Isolation and identification of C-type natriuretic peptide in human monocytic cell line, THP-1

. Vol. 189, No. 2, 1992 December

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15, 1992

ISOLATION

AND IDENTIFICATION IN HUMAN

OF C-TYPE

MONOCYTIC

CELL

NATRIURETIC

LINE,

697-704

PEPTIDE

THP-1

Yushiro ISHIZAKA’ ‘2, Kenji KANGAWA’, Naoto MINAMIN03, Koichi ISHI14, Satoshi TAKAN04, Tanenao ET02 and Hisayuki MATSU03# Departments of ‘Biochemistry and 21nternal Medicine, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, Japan 3National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565, Japan 4Central Research Laboratories, Ajinomoto Co. Inc., Kawasaki 210, Japan

Received

October

26,

1992

SUMMARY: In a survey for unknown bioactive peptides in mammalian cell lines, we isolated peptides exhibiting a strong relaxant effect on chick rectum from a phorbol ester-supplemented culture medium of the human monocytic cell line, THP-1. The peptide was deduced to be a C-terminal 29-residue peptide derived from a human C-type natriuretic peptide (CNP) precursor. CNP mRNA was also detected in the THP-1 cells, and expression of CNP gene and CNP concentration in the culture medium was found to be highly augmented by the stimulation of phorbol ester. Production of CNP in THP-1 cells suggests that CNP also functions as a local regulator in the blood cell-vascular system, although CNP has previously been recognized as a neuropeptide functioning in the central nervous system. B 1992 Academic Press, Inc.

C-type natriuretic peptide (CNP) is a third member of the natriuretic peptide family (1). In contrast to A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) which are mainly expressed in heart tissue, CNP is shown to be localized in the central nervous system and is now thought to function as a neuropeptide in the central regulation of body fluid volume and blood pressure. On the other hand, CNP specific receptor, guanylate cyclase B (GC-B), is widely distributed in the peripheral tissue as well as in the central nervous system (2,3). Furthermore, GC-B is mainly expressed in cultured vascular smooth muscle cells, and exogenously administered CNP is found to suppress proliferation of cultured vascular smooth muscle cells (4). These data strongly suggest that CNP is also produced in peripheral tissue and

#To whom correspondence should be addressed. Abbreviations: CNP, C-type natriuretic peptide; ANP, A-type (atrial) natriuretic peptide; BNP, B-type (brain) natriuretic peptide; GC, guanylate cyclase; TPA, 12-O-tetradecanoylphorbol13-acetate; FCS, fetal calf serum; HPLC, high performance liquid chromatography; TFA, trifluoroacetic acid; Mr, relative molecular mass.

697

Copyright 0 1992 All rights of reproduction

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functions as a hormone or a local regulator, although we have not been able to detect CNP in circulating blood at significant concentrations (5). During a survey for unidentified peptides in a series of human cell lines, we found potent chick rectum relaxant activity in the culture medium of a phorbol ester-treated human cell line, THP-1, established from acute monocytic leukemia (6). The chick rectum active peptide thus isolated was identified by peptide sequencing to be human CNP, the structure of which has heretofore been deduced only from genomic cloning (7).

The present identification of CNP in

the THP-1 cell line may provide a new insight for so far unclarified functions of CNP. MATERIALS

