The HMC-1 Human Mast Cell Line Expresses the Hepatocyte Growth Factor Receptor c-met

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

239, 740–745 (1997)

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The HMC-1 Human Mast Cell Line Expresses the Hepatocyte Growth Factor Receptor c-met Koji Yano,* Kazuhiko Nakao,* Koichi Sayama,‡ Keisuke Hamasaki,* Yuji Kato,*,1 Keisuke Nakata,† Nobuko Ishii,† Joseph H. Butterfield,§ and Stephen J. Galli‡ *The First Department of Internal Medicine and †Health Research Center, Nagasaki University School of Medicine, Nagasaki, Japan; ‡Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; and §Department of Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota

Received September 22, 1997

Hepatocyte growth factor (HGF) was originally characterized as a strong inducer of liver regeneration. However, it is now clear that HGF and its receptor, the proto-oncogene c-met, can be expressed in many other tissues, and that HGF can mediate diverse biological activities. We investigated the expression and function of c-met in a human mast cell line (HMC-1). We found that HMC-1 cells express c-met and that c-met expression can be upregulated by treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Although HGF did not detectably influence the proliferation or morphology of HMC-1 cells, HGF inhibited the cells’ ability to release tumor necrosis factor-alpha (TNF-a) in response to stimulation with PMA and the calcium ionophore, A23187. These results add the inhibition of TNFa production to the other recognized effects of HGF/cmet on cellular function. q 1997 Academic Press Key Words: mast cell; c-met proto-oncogene; tumor necrosis factor-alpha.

HGF (1, 2), also known as scatter factor (3, 4), was originally described as a potent mitogen for hepatocytes in primary culture. More recently, HGF has been shown to have effects on a wide variety of cell types, in which it can elicit mitogenic responses, enhance motility or induce morphological changes (reviewed in 5, 6). The multiple activities induced by HGF are mediated through its binding to and activation of a transmembrane tyrosine kinase receptor, which is the product of the c-met proto-oncogene (7, 8). The c-met receptor is widely expressed and can be found on the surface of hepatocytes, fibroblasts, keratinocytes, and melano1 To whom correspondence should be addressed. The First Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki 852, Japan. Fax: 81-95-8497270. E-mail: katoy@net. nagasaki-u.ac.jp.

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cytes. Moreover, many tumor cell lines, including those derived from certain hepatomas, melanomas, gastrointestinal tumors, or thyroid or lung cancers, overexpress the HGF receptor (6, 9-12). Although most studies of the c-met receptor have focused on epithelial cells, it has recently been shown that certain hematopoietic progenitor cells or leukemic cells can also express the c-met receptor. For example, some human lymphoma cells, especially those of Hodgkin’s disease origin, express c-met mRNA and protein (13). c-Met expression has also been detected on human bone marrow CD34/ progenitor cells (14), and on mouse and human monocytic leukemic cell lines, such as NFS-60 (15, 16) and THP-1 (17). These findings have raised the possibility that c-met / HGF interactions may have roles in hematopoiesis or oncogenesis (18). However, to the best of our knowledge, the expression of c-met by mast cell lines has not yet been studied. The aims of the present study were to investigate whether the only available human mast cell line, namely HMC-1 cells (19), can express c-met mRNA and protein. We found that HMC-1 cells express c-met, that c-met expression can be upregulated by treatment of the cells with the phorbol ester, PMA, and that HGF can inhibit the ability of these cells to release TNF-a in response to stimulation with PMA and calcium ionophore. MATERIALS AND METHODS Reagents and cells. Recombinant human HGF(rh HGF) which had been derived in Chinese hamster ovary cells and had been purified with fast protein liquid chromatography as described before (18), was provided by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan). A polyclonal antibody against the C-terminal region of human met protein was purchased from Santa Cruz Biotechnology, Inc. (Cat. No. sc-161; Santa Cruz, CA). Phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore, A23187, were purchased from Sigma Chemical Co. (St Louis, MO). HMC-1, a human mast cell leukemia cell line that was originally established from the peripheral blood of a patient with mast cell leukemia (19), and Hep G2, a human hepatoblastoma cell line (20),

