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Endocrinology Vol. 139, No. 3 852-858
Copyright © 1998 by The Endocrine Society

ARTICLES

Expression Profile of Receptor-Type Protein Tyrosine Kinase Genes in the Human Thyroid

Kunihiko Tanaka, Yuji Nagayama, Toru Nakano, Noboru Takamura, Hiroyuki Namba, Shuji Fukada, Kanji Kuma, Shunichi Yamashita and Masami Niwa

Departments of Pharmacology 1 (K.T., Y.N., M.N.) and Nature Medicine, Atomic Bomb Disease Institute (N.T., H.N., S.Y.), Nagasaki University School of Medicine, Nagasaki 852-8523; The Shionogi Research Laboratories, Shionogi Co. (T.N.), Osaka 553; and Kuma Hospital (S.F., K.K.), Kobe 650, Japan

Address all correspondence and requests for reprints to: Yuji Nagayama, M.D., Department of Pharmacology 1, Nagasaki University School of Medicine, 1–12-4 Sakamoto, Nagasaki, 852-8523, Japan. E-mail: nagayama{at}net.nagasaki-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein tyrosine kinases (PTKs) play a role in regulating the growth and differentiated functions of thyroid cells and are probably involved in tumorigenesis of papillary-type thyroid carcinoma. To better understand the roles of PTKs in the physiology and pathophysiology of the thyroid, we analyzed the expression profile of receptor-type PTKs in normal human thyroid tissues. Highly conserved regions in the catalytic domains of receptor-type PTKs were amplified by RT-PCR using degenerate oligonucleotide primers. Nucleotide sequencing of about 100 clones identified 21 PTKs, including 16 receptor type and 5 nonreceptor type; no novel PTK was identified. Insulin-like growth factor I receptor, platelet-derived growth factor receptor (PDGFR), TrkE, Axl, epidermal growth factor receptor, etc., appear to be the most abundant receptor-type PTKs in the thyroid; many of which (PDGFR, TrkE, Axl, etc.) have never previously been demonstrated to be expressed in the thyroid. The expression of messenger RNAs (mRNAs) for PDGFR, axl, and trkE in normal thyroid cells was confirmed by Northern blot analysis, and interestingly, the expression levels of PDGFR and trkE mRNAs were decreased in all three thyroid carcinoma cell lines examined (FRO, WRO, and NPA), whereas axl mRNA and protein were overexpressed in 2 of 3 thyroid carcinoma cell lines (FRO and WRO) compared with that in normal tissue. The axl gene was, however, neither amplified nor rearranged. The biological activity of the ligand for Axl, the product of growth arrest-specific gene 6 (Gas6), was then evaluated, demonstrating modest mitogenic activity in thyroid carcinoma cells overexpressing Axl. Furthermore, gas6 mRNA was expressed in FRO cells.

Thus, we here identify a variety of PTKs expressed in the thyroid gland, many of which may participate in the regulation of thyroid cell function. Variable expression levels of some PTKs in normal and cancerous cells suggest that there may be an imbalance and disarray of phosphorylation events in thyroid carcinoma cells. Furthermore, Gas6 is identified as a novel growth factor for thyroid carcinoma cells overexpressing Axl receptor tyrosine kinase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROTEIN tyrosine kinases (PTKs) are believed to play a pivotal role in regulating the growth and differentiated functions of eukaryotic cells as components of signal transduction pathways (1). The PTK family is classified into receptor type and nonreceptor type (cytoplasmic type), and includes cellular receptors for certain growth factors and protooncogenes (2).

In thyroid cells, activation of PTK cascades, for example, by epidermal growth factor (EGF) and fibroblast growth factor (FGF) are closely linked to cell proliferation and dedifferentiation (3). On the other hand, TSH, a pituitary glycoprotein hormone that is indispensable for stimulating both thyroid cell proliferation and differentiation and largely functions through the cAMP-protein kinase A (serine/threonine kinase)-dependent signal transduction cascade and, to a lesser extent, the Ca2+/protein kinase C-dependent pathway (3), increases tyrosine phosphorylation of cellular proteins (4). Thus, the complex network of various growth factors regulates the growth of thyroid cells via tyrosine phosphorylation cascades.

