Molecular Human Reproduction, Vol. 9, No. 11, 701-707,
November 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Clinical implications of coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine leiomyoma
Submitted on May 8, 2003; accepted on July 9, 2003
Department of Obstetrics and Gynecology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City, 500-8705, Japan
1 To whom correspondence should be addressed. e-mail: jf{at}cc.gifu-u.ac.jp
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Abstract |
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The expression of Gas6, the protein product of the growth arrest-specific gene 6 (gas6), a member of the vitamin K-dependent protein family, and the receptor tyrosine kinases Axl and Sky and their mRNAs in uterine leiomyoma and normal uterine myometrium tissues were investigated by competitive RT–PCR–Southern blot analysis using recombinant RNA and immuno histochemical analysis respectively. There was no significant difference between the histoscores and levels of Sky mRNA in uterine leiomyoma and normal uterine myometrium, although the levels of Gas6 and Axl mRNAs in uterine leiomyoma were significantly higher than in normal uterine myometrium in each case. It is suggested that Gas6 and Axl signal transduction is aberrantly stimulated in uterine leiomyoma, possibly related to its growth.
Key words: growth arrest-specific gene 6/receptor tyrosine kinase Axl/uterine leiomyoma
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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 that negatively regulates blood coagulation (Manfioletti et al., 1993). Similar to protein S, Gas6 is composed of defined structural motifs: a
-carboxylated N-terminus (Gla domain), four tandem repeats of an epidermal growth factor (EGF)-like domain, and a large C-terminal domain with similarity to sex hormone-binding globulin. 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 was 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 to 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. Recent studies suggest a role for these receptors in developmental processes, in the function of the haematopoietic and nervous systems, and in tumorigenesis (Crosier and Crosier, 1997).
Gas6 acts as a growth-potentiating factor for thrombin-induced proliferation of vascular smooth muscle cells (Nakano et al., 1995, 1997). Additionally, Gas6 has been shown to be a novel chemoattractant that induces Axl-mediated migration of vascular smooth muscle cells (Fridell et al., 1998). Gas6 is also able to induce cell cycle re-entry and protect serum-starved NIH3T3 cells from apoptotic cell death (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). Gas6 is expressed in intestine, testicular somatic cells, pulmonary endothelium and uterine endometrium, and in uterine endometrial cancers (Prieto et al., 1999; Chan et al., 2000; Healy et al., 2001; Wimmel et al., 2001; Sun et al., 2002, 2003).
Axl is expressed in breast, skeletal muscle, heart, haematopoietic tissue, testis, ovarian follicles and uterine endometrium (Faust et al., 1992; Graham et al., 1994; Neubauer et al., 1994; Berclaz et al., 2001; Wimmel et al., 2001; Sun et al., 2002). Axl expression occurred in 
60% of non-small cell lung cancer cell lines and appears to be a consequence of cellular adhesion, and possibly influences differentiation in lung cancers (Wimmel et al, 2001). Axl gene expression is observed in cutaneous malignant melanoma (Quong et al., 1994). Axl was localized in the membrane of human breast cancer cells, and the number of cells expressing Axl was found to be higher in cancerous tissue than in normal breast (Berclaz et al., 2001). In our previous study, the levels of Gas6 and Axl mRNA in well-differentiated uterine endometrial cancers were significantly higher than those in normal uterine endometria. The coexpression of Gas6 and Axl, as a unique differentiation pathway, might be involved in the development and growth of well-differentiated uterine endometrial cancers (Sun et al., 2003).
While Sky is expressed preferentially in the central nervous system, it is expressed at lower levels in kidney, haematopoietic cells, testis and ovary (Fujimoto and Yamamoto, 1994; Graham et al., 1994; Lai et al., 1994; Neubauer et al., 1994; Ohashi et al., 1995; Prieto et al., 1999; Yamagita et al., 2001).