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were cultured in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 10 % fetal bovine serum (JRH Boisciences, Lenexa, KS), 2 mmol of L-glutamine, 100 units/ml of penicillin, and 100 mg/ ml of streptomycin at 37 7C, 5% CO2 in a humidified incubator. Cell treatment and rhHGF stimulation. For RT-PCR of c-met, HMC-1 cells were incubated with or without 0.1 mmol of PMA for 6 h. For TNF-a analysis by ELISA, HMC-1 cells were treated with vehicle only, with HGF at 20 ng/ml, or with HGF (0, 2, 10, or 20 ng/ ml ) plus PMA (0.1 mmol) and the calcium ionophore A23187 (0.2 mmol) for 18 h. For analysis of TNF-a mRNA by Northern blot, HMC1 cells were treated as above for 18 h, or with vehicle only or with PMA (0.1 mmol) for 48 h before exposure to HGF (20 ng/ml) for an additional 18 h. Total RNA was extracted from the cell pellet using ISOGEN (Nippon Gene, Toyama, Japan), according to the manufacturer’s instructions. Assessment of mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting. To assess relative amounts of mRNA, we performed RT-PCR using ten fold serial dilution of the specimens as previously described (21, 22). Briefly, tenfold serial dilutions (1:10, 1:100, 1:1000) of total RNA were made in diethyl pyrocarbonate (DEPC, Sigma)-treated H2O. The cDNA was synthesized from total RNA with oligo d(T) probe using reverse transcriptase (Gibco BRL) and was then amplified for 1 min at 94 7C, 1 min at 58 7C , and 2 min at 72 7C, 38 cycles for c-met or TNFa, and 20 cycles for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in a 50 ml volume reaction using TaKaRa PCR kit (TaKaRa Shuzo Co. Ltd., Siga, Japan). Three pairs of oligonucleotide primers were used. Human c-met sense; 5*-ACAGTGGCATGTCAACATCGCT-3 * antisense; 5*-GCTCGGTAGTCTACAGATTC-3 * (23), human TNF-a sense; 5*-GAGTGACAAGCCTGTAGCCCATGTTGTAGC-3 *, antisense; 5*-GCAATGATCCCAAAGTAGACCTGCCAAGAC-3 * (24), G3PDH sense; 5*-ACCACAGTCCATGCCATCAC-3 *, antisense; 5*-TCCACCACCCTGTTGCTGTA-3 * (25). The expected sizes of the amplified DNA fragments for c-met, TNF a, and G3PDH were 656, 444, and 452 bp, respectively. The PCR products were electrophoresed on 1.0% agarose gel stained with ethidium bromide and analyzed by UV illuminator. The PCR products were then subjected to Southern hybridization. Western immunoblot analysis. HMC-1 cells (1 1 107) were incubated with or without 0.1 mmol of PMA for 48 h. Cells were then washed twice with ice-cold phosphate-buffered saline twice and lysed with 100 ml of RIPA buffer (1% NP40, 0.1% SDS, 10 mg/ml phenylmethylmethylsulfonyl fluoride, 30 ml/ml aprotinin, and 10 mg/ml of sodium orthovanadate in phosphate buffered saline). The extract was kept on ice for 30 min, passed through 23 G needle, and centrifuged for 20 min. The protein concentration of the lysate was determined using a protein assay (Bio-Rad Laboratories, Hercules, CA). Samples were then separated on 6% SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The proteins were transferred to a nitrocellulose membrane (Amersham Life Science Inc., Arlington Heights, IL). Nonspecific binding of the membrane was blocked with 5% skim milk for 1 h at room temperature. The membrane was then probed with an rabbit polyclonal anti-met Ab (Santa Cruz) which is known to be very specific for c-met protein (17, 26), at room temperature, overnight. The signals were then visualized by the enhanced chemiluminescence system (Amersham). Northern blot. Twenty micrograms of total RNA was fractionated on a 1.0 % agarose formaldehyde gel and blotted onto a nitrocellulose membrane (Amersham). A cDNA probe for TNF-a, which was generated by RT-PCR of HMC-1, as described above, was labeled with [32P]-dCTP using Bca Best random labeling kit (TaKaRa Shuzo). The membrane was hybridized with the labeled cDNA probe as described elsewhere (27). After hybridization, the membrane was washed with final stringency at 0.1% SDS, 0.2 1 SSC, 42 7C. The hybridized membrane was then exposed to an X-ray film (Konica Co., Tokyo, Japan) for 4 days.