Thyroid carcinomas are classified pathologically as papillary, follicular, or anaplastic carcinomas of thyroid follicular epithelial cell origin and as medullary carcinoma of parafollicular cell origin. Among these, papillary thyroid carcinomas are often characterized by the aberrant expression of activated receptor-type PTKs such as ret and trk protooncogenes consequent to chromosomal rearrangements (5, 6). Each has been found to be activated in 15–20% of this type of carcinoma, with some geographic differences (7, 8). Further, overexpression of receptor-type PTKs, including MET [a receptor for hepatocyte growth factor (HGF)] and EGF receptor, has also been identified in thyroid carcinomas of the papillary type and of other types as well (5, 9). These data strongly implicate abnormal tyrosine kinase activity in the pathogenesis of thyroid carcinomas, particularly of the papillary type.

In the present studies, to better understand the roles of PTKs in the physiology and pathophysiology of thyroid cells, the expression profile of receptor-type PTKs was elucidated in normal human thyroid tissues by a means of a RT-PCR-based cloning technique using degenerate oligonucleotide primers corresponding to the conserved regions of receptor-type PTKs. We here report that 21 PTKs could be isolated, including 16 receptor type and 5 nonreceptor type. Furthermore, among them, Axl receptor tyrosine kinase was overexpressed in two thyroid carcinoma cell lines, and growth arrest-specific gene 6 (Gas6), the ligand for Axl, has mitogenic activity for these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Human thyroid carcinoma cell lines, FRO, WRO, and NPA (10), were cultured with RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS and the appropriate antibiotics. Human thyroid follicular epithelial cells in primary culture, obtained as previously described (11), were cultured in Ham’s F-12 (Life Technologies) with 5% FBS.

Analysis of the expression profile of receptor-type PTKs in normal thyroid tissues
Oligonucleotide primers were synthesized corresponding to the amino acids HRDLAARN and DVWS(F/Y)G(I/V), which are highly conserved sequences in the catalytic subdomains VI and IX of receptor-type PTK catalytic domains (2) (Fig. 1Go). The sense primer was 5'-CA(C/T)(A/C)GIGA(C/T)(C/T)TIGCIGCI(A/C)GIAA-3', and the antisense primer was 5'-A(C/T)ICC(A/G)(A/T)AI(C/G)(A/T)CCAIAC(A/G)TC-3', where I indicates inosine. The amino acid sequence DLAAR is included in the upstream primer to exclude src-related cytoplasmic PTKs and serine/threonine kinases, in which the corresponding sequences are DLRAA and DLKPE, respectively. An invariant DFG triplet and a potential autophosphorylation site(s) (Tyr) would be included in the amplified region. This method has previously been proven to be valid for cloning various receptor-type PTK complementary DNAs (cDNAs) (12, 13).



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  		Figure 1. Amino acid sequences in the catalytic domains of receptor-type PTKs identified in the present studies and nucleotide sequences for the degenerate primers used. Highly conserved amino acids are boxed. The degenerate oligonucleotide primers whose nucleotide sequences are shown at the bottom correspond to amino acid sequences HRDLAARN and DVWS(F/Y)G(I/V).