Uterine leiomyoma is the most common benign smooth muscle cell tumour of the myometrium, occurring in as many as 30% of women over 35 years of age (Vollenhoven et al, 1990). Leiomyoma growth is dependent on ovarian steroids (West et al., 1987). On the other hand, a growing body of evidence suggests that the action of estrogen may be mediated in part by local growth factors, such as EGF and insulin-like growth factor 1 (IGF-1), produced by the target cells (Huet-Hudson et al., 1990; Murphy and Ghahary, 1990; Nelson et al., 1992). Furthermore, it is intriguing whether the coexpression of Gas6 and Axl, as a unique differentiation pathway, is involved in the development and growth of leiomyoma. Therefore, the aim of the present study was to investigate Gas6 and its receptors Axl and Sky related to specific growth in uterine leiomyoma, and evaluate their plausible growth potentials and mechanisms.
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Patients
The specimens of uterine leiomyoma and the corresponding normal uterine myometrium in each case were obtained by hysterectomy from 30 patients (age 35–42 years) with uterine leiomyoma, at the Department of Obstetrics and Gynecology, Gifu University School of Medicine from October 2000 to March 2002. 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. The remainder of each specimen was immediately frozen in liquid nitrogen and later prepared for the subsequent experiments.
Competitive RT–PCR–Southern blot analysis with preparation of internal standard recombinant RNA (rcRNA)
Following the procedures described in one of our previous studies (Sun et al., 2002), the synthesis of internal standard rcRNA was performed. DNA construction of the internal standard was synthesized by PCR from a BamH/EcoRI fragment of v-erbB (Clontech Laboratories, Palo Alto, CA, USA) with two sets of oligonucleotide primers containing gas6-, Axl- or Sky-specific primer sequences and T7 promoter (Janssen et al., 1991; Manfioletti et al., 1993; Vanden et al., 1993; Ohashi et al., 1994).
Total RNA was isolated from the tissue 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, the total RNA (3 µg) and a series of diluted rcRNA (1–103 fmol) 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 (dNTPs) and 30 pmol of 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'-GGTGGCTG TGAAGACGATGA-3' (Axl-5'; 1820–1839), 5'-CTCAGATACTCCATGCC ACT-3' (Axl-3'; 2103–2122), 5'-CACTGAGCTGGCTGACTAAGCCCC-3' (Sky-5'; 2719–2742) and 5'-AATGCATGCACTTAAGCAGCAGGG-3' (Sky-3'; 3039–3062) 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 dNTPs. 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 using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression (GAPDH-5', 71–96, 5'-TGAAGGTCGGAGTC AACGGATTTGGT-3' and GAPDH-3', 1030–1053, 5'-CATGTGGGCCATG AGGTCCACCAC-3').
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 10x standard sodium citrate solution (SSC; 20x SSC: 0.33 mol/l NaCl, 0.33 mol/l sodium citrate, pH 7.0). After blotting, the membrane was dried at 75°C and then cross-linked by ultraviolet 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'-TTGATGGAAG TGGGCCAGTC-3') and biotinylated internal standard gene-specific oligonucleotide probe (5'-TGTTATACAGGGAGTGAAA-3') simultaneously to detect specific genes, or hybridized with biotinylated gas6-, Axl- or Sky-5' (10 pmol/µl; Rikaken Co. Ltd) to detect their precise intensities at 42°C for 24 h. The membrane was washed with 2x SSC for 15 min at room temperature, then with 2x SSC for 15 min at 42°C, and finally with 0.5x SSC 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).
Northern blot analysis
Total RNA (30 µg) was denatured at 65°C for 15 min, size-fractionated by electrophoresis through 1% agarose-formaldehyde gel, and blotted onto a nylon membrane (Amersham, Amersham, UK) by capillary transfer for 20 h using 20x SSC. After blotting, the membrane was dried at 80°C for 2 h. The DNA probe was labelled for Gas6, Axl and Sky mRNAs with alkaline phosphatase using Gene Images Labelling and Detection Kit (Amersham). The membrane was incubated in the AlkPhos Direct hybridization buffer and the labelled probe was added (10 ng/ml) to the buffer. After hybridization at 55°C for 24 h, the membrane was washed twice with primary wash buffer [2 mol/l urea, 0.1% sodium dodecyl sulphate (SDS), 50 mmol/l Na phosphate, pH 7.0, 150 mmol/l NaCl, 1 mmol/l MgCl2, 0.2% blocking reagent] for 10 min at 55°C, and then washed twice with secondary wash buffer (50 mmol/l Tris base, 0.1 mol/l NaCl, 2 mmol/l MgCl2) for 10 min at room temperature. The detection reaction was visualized using CDP-Star detection reagent (Amersham). The membrane was exposed to Kodak XAR-5 films (Eastman Kodak) for 1 h.