AND METHODS

Cell culture: Human acute monocytic leukemia cell line (THP-1) was maintained in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS) at 37 “C under humidified 5% CO,/95% air. The cells (1 X 106/ml) were then cultured for two days in serum-free Eagle’s minimal essential medium supplemented with 100 rig/ml of 12-O-tetradecanoylphorbol-13acetate (TPA) for isolation of peptides. For measurements of CNP concentration in the medium, the cells (1 X 106/ml) were cultured for 2 days in duplicate in RPM1 1640 medium with or without 100 rig/ml of TPA or 10% FCS. Isolation: The culture medium (10 liters) thus obtained was filtered through a GF/B filter (Whatman), and the resulting filtrate was directly loaded onto a reverse phase C-18 column (90m1, Chemco LC-SORB SPW-C-ODS). After thoroughly washing with 0.1% trifluoroacetic acid (TFA), the adsorbed materials were eluted with 60% CH3CN in 0.1% TFA. The eluate was then evaporated in vacuum to dryness. The residual materials were dissolved in 1M CH3COOH and then boiled for 10 min to abolish intrinsic proteolytic activity. The solution was loaded onto an SP-Sephadex C-25 column (H+-form, 1.5 X 6 cm, Pharmacia). Succesive elutions with 1M CH&OOH, 2M pyridine, and then 2M pyridine-acetate (pH 5.0) afforded three respective fractions of SP- I, SP- II and SP- ill. After lyophilization, the SP- ill fraction (dry weight: 10 mg) was subjected to gel filtration on a Sephadex G-50 column (fine, 1.8 X 134 cm, Pharmacia). Column effluents were monitored by measuring absorbance at 280 nm, and an aliquot of each fraction was submitted to a bioassay for chick rectum relaxant activity. Fractions eliciting rectum relaxant activity were lyophilized and subjected to ion exchange high performance liquid chromatography (HPLC) on a TSK gel CM-2SW column (4.6 X 250 mm, Tosoh) using a gradient elution of HCOONH4 (pH 6.5) from 10 mM to 1 M in the presence of 10% CH3CN. Rectum active fractions were each subjected to reverse phase HPLC on a ~Bondasphere Cl8 column (2.1 X 150 mm, 3OOA, Waters) with a linear gradient elution of CH3CN from 10% to 60% in 0.1% TFA. Purity of the isolated peptides was confirmed by another reverse phase HPLC system (2.1 X 150 mm, Vydac 219TP5215 phenyl column). Column effluents from reverse phase HPLC were monitored by measuring absorbance at 210nm and 280nm, simultaneously. Bioassay: Chick rectum relaxant activity was measured according to the described method by using strips of freshly isolated chick rectum bathed in Krebs-Henseleit solution (8). Sequence analyses: Amino acid sequence analyses of the peptides were performed by a gas-phase sequencer equipped with phenylthiohydantoin (PTH)-amino acid analysing HPLC system (Model 470A/120A, Applied Biosystems). PTH-amino acids were detectable as low as 0.1 pmol, and 5 pmol of standard PTH-amino acid mixture was used as a calibration mixture. Radioimmunoassavs (RIAs) for CNP, ANP and BNP: RIA for CNP was performed using an antiserum (#171-4) raised aginst CNP-22 as reported previously (9). ANP and BNP showed 0.015% and 0.46% crossreactivity in this RIA system, respectively. RIAs for ANP and BNP were carried out as reported (10,ll). RNA extraction and blot analvsis: From THP-1 cells cultured for 2 days with 100 rig/ml of TPA (1.5 X 107 cells) and without TPA (3.0 X 107 cells), total RNA was extracted from each

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by the acid guanidium thiocyanate-phenol-chloroform method (12). Poly(A)+RNA was isolated by using an oligotex-dT30 (Nippon Roche). Poly(A)+RNA (4 pg) was denatured with glyoxal and dimethylsulfoxide, and was electrophoresed on a 1% agarose gel. RNA was then transferred to a nylon membrane (Zeta Probe membrane, Bio-Rad) and fixed by ultraviolet irradiation (Stratalinker, Stratagene). The membrane was prehybridized and hybridized at 37 “C in 6X SSPE containing 40% formamide, 5X Denhardt’s solution, O.S%SDS, and denatured salmon sperm DNA at 100 ,ug/ml. A full-length rat CNP cDNA was labeled by the random-primed method and used for the hybridization (13). The blot was washed at 37°C once in 2XSSC/O.l% SDS, once in 0.5XSSC/O.l% SDS, and finally twice in O.lXSSC/O.l% SDS. Autoradiography was carried out at -80°C for 3 days. RESULTS AND DISCUSSION A two day-culture medium of THP-1 cells stimulated with TPA was extracted with a reverse phase C-18 column and then separated by SP-Sephadex ion exchange chromatography. The bulk of chick rectum relaxant activity in the culture medium was efficiently condensed in a basic peptide fraction (SP- III). In Sephadex G-50 gel filtration of the SP-III fraction, relaxant activity was observed only in fractions #46-51 corresponding to relative molecular mass (Mr) 3K (Fig. 1). The relaxant activity was further separated by CM ion exchange HPLC into 4 fractions (fractions A, B, C, and D), as shown in Fig. 2. Fraction D was further purified by reverse phase HPLC on a C-18 column to a homogeneous state (Fig. 3a), and purity of the

5 F

10

20

30

40

Fraction

Figure 1.