FIG. 1. HMC-1 cells express c-met mRNA that can be upregulated by PMA. HMC-1 cells were incubated with or without PMA for 6 h. Ten fold serial dilutions of the samples of total RNA were subjected to RT-PCR for c-met and G3PDH. The gel was transferred to a nitrocellulose membrane, and then probed with [32P]-labeled probe. (A) Agarose gel, with visualization of PCR products by ethidium bromide. (B) Southern hybridization of c-met PCR products.

Cytokine determination. TNF-a and IL-6 release was determined by a specific enzyme-linked immunosorbent assay (ELISA, Otsuka Pharmaceutical Co. Ltd.) with a detection limit of 20 pg/ml. Data analysis. Data are expressed as mean { SEM. Student’s t test (2-tailed) was utilized for comparison between two samples, and p values less than 0.05 were considered significant.

RESULTS HMC-1 Cells Express c-met mRNA and Levels of Transcript Can Be Increased by PMA Treatment RT-PCR using c-met primers revealed that HMC-1 cells express c-met transcripts (Fig. 1). The PCR products were then isolated and nucleotide sequence was analyzed. Sequence data confirmed that the 656 bp PCR product matched the predicted c-met cDNA sequence (23) (data not shown). The RT-PCR products were detectable at the 1:10 dilution in the control sample (not treated with PMA), whereas the PCR products of the sample of PMA-treated cells were observed down to the 1:100 dilution. By contrast, the PCR products of the housekeeping gene G3PDH appeared to be observed equally in all samples (down to the 1:10 dilution), indicating that all of the samples contained approximately the same amounts of total RNA (Fig. 1A). The relative amounts and identities of the PCR products were further confirmed by Southern hybridization using [32P]-labeled probes (Fig. 1B). As in Fig. 1A, the c-met PCR products of the samples of PMA-treated cells were detected down to the 1:100 dilution, and the c-met PCR products of the control samples were visual-

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FIG. 2. HMC-1 cells express c-met protein that can be increased by PMA treatment. HMC-1 cells were incubated with or without 0.1 mmol of PMA for 48 h. HMC-1 cells and Hep G2 cells (positive control for expression for c-met protein) were lysed, thirty microgram of lysates were subjected to SDS-PAGE. (A) A blot in Non-reducing conditions showing 190 kDa bands, where (B) A blot in Reducing conditions showing 140 kDa bands. In each case, amounts of c-met product were increased by PMA treatment.

microscopy; nor did such treatment for 24 or 48 h have any detectable effect on DNA synthesis, as assessed by [3H] thymidine incorporation (Data not shown). We also assessed whether HGF might influence cytokine production by HMC-1 cells, focusing on two cytokines, tumor necrosis factor-alpha (TNF-a) and interleukin 6 (IL-6), which are thought to represent important mast cell products under certain physiological or pathological conditions. Treatment of HMC-1 cells with HGF (from 2-100 ng/ml for 0-48 h) did not induce detectable release of TNF-a or IL-6 (e.g., Figs. 3 A, B). By contrast, as previously reported (28), stimulation of HMC-1 cells for 18 h with PMA (0.1 mmol) plus A23187 (0.2 mmol) induced substantial amounts of TNF-a and IL-6 production (Figs. 3 A, B). Moreover, when HMC1 cells were cultured with PMA plus A23187, the addition of HGF (2,10, or 20 ng/ml) resulted in a dose-de-