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). Four micron sections of formalin-fixed paraffin-embedded tissues were cut with a microtome and dried overnight at 37°C on a silicanized 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). The following steps were used. 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 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 semi-quantitatively evaluated as described by McCarty et al. (McCarty et al., 1985). Each stained section was given a histochemical score (histoscore) calculated by the formula: 
(i + 1) x P i, in which i = nuclear staining intensity (range 1–4, 0 indicates no staining) and Pi = percentage of stained nuclei of cells ( McCarty et al., 1985).
Western blot analysis
Frozen tissues of uterine leiomyoma (wet weight, 10–20 mg) were homogenized in a homogenizing buffer [5 mmol/l Tris–HCl (pH 7.4), 5 mmol/l NaCl, 1 mmol/l CaCl2, 2 mmol/l O,O'-Bis (beta-aminoethyl) ethyleneglycol-N,N,N',N'-tetraacetic acid, 1 mmol/l MgCl2, 2 mmol/l DTT, 25 µg/ml aprotinin and 25 µg/ml leupeptin] with a Polytron homogenizer (Kinematics, Luzern, Switzerland). This suspension was incubated for 30 min on ice, and centrifuged in a microfuge at 10 000g for 30 min to obtain the supernatant. The protein concentration of samples was measured by the method of Bradford (Bradford, 1976). Sample concentrations were adjusted to 10 µg/30 µl in lysis buffer. Thirty microlitres of each sample was mixed with 20 µl of sample buffer containing 10% glycerol, 60 mmol/l Tris–HCl, pH 6.8, 2% SDS and 0.02% bromophenyl blue, boiled for 5 min, and subjected to SDS–PAGE on 4% polyacrylamide gel. The proteins were then transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore Corporation, Bedford, MA, USA). The blots were incubated with the primary antibodies against Gas6, Axl and Sky, each at a dilution of 1:500, overnight at 4°C after blocking for non-specific staining with 5% bovine serum albumin in Tris-buffered saline–Tween-20. The reaction was visualized with an enhanced chemiluminescence detection system using an ECL kit (Amersham).
Statistics
We performed total RNA isolation and competitive RT–PCR–Southern blot analysis in triplicate in three different parts of each individual sample. 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 when P was <0.05. Data were expressed as mean ± SD.
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Results |
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In the Northern blot analysis, bands signal-specific to Gas6, Axl or Sky were detected as shown in Figure 1. Therefore, the levels of Gas6, Axl and Sky mRNAs can be determined by RT–PCR. In the competitive RT–PCR–Southern blot analysis for Gas6, Axl or Sky mRNAs, only two predicted PCR products from samples and the rcRNA were detected without non-specific products in all samples analysed. The level of Gas6, Axl or Sky mRNAs was determined using a standard curve of a serial dilution of rcRNA by competitive RT–PCR–Southern blot analysis, as detailed in one of our previous studies (Sun et al., 2002) (Figure 2).
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The levels of Gas6 and Axl mRNAs in uterine leiomyoma (243.3 ± 39.0 and 126.3 ± 30.6 fmol/µg of total RNA respectively) were significantly higher (both, P < 0.01) than those in normal uterine myometrium (106.8 ± 28.2 and 62.1 ± 17.4 fmol/µg of total RNA respectively), while there was no significant difference between the levels of Sky mRNA in uterine leiomyoma (5.5 ± 1.1 fmol/µg of total RNA) and in normal uterine myometrium (5.7 ± 1.4 fmol/µg of total RNA), as shown in Figure 3. The level of Sky mRNA was only
10% of the levels of Gas6 and Axl mRNAs in both uterine leiomyoma and normal uterine myometrium.