 
Total RNA was extracted from the four normal thyroid tissues obtained at the time of total thyroidectomy in four patients with papillary thyroid carcinomas by the acid guanidinium thiocyanate-phenol-chloroform method (Isogen, Wako Pure Chemical Co., Osaka, Japan). First strand cDNA was synthesized from 500 ng total RNA using Moloney murine leukemia virus reverse transcriptase (Life Technologies) and random primers, a fraction of which was then amplified using 25 pmol of each primer, 200 µM of each deoxy-NTP, and 2 U Taq polymerase (Takara Biochemical Co., Osaka, Japan) in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin in a final volume of 50 µl. Thirty-five cycles of denaturation (94 C for 90 sec), annealing (45 C for 90 sec), and extension (72 C for 60 sec) were conducted with an automated thermal cycler. All reactions were initiated with 5-min incubation at 95 C and terminated with 7-min incubation at 72 C. The approximately 210-bp products were gel-purified with the Qiaex II Kit (Qiagen, Chatsworth, CA) and subcloned into pCRII-T vector (Invitrogen, La Jolla, CA). Clones were sequenced by the dideoxynucleotide chain termination methods (14) using a Thermo Sequenase Core Sequencing Kit (Amersham, Arlington Heights, IL) and a Hitachi SQ-500DNA Sequencer (Hitachi Electronics Engineering Co., Tokyo, Japan).

Northern blot analysis
Total RNA was extracted from three thyroid carcinoma cell lines and normal thyroid cells cultured in the presence or absence of 10 U/liter bovine TSH (Sigma Chemical Co., St. Louis, MO) for 3 days by the acid guanidinium thiocyanate-phenol-chloroform method. Total RNA (25 µg) was subjected to electrophoresis in 1% agarose-2.2 M formaldehyde gels and blotted onto Hybond nylon membrane paper (Amersham) by standard techniques (14), using 10 x SSC (standard saline citrate). After prehybridization at 42 C for more than 4 h in 50% formamide, 5 x SSPE, 1% SDS, and 100 mg/liter denatured salmon sperm DNA, hybridization was performed overnight at 42 C with an {alpha}-32P-labeled cDNA probe of the PCR product in the same buffer. The final washing was performed in 0.1 x SSC and 0.1% SDS at 55 C, followed by autoradiography. The blot was stripped by boiling in 0.1% SDS and reprobed with cyclophilin cDNA to estimate the relative amounts of messenger RNA (mRNA) on the blot. mRNA sizes were estimated relative to 28S and 18S ribosomal RNA. The cDNA probe was labeled with [{alpha}-32P]deoxy-CTP by the Multiprime DNA Labeling System (Amersham) to a specific activity of > 1 x 109 cpm/µg DNA. Densitometric analysis was performed with a Fujix Bioimaging Analyzer BAS-2000 (Fuji, Tokyo, Japan).

Immunoblot analysis
The cells or normal thyroid tissue were suspended in 1 ml ice-cold buffer containing 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 20 mg/liter leupepsin, and 0.5 mM phenylmethylsulfonylfluoride (Sigma); homogenized; and centrifuged at 15,000 x g for 30 min at 4 C. The pellet was dissolved in 100 µl of the same buffer. Protein concentrations were determined according to the method of Bradford using reagents provided by Bio-Rad (Protein Assay Kit, Richmond, CA). Samples were boiled for 3 min in the presence of SDS-PAGE sample buffer. Equal amounts of proteins per sample (40 µg) were applied to a 7.5% acrylamide separating gel and transferred to a nylon membrane (Clear Blot Membrane-p, Atto Co., Tokyo, Japan). The membrane was blocked with 5% skim milk in TBS (150 mM NaCl and 20 mM Tris-HCl, pH 7.4) overnight at 4 C and then immunoblotted with polyclonal goat antiserum against human Axl [Santa Cruz Biochemistry (Santa Cruz, CA); dilution, 1:200] at room temperature for 1 h. After extensive washing with TBS and 0.05% Triton X-100, the immunoblots were incubated with a biotinylated protein A (Amersham; dilution, 1:500) at room temperature for 1 h and washed extensively with TBS and 0.05% Triton X-100. Detection of Axl was performed with streptavidin-horseradish peroxidase (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine/H2O2.

