas6 and Axl, in ovarian endometriosis. Signal transduction pathways of Gas6 and Axl may possibly be involved in the development and growth of ovarian endometriosis.
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The authors wish to thank Mr John Cole for proofreading the English of this manuscript.
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1 To whom correspondence should be addressed. E-mail: d2105006{at}guedu.cc.gifu-u.ac.jp
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Submitted on July 17, 2001; resubmitted on November 5, 2001; accepted on February 21, 2002.
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  M. Gallicchio, S. Mitola, D. Valdembri, R. Fantozzi, B. Varnum, G. C. Avanzi, and F. Bussolino Inhibition of vascular endothelial growth factor receptor 2-mediated endothelial cell activation by Axl tyrosine kinase receptor Blood, March 1, 2005; 105(5): 1970 - 1976. [Abstract] [Full Text] [PDF]
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 W. S. Sun, J. Fujimoto, and T. Tamaya Coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine endometrial cancers Ann. Onc., June 1, 2003; 14(6): 898 - 906. [Abstract] [Full Text] [PDF]
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