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In the immunohistochemical study, positive staining of Gas6, Axl and Sky was found predominantly in all uterine leiomyoma samples, and in smooth muscle cells of all normal uterine myometrium specimens analysed (Figure 4). The histoscores of immunohistochemical staining for Gas6 and Axl in uterine leiomyoma (367 ± 43 and 344 ± 28 respectively) were significantly higher (P < 0.01) than those in normal uterine myometrium (220 ± 38 and 233 ± 40 respectively), while there was no significant difference between Sky histoscores in uterine leiomyoma (140 ± 26) and in normal uterine myometrium (133 ± 28), as shown in Figure 5. In Western blots, the intensities of Gas6 and Axl in uterine leiomyoma (190 ± 44 and 163 ± 46 respectively) were significantly higher (P < 0.01) than in normal uterine myometrium (85± 18 and 81 ± 21 respectively), while there was no significant difference between the intensity of Sky in uterine leiomyoma (69 ± 19) and in normal uterine myometrium (66 ± 22), as shown in Figure 6. In Western blotting for Gas6, Axl and Sky in normal uterine myometrium and uterine leiomyoma, a single band identified with the size of Gas6, Axl or Sky was detected, as shown in Figure 7 A, where Western blot bands of three representative cases are shown. There was a significant positive correlation between the intensity of Western blots and histoscores for Gas6, Axl and Sky (Figure 7B).
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Discussion |
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In the present study, the expressions of Gas6 and Axl were definitely detected in uterine leiomyoma and normal uterine myometrium in each case. 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 might have an original ligand other than Gas6. In the present study, the percentage of Sky mRNA levels to Axl mRNA levels was 8% in normal uterine myometrium and 4% in uterine leiomyoma. 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 uterine leiomyoma and normal uterine myometrium appears to be exerted mainly via Axl. The different pattern of expression suggests that the presence of a distinct role for each receptor. 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 was expressed at 10-fold higher levels in peritoneal metastatic nodules than in other normal and malignant tissues. This indicates that overexpression and possibly a differential processing event of tyrosine kinase receptors may be involved in colon cancer, and that they are potential markers for the progression of this disease (Craven et al., 1995). Gas6 is identified as a novel growth factor for thyroid carcinoma cells overexpressing Axl receptor tyrosine kinase (Tanaka et al., 1998).
The levels of Gas6 and Axl in uterine leiomyoma were significantly higher than those in normal uterine myometrium in each case. However, Gas6 and Axl levels, both in normal myometrium and in uterine leiomyoma, showed no significant alteration during the menstrual cycle (Sun et al., 2002). The levels of Gas6 and Axl in endometriotic endometria and in well-differentiated uterine endometrial cancers were significantly higher than in normal endometria; however, Gas6 and Axl levels in normal endometria showed no significant alteration during the menstrual cycle. Therefore, Gas6 might maintain continuous basic proliferation activity during the menstrual cycle in normal endometria and higher activity in endometriotic endometria and in well-differentiated uterine endometrial cancers, regardless of sex steroid influence, probably related to the unique differentiation in uterine endometrium (Sun et al., 2002, 2003). Gas6 and Axl might not be linked to sex steroidal regulation directly either in myometrium or in leiomyoma. This evidence is plausibly related to aberrantly stimulated growth of uterine leiomyoma as a unique differentiation pathway in Gas/Axl function.
In conclusion, the present study is the first to demonstrate the coexpression of Gas6 and receptor tyrosine kinases Axl and Sky in normal uterine myometrium and uterine leiomyoma and the higher expression of Gas6 and Axl in uterine leiomyoma. Therefore, the signal transduction of Gas6 and Axl might be involved in the development and growth of uterine leiomyoma.
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Acknowledgements |
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The authors wish to thank Mr John Cole for proofreading the English of this manuscript.
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References |
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