Southern blot and dot blot analysis of genomic DNA
High mol wt genomic DNA was extracted from thyroid carcinoma cell lines and normal thyroid tissue by standard techniques (14). DNA (10 µg) was digested with 30 U EcoRI, HindIII, or PstI. After electrophoresis in 0.7% agarose gel, the restriction fragments were denatured with 0.5 N NaOH-1.5 M NaCl and neutralized with 0.5 M Tris-HCl (pH 7.5)-1.5 M NaCl. The DNA was then blotted and hybridized as described for Northern blot analysis. The 3.4-kilobase full-length axl cDNA (15) was used as a probe.

Dot blot analysis was also performed as previously described (16). Serial dilutions of genomic DNA were spotted onto the nylon membrane and then probed for the axl cDNA as described above. Densitometric analysis was performed with a Fujix Bioimaging Analyzer BAS-2000.

Cell survival and mitogenic activity of Gas6
The cells were plated at a density of 2 x 104 (for mitogenic assay) or 2 x 105 (for cell survival assay) in 48-well multiplates. The next day (day 0), the cells were washed extensively and treated with medium containing 0% or 0.5% FBS and recombinant human Gas6 (17, 18). Viable cells were counted using the trypan blue exclusion test on days 2.5 and 5.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first took advantage of the RT-PCR method using degenerate oligonucleotide primers to amplify the conserved regions in the catalytic domains of receptor-type PTKs from normal human thyroid mRNA (Fig. 1Go). As shown in Fig. 2Go, lane 3, a single DNA fragment with the expected size of approximately 210 bp was successfully amplified. No nonspecific amplification was observed. PCR with total RNA without RT also demonstrated no PCR products (Fig. 2Go, lane 2), suggesting no genomic DNA contamination in the total RNA isolated or the existence of an intronic sequence(s) in genomic DNAs between the two primers. The PCR products were then subcloned, and together, 102 clones from 4 tissues were screened (20–30 clones for each). Sequencing analysis and homology search against the BLAST Search DataBank yielded 21 distinct cDNA clones, representing 16 receptor-type PTKs and 5 nonreceptor-type PTKs. No novel kinase was identified, except for a 16-bp deletion (nucleotides 1584–1599) identified in 2 of 3 ryk cDNA clones from 1 tissue (termed ryk-del, Fig. 3Go). This deletion seems to be heterozygous and causes a frame shift and premature termination, disrupting a potential autophosphorylation site. It is presently not known whether this type of mutation causes gain or loss of function without in vitro mutagenesis and functional analysis. Ryk is an orphan receptor (19).



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  		Figure 2. Electrophoresis of PCR products on a 2% agarose gel. Total RNA was extracted from the normal thyroid tissues, reverse transcribed, and amplified by PCR (35 cycles of denaturing at 94 C for 90 sec, annealing at 45 C for 90 sec, and extension at 72 C for 60 sec). Lane 1, One-kilobase DNA ladder (Life Technologies); lane 2, PCR without reverse transcription; lane 3, RT-PCR.

 


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  		Figure 3. Nucleotide and deduced amino acid sequences of wt-ryk and mutant ryk (ryk-del) with a 16-bp deletion (nucleotides 1584–1599). Y indicates a putative autophosphorylation site. A novel amino acid sequence that appeared by the frame shift is shown in italics.

 
The frequencies of PTK cDNAs identified are summarized in Table 1Go. The most abundant receptor-type PTK appears to be insulin-like growth factor I (IGF-I) receptor, followed by platelet-derived growth factor receptor (PDGFR), TrkE, Axl, EGF receptor, HEK4, HEK8, Ryk, c-erbB2, Met, flt4, RON, Erk, KDR, Tie, and TKT. The nonreceptor-type PTKs identified include c-Abl, JAK1, Syk, FAK, and Tyk2, all of which contain the sequence DLAAR mentioned above.