50

60

70

number

Gel filtration of basic peptide fraction obtained from culture medium of TPA-treated THP-1 cells. Column: Sephadex G-50 (fine), 1.8 X 134 cm. Sample: SP- IIf fraction of culture medium of THP-1 cells. Eluent: 1M CH$OOH. Fraction size: 5 ml/tube. Chick rectum relaxant activity is indicated by black columns and ir-CNP by white columns.

699

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r

z 0.001 c zz c? $ D5 0.00: 5 2 0

L

0

SO

40

Time

Figure 2.

IX

(min)

Ion exchange HPLC of chick rectum active fraction of Mr 3K. Sample: Fractions (#46-51) exhibiting chick rectum relaxant activity in Fig. 1. Column: TSK gel CM-2SW, 4.6 X 250 mm (Tosoh). Flow rate: 1.0 ml/min. Solvent system: Linear gradient elution from A to 50% B (80 min) followed by that from 50% B to 100% B (20 min). (A) 1OmM HCOONH4 (pH 6.5): CH-JN = 9O:lO (v/v), (B) l.OM HCOONH4 (pH 6.5): CH&N = 9O:lO (v/v). Chick rectum relaxant activity is indicated by black columns and ir-CNP by white columns.

peptide was confirmed by another HPLC using a phenyl column (Fig. 3b). The isolation yield of the peptide was estimated to be 10 pmol starting from 10 liters of culture medium. By sequence analysis from the N-terminus, PTH-amino acids were identified up to the 21st step except for the 13th step (Cys), and the amino acid sequence thus determined was found to be identical to the established C-terminal

sequence of human CNP precursor

(prepro-CNP[98-1181)

(7). Since the peptide elicited chick rectum relaxant activity as well as

CNP immunoreactivity

comparable to that of CNP-22, one of the endogenous and active

molecular forms, it was deduced to be a C-terminal 29 amino acid peptide of CNP precursor (CNP-29), as shown in Fig. 4. This is the first evidence for the presence of human CNP as a peptide, since human CNP has so far been deduced by genomic cloning and identified as an immunoreactive compound (5). After isolation of CNP-29, we measured immunoreactive (ir-) CNP in each purification immunoreactivity.

step and found that all rectum active fractions exhibited CNP

In fact, N-terminally

shortened peptides of CNP-29 (CNP-23, CNP-25 and

CNP-24) were each isolated from fractions A, B and C in ion exchange HPLC (Fig. 2). These data indicate that proteolytic processing is different in the THP-1 cell system from that in mammalian brain where CNP-53 and CNP-22 are major endogenous molecular forms (9,14). 700

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a

I 0 Time

10

20

Tlme(min)

(min)

Figure 3. Final purification of CNP-29 by reverse phase HPLC. (a) Sample: Fraction D exhibiting chick rectum relaxant activity in Fig. 2. Column: PBondasphere C18, 2.1 X 150 mm (Waters). Flow rate: 0.3 ml/min. Solvent system: Linear gradient elution of CH,CN from 10% to 60% in 0.1% TFA over 80 min. (b) Sample: Peak with chick rectum relaxant activity in Fig. 3a. Column: 219TP5215 phenyl, 2.1 X 150 mm (Vydac). Flow rate: 0.3 ml/min. Solvent system: Linear gradient elution of CH&N from 5% to 60% in 0.1% TFA over 40 min. Chick rectum relaxant activity is observed in black column regions.

In Fig. 5, we examined day culture fmol/ml. medium

without

TPA

In contrast,

effects of TPA on biosynthesis

stimulation,

in the presence

up to 150 fmol/ml

ir-CNP

concentration

of 100 @ml

of TPA,

for 2 days, and its concentration

of CNP in THP-1 in the medium ir-CNP

cells.

in the

by the addition

CNP- 53 4 POAAGGGOKKGDKAPGG;GANLKGDRSRLL~DLRVDT;SRAA CNI- 29 CNI'- 22 t 4 100 I WARLLOEHPNAaYKGANmGLSKGCFGLKLDRlGSMSGLGC

CNP- 29 :

Figure 4.