ized only to the 1:10 dilution (Fig. 1A upper), whereas all of the samples contained approximately the same amounts of G3PDH products (Fig. 1B lower). These results indicate that HMC-1 cells express c-met mRNA and that the levels of c-met mRNA can be upregulated by treatment with PMA. HMC-1 Cells Express c-met Protein To clarify whether the c-met mRNA was translated to protein, we analyzed HMC-1 cell lysates in Western blots. As a positive control, the same amount of a lysate of Hep G2 cells was also subjected to SDS-PAGE immunoblot. As shown in Fig. 2, immunoreactive 190 kDa cmet protein was very faintly detectable in control HMC-1 cells under non-reducing conditions, but was not detectable under reducing conditions; no 140 kDa band was visualized. However, treatment of the cells with PMA for 48 h markedly increased c-met protein expression. These findings indicate that PMA stimulation of HMC-1 cells can increase the expression of both c-met mRNA and c-met protein. HGF Inhibits TNF-a Release by HMC-1 Cells Which Have Been Stimulated with PMA and the Calcium Ionophore, A23187 HGF can have diverse effects on certain hematopoietic cell lines, including the induction of proliferation and/ or alterations in morphology. We therefore explored whether HGF might elicit such effects in HMC-1 cells. However, HGF treatment (from 2 to 100 ng of HGF/ ml) for 1-7 days had no detectable effect on HMC-1 cell number or viability, as assessed by trypan blue exclusion, or on the cells’ morphology, as observed by light

FIG. 3. Effect of HGF on the PMA/A23187-induced production of immunoreactive IL-6 and TNF-a protein by HMC-1 cells. Data are expressed as mean { SEM (nÅ3; results are representative of those obtained in three similar experiments). HMC-1 cells were treated for 18 h with vehicle only (control), HGF at 20 ng/ml, or with HGF (2, 10, or 20 ng/ml ) plus PMA (0.1 mmol) and calcium ionophore A23187 (0.2 mmol) (/). Supernatants were assayed by ELISA for TNF a (A) and IL-6 (B). (A) ** : põ0.01 vs. PMA/A23187-treated cells which had not been treated with HGF. (B) No effect of HGF treatment on IL-6 production was observed.

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signal transduction are impaired by protein kinase C (PKC) activation or ionomycin treatment (29, 30). We therefore treated HMC-1 cells with PMA (0.1 mmol) for 48 h before exposure to HGF (20 ng/ml) for an additional 18 h in the absence of PMA, conditions that would favor PKC depletion and effective signal transduction via c-met (29). Under these conditions, HMC1 cells released only minimal amounts of TNF-a (up to 38 pg/ml), and no effect of HGF on these low levels of TNF-a production was detected (data not shown). However, compared to cells treated with vehicle (0) or PMA alone, cells treated with PMA and HGF exhibited very low levels of TNF-a mRNA (Fig. 4B). These results indicate that, under some conditions, HGF treatment can result in decreased levels of TNF-a mRNA in HMC1 cells. DISCUSSION

FIG. 4. (A) HMC-1 cells were treated with vehicle (0) or with PMA (0.1mmol) and calcium ionophore A23187 (0.2 mmol) with or without HGF (at 20 ng/ml) for 6 h. Total RNA was extracted, and 20 mg of RNA of each sample was subjected to Northern blot analysis. Treatment with PMA/A23187 and HGF for 6 h resulted in a slight decrease in levels of TNF a mRNA. Results are representative of those obtained in three similar, independent experiments. (B) HMC1 cells were treated with PMA (0.1 mmol) for 48 h before exposure to HGF (20 ng/ml) for additional 18 h. Compared to cells treated with vehicle (0) or PMA alone, cells treated with PMA and HGF exhibited very low levels of TNF a mRNA.

pendent and significant (up to 64.7%) inhibition of TNF-a production compared to that from cells in which HGF was not added. Thus, PMA/A23187 challenge of cells incubated with 0, 2, 10, or 20 ng of HGF/ml released 2550{187, 2137{73, 1160{89, or 1131{163 pg of TNF-a/ml (mean{SEM), respectively (Fig. 3 A). HGF Reduces Levels of TNF-a mRNA in HMC-1 Cells Which Have Been Stimulated with PMA and the Calcium Ionophore, A23187 Fig. 4 A shows Northern blot results for HMC-1 cells which had been stimulated in conditions similar to those of Figs. 3; i.e. simultaneous treatment by PMA (0.1 mmol)/A23187 (0.2 mmol) with or without HGF (20 ng/ml). PMA plus calcium ionophore treatment did not alter 2 kb TNF-a mRNA levels, whereas 20 ng/ml of HGF slightly decreased it. However, the effect of HGF under these conditions seemed rather modest. In a gastric cancer cell line, c-met tyrosine phosphorylation and