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  		Table 1. Summary of the frequencies of PTK cDNAs isolated from the normal thyroid glands determined by RT-PCR

 
Although the receptors for IGF-I, EGF, and HGF have previously been reported to be expressed in the thyroid (3, 9, 20, 21, 22), to our knowledge, expression of other receptor-type PTKs, such as Axl, TrkE, HEKs, Ryk, etc., has never previously been reported in the thyroid. To confirm the expression of these PTKs in thyroid follicular cells and, if expressed, to examine whether TSH regulates their steady state expression level, Northern blot analysis was performed with 25 µg total RNAs from normal human thyroid follicular cells cultured in the presence or absence of TSH (10 U/liter for 3 days) and three thyroid carcinoma cell lines (FRO, WRO, and NPA). As shown in Fig. 4Go, mRNAs for PDGFR, trkE, and axl could be readily detected in normal thyroid cells; interestingly, expression levels of PDGFR and trkE mRNAs were down-regulated in all three thyroid cancer cell lines examined, while axl mRNA was overexpressed in two of three thyroid cancer cell lines (FRO and WRO). Expression of HEK8 and ryk mRNAs was faintly detected in carcinoma cell lines. Steady state levels of expression of any PTKs were not regulated by TSH in the normal thyroid follicular cells in primary culture. mRNAs for HEK4, c-erbB2, etc. were not detected (data not shown).



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  		Figure 4. Northern blot analysis of PTK mRNA expression in normal thyroid cells cultured in the presence or absence of TSH and in thyroid carcinoma cell lines, FRO, WRO, and NPA. Twenty-five micrograms of total RNAs were fractionated on 1% agarose-formaldehyde gel, transferred onto a nylon membrane, and sequentially hybridized to radiolabeled PCR products (PDGFR, axl, trkE, HEK8, and ryk) and cyclophilin cDNA.

 
As Axl has been reported to have a transforming activity in NIH-3T3 cells (15), and the ligand for Axl has recently been identified as the product of gas6 (23), which shows mitogenic and/or survival activities in some cells (23, 24, 25), we then studied the Axl/Gas6 system in more detail. Firstly, to confirm Axl overexpression at the protein level, immunoblots were performed with a polyclonal antibody against human Axl, demonstrating that Axl protein was overexpressed in FRO and WRO cells compared with that in normal thyroid tissue (Fig. 5AGo), consistent with the data from Northern blot analysis. Axl appears to be a doublet of approximately 140 kDa, as previously reported (15, 26). No signal was detected in NPA cells (data not shown). Secondly, to determine whether overexpression was due to gene amplification or any structural rearrangement of the gene, Southern and dot blot analyses of the axl gene were employed; no evidence for gene amplification or rearrangement was found (Fig. 5Go, B and C, and data not shown).



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  		Figure 5. A, Western blot of Axl in thyroid carcinoma cells and a normal thyroid tissue. Forty micrograms of crude membrane proteins from FRO and WRO cells and a normal thyroid tissue were subjected to immunoblotting with polyclonal goat antisera against human Axl. B, Dot blot analysis of the axl gene. Serial dilutions of genomic DNA were spotted onto nylon membrane and probed for radiolabeled axl cDNA. 1:1 corresponds to a concentration of 2 µg genomic DNA. C, Southern blot analysis of the axl gene digested with EcoRI. Ten micrograms of genomic DNA were digested with 30 U EcoRI and resolved in 0.7% agarose gel. The products were transferred onto a nylon membrane and hybridized to radiolabeled axl cDNA.

 
To examine the functional properties of Gas6 on thyroid cell proliferation and survival, thyroid carcinoma cells were cultured in medium containing 0% or 0.5% FBS in the presence or absence of 15 nM Gas6 (Fig. 6Go). All cells cultured in serum-starved medium died even in the presence of Gas6, suggesting no survival effect of Gas6 on thyroid carcinoma cells regardless of the overexpression of Axl (Fig. 6Go, A–C). However, Gas6 induced a modest, but significant, cell proliferation of FRO and WRO, but not NPA, cells cultured with medium containing 0.5% FBS (Fig. 6Go, D-F). A dose-dependent effect of Gas6 was also confirmed in FRO and WRO cells (Fig. 6Go, G–I). These data demonstrate the mitogenic effect of Gas6 on Axl-overexpressing thyroid carcinoma cells, although the degree of Gas6 induction of cell proliferation observed in FRO and WRO cells was much smaller than that induced by 10% FBS (data not shown).



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  		Figure 6. Mitogenic activity of Gas6 in thyroid carcinoma cell lines overexpressing Axl receptor tyrosine kinase. Thyroid carcinoma cells were cultured in medium containing 0% or 0.5% FBS in the presence or absence of 15 nM recombinant human Gas6 for up to 5 days in A–F or in the presence of various concentrations of Gas6 (0 to 15 nM) for 5 days in G–I. Viable cells were then counted using the trypan blue exclusion test. The data are representative of two separate experiments; each point represents the mean ± SE of triplicate determinations, expressed as a percentage relative to the value on day 0. *, P < 0.01; ** P < 0.05 (vs. cells cultured without Gas6, by unpaired Student’s t test).

 
Finally, Northern blot analysis of Gas6 mRNA expression was performed to answer the question of whether Gas6 exerts its mitogenic action in an autocrine fashion. As shown in Fig. 7Go, gas6 mRNA was faintly detected only in FRO cells, suggesting the possibility of an autocrine mechanism for the Axl/Gas6 system in this carcinoma cell line. No increase in gas6 mRNA expression by serum starvation, however, was observed.



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  		Figure 7. Northern blot analysis of gas6 mRNA expression in thyroid carcinoma cell lines cultured in the presence of 0.5% or 10% FBS. Twenty-five micrograms of total RNAs were fractionated on 1% agarose-formaldehyde gel, transferred onto a nylon membrane, and sequentially hybridized to radiolabeled gas6 cDNA and cyclophilin cDNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present studies, we first clarified the expression profile of PTK genes in normal human thyroid tissues. Sequencing analysis of approximately 100 clones amplified from the thyroid cDNAs with oligonucleotide primers corresponding to the conserved regions of known PTKs identified 16 receptor-type PTKs and 5 nonreceptor-type PTKs. To our knowledge, other receptor-type PTKs known to be expressed in the thyroid gland include FGF receptor (9), c-Kit (27), and insulin receptor (3), suggesting that at least 19 receptor-type PTKs appear to be expressed in the thyroid glands. Although we cannot exclude the possibilities that some clones obtained at very low frequency may be derived from other components of the thyroid tissues (vascular endothelial cells, parafollicular cells, etc.) and/or may be illegitimate transcripts, a variety of PTKs probably participate in the regulation of thyroid cell proliferation and/or differentiated functions.

Among these 19 receptor-type PTKs, receptors for IGF-I, FGF, and EGF are well known to be expressed in the thyroid gland. Their ligands are also secreted by thyroid cells as autocrine factors (3, 9, 20, 21, 22). IGF-I is probably one of the most extensively studied growth factors in the thyroid; IGF-I stimulates tyrosine phosphorylation of intracellular proteins by itself and synergistically with TSH and EGF (4, 9, 20). Further, HGF has been reported to have the strongest mitogenic activity on dog thyroid cells (21).

Northern blot analysis of the expression of mRNAs for other PTKs identified here demonstrated that mRNAs for PDGFR, trkE, and axl are abundantly expressed in normal thyroid cells. However, the expression levels of these mRNAs do not necessarily parallel the frequency of the cDNA clones in PCR analysis, suggesting that the PCR cloning strategy we used here might have given us a biased representation of PTKs expressed in the thyroid glands. Nevertheless, interestingly, our data clearly show that axl is overexpressed in two thyroid carcinoma cell lines, whereas PDGFR and trkE mRNAs are down-regulated in all three cell lines. These data reveal the variable expression levels of these PTKs in thyroid carcinoma cell lines, suggesting that there may be an imbalance and disarray of phosphorylation events in cancer cells.