We found that the HMC-1 mast cell line expressed c-met mRNA and protein, and that levels of c-met expression in HMC-1 cells could be enhanced when the cells were stimulated with PMA. Although the expression of c-met by mast cells or mast cell lines has not previously been reported, PMA treatment has been shown to upregulate the expression of c-met in other cell types, including cell lines of hematopoietic origin (13, 31, 32). Furthermore, our Western blot analysis, which detected a c-met immunoreactive protein of Ç190 kDa in non-reducing conditions and of Ç140 kDa in reducing conditions, suggests that HMC-1 cells express primarily the major, originally described, isoform of c-met (7, 8), rather than alternative c-met isoforms that can be generated by proteolytic cleavage or alternative splicing (33, 34). Both the amount of c-met mRNA expressed by HMC1 cells (which was not detectable in Northern blots) and the amount of c-met protein in these cells (which was substantially less than that present in Hep G2 cells) was quite small. Nevertheless, the c-met protein expressed by HMC-1 cells appeared to be functionally active, in that incubation of the cells with the c-met ligand, HGF, resulted in a significant impairment of the ability of HMC-1 cells to release TNF-a upon stimulation with PMA and the calcium ionophore, A23187. When compared to vehicle- or PMA-treated HMC-1 cells, PMAtreated HMC-1 cells which had also been treated with HGF expressed much lower levels of TNF-a mRNA. This result suggests that under some conditions, HGF can decrease transcription of TNF-a mRNA and/or increase the rate at which the message is degraded. Although HGF has been shown to induce a number of different responses in the various cell types which express c-met, including effects on proliferation, motility, and morphology, we are unaware of prior reports that HGF can reduce cytokine production by such cells. Notably, this effect was observed with TNF-a but not

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with IL-6. This result indicates that the regulation of the expression of these two cytokines by HMC-1 cells can be differentially regulated. Mast cells are normally distributed throughout connective tissues where they are thought to play an important role as effector cells in immediate hypersensitivity responses and host immunity to parasites, as well as in a broad spectrum of chronic inflammatory conditions (35-37). Mast cells can also contribute to innate or natural immunity to certain pathogens (37-39). In all of these settings, mast cells are regarded as potential sources of cytokines, including TNF-a and IL-6, which in turn can influence the function of other resident cells or recruited inflammatory cells (36, 37). Because the HMC-1 cell line is the only long-term human mast cell line, it has been widely used for studies of cytokine production and other activities of human mast cells (22, 28, 40-43). This cell line was derived from a patient with mast cell leukemia and it expresses features which in some respects resemble those of very immature cells in the mast cell lineage (21); i.e., it expresses a mast cell-associated protease and the c-kit receptor, but neither detectable levels of FceRI, the high affinity IgE receptor, nor high levels of histamine (19, 40, 41, 43). Moreover, the c-kit protein expressed by HMC-1 cells is constitutively activated as a result of two point mutations in the corresponding gene (44). Thus, HMC-1 cells express several differences from mature human mast cells. We have also detected c-met mRNA by RT-PCR in three different preparations of human umbilical-cord blood-derived mast cells, which were generated after 16, 12 or 12 weeks of culture in stem cell factor, interleukin-6, and prostaglandin E2 (as described in ref. 22), and which were of purities of 98%, ú99%, and ú99%, respectively, by Kimura stain (22). This finding suggests that non-neoplastic human mast cells may also be able to express c-met, at least under some circumstances. Further studies of normal human mast cells may be difficult, given our findings in HMC-1 cells and umbilical cord-blood-derived mast cells suggesting that mast cells may express relatively low levels of c-met, as well as the problems in obtaining large numbers of normal human mast cells for biochemical analysis. Nevertheless, the present results suggest that such studies may be of interest in extending our view of the biology of HGF and its receptor. They may also provide new insights into the regulation of mast cell TNF-a production. ACKNOWLEDGMENTS We thank Cellular Technology Institute, Otsuka Pharmaceutical Co., for the kind gifts of rhHGF and cytokine ELISA; Dr. H. Yamasaki at The First Department of Internal Medicine, Nagasaki University School of Medicine, for his helpful advice, M. Miyamoto at Beth Israel Deaconess Medical Center and T. Yoshikawa at Nagasaki University for their technical help, and M. Motomura for photography.

This work was supported in part by United States Public Health Services Grant P50 HL-56383, Project 4 (S. J. Galli).

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