PDGFR has previously been reported to be absent in normal thyroid cells and aberrantly expressed in an anaplastic carcinoma cell line (28), although we are aware of one report describing the mitotic effect of PDGF on a normal rat thyroid cell line, FRTL5 cells (20). TrkE is a member of the Trk family, whose ligand is probably nerve growth factor (29). It will be intriguing to elucidate the binding and biological activities of PDGF and nerve growth factor in thyroid cells in the future.

Axl (also called Ufo, AR, or Tyro-7) receptor tyrosine kinase together with Sky/Brt/Dtk/Rse/Tif/Tyro-3 and Mer/Eyk/Tyro-12 comprise a new family of PTKs whose extracellular domain contains a combination of fibronectin type III repeats and Ig motifs (18). The cDNA for axl has originally been identified as a transforming gene from human myeloid leukemia cells by DNA transfection/transformation assay (15, 30), whose transforming ability is probably due to normal receptor overexpression rather than genetic rearrangement or mutation (18, 26). Indeed, its overexpression has recently been documented in some carcinomas, including metastatic sarcomas; a cervix carcinoma cell line, HeLa; and metastatic colon cancers (26, 31, 32). The ligand for Axl has recently been identified as Gas6 (23), which also binds to Sky and Mer (18). Gas6 was first identified by Manifioletti et al. (33) as a gene induced by serum deprivation in NIH3T3 cells. Gas6 was later purified from vascular smooth muscle cells as a growth-potentiating factor for Ca2+-mobilizing growth factors (17). Gas6 also has mitogenic activities for NIH-3T3 cells (23, 25) and Schwann cells (34), and prevents serum-starved NIH-3T3 cells (25) and vascular smooth muscle cells (24) from cell death. Thus, Gas6 exerts its mitogenic and survival activities as an autocrine factor. These unique characteristics of the Axl/Gas6 system led us to evaluate the functional significance of Gas6 in thyroid carcinoma cells overexpressing Axl. Although we did not find cell survival activity of Gas6 in thyroid carcinoma cells incubated in serum-free medium, Gas6 did stimulate cell proliferation of FRO and WRO cells, both of which overexpress Axl, cultured in low serum medium. This report is the first to describe the mitogenic effect of Gas6 on thyroid carcinoma cells, although its effect is modest, as previously reported in Schwann cells and NIH-3T3 cells (23, 34). Although TSH can mobilize intracellular Ca2+, the growth-potentiating ability of Gas6 for TSH could not be evaluated in these cells, because these cells have lost their endogenous TSH receptor (35) (data not shown). Further, it is suggested that the Axl/Gas6 system may act in an autocrine fashion in FRO cells.

We also found overexpression of Axl in approximately 50% of surgically removed human thyroid carcinomas by immunohistochemistry (unpublished data), suggesting the possible pathological role of Axl in tumorigenesis of human thyroid carcinomas.

In summary, we here analyzed the expression profile of receptor-type PTKs in the thyroid using a PCR-based cloning technique and identified 16 receptor-type PTK mRNAs. Our data reveal variable expression levels of some PTKs in thyroid carcinoma cells, suggesting an imbalance and disarray of phosphorylation events in carcinoma cells. Furthermore, 2 thyroid carcinoma cell lines are found to overexpress Axl receptor tyrosine kinase, and Gas6, the ligand for Axl, is identified as a novel growth factor for thyroid carcinoma cell lines overexpressing Axl.


   Acknowledgments
 
We thank Prof. E. T. Liu for providing the full-length axl cDNA, and Prof. Basil Rapoport for revising the manuscript.

Received August 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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