1*0

I

+KGANKKGL~KGCFGLKLDZIGSMSGLG? 7777777-7777 TT-77-7-

Amino acid sequences of prepro-CNP and CNP-29. One letter notation for amino acid is used. Arrows under amino acid residues of CNP-29 indicate the residues identified by sequencing. Arrows and boxes indicate putative processing sites for generating CNP-53, CNP-29, and CNP-22. In addition to CNP-29, CNP-23 @repro-CNP[104-126]), CNP-24 (prepro-CNP [103-1261) and CNP-25 (prepro-CNP[102-1261) were identified in the present study. 701

2

was as low as 1

was accumulated

was not affected

After

of

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a

I$:;; -4.40 -2.37 -1.35

-0.24

Figure 5.

TPA

(-1

f-1

(+)

(+)

FCS

(+)

(-)

(+)

(-)

TPA

(-)

(t-1

Effects of TPA on peptide and mRNA levels of CNP in THP-1 cells. (a) Immunoreactive CNP concentration of culture medium of THP-1 cells after two-day incubation with and without TPA (100 @ml) or FCS (10%). @) Northern blot analysis of CNP mRNA of THP-1 cells with and without 2-clay stimulation of TPA in the absenceof FCS Numbers on the right side are molecular sizesshown in kilobases.

10% FCS (Fig. Sa). AS shown in Fig. 5b, a significant level of CNP mRNA was not detected in the THP-1 cells without TPA stimulation. By the 2 day-stimulation with TPA, an intense band was observed at the 1.2 kilobase (kb) region which corresponded to that identified as rat CNP mRNA (13). These data demonstrated that THP-1 cells have a potential ability to synthesize CNP and that expression of CNP gene and CNP production of THP-1 cells were

highly

augmented by TPA stimulation. In order to check the possibility that ANP and BNP are also produced by THP-1 cells, the culture medium was condensed and submitted to RIAs for ANP and BNP. However, even under TPA-stimulated

conditions, not more than 1 fmol/ml of ANP or BNP immunoreactivity

was observed in the culture medium, indicating that THP-1 cells selectively synthesize CNP among the three natriuretic peptides. THP-1 cell line, established from human acute monocytic leukemia, has been identified as a monocyte-derived TPA stimulation (6,15).

cell line and is known to differentiate into macrophage-like cells by This cell line is also known to synthesize activin and leukemia

inhibitory factor, and to express scavenger receptor by TPA stimulation (16-18). study, THP-1

cell is shown to synthesize CNP.

In the present

Its production is augmented more than

lOO-fold by TPA stimulation but not at all by the addition of FCS (Fig. Sa). Furthermore, we have very recently confirmed the presence of CNP immunoreactivity

in the TPA-supplemented

culture medium of HL-60 cells, another leukemia cell line which is also differentiated into macrophage-like cells by TPA stimulation (to be published). These results suggest that THP-1 cells might actively produce CNP after differentiation

into macrophage-like

cells by TPA

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It should be confirmed whether normal macrophages can produce CNP, since

THP-1 cell is an established cell line and THP-l-derived

macrophage-like cells might have

different properties. Macrophages are well known as phagocytic cells but are also secretory cells producing growth factors and cytokines.

In the injured artery, monocytes first adhere to the surface of

injured endothelial cells and then invade the subendothelial space. During the latter process, monocytes are transformed into activated macrophages, which in turn start secreting growth factors and stimulating the growth of vascular smooth muscle cells. On the other hand, T-cell derived interferon-7

is known as a physiological growth inhibitory factor of vascular smooth

muscle cells (19). We have recently found that CNP has a growth inhibitory effect on cultured rat vascular smooth muscle cells (4). Thus, if activated macrophages could synthesize and secrete CNP, as in the case of the TPA-stimulated THP-1 cells, CNP should be recognized as a new factor in regulating proliferation (contractile-type)

of vascular smooth muscle cells in vivo.

Normal

vascular smooth muscle cells mainly express GC-A, a receptor specific for

ANP and BNP, while cultured vascular smooth muscle cells, corresponding to synthetic-type cells in the atherosclerotic region, actively express CNP specific GC-B.

All of these facts

support the hypothesis that CNP secreted from activated macrophages participates as a local mediator in the growth regulation of vascular smooth muscle cells in the development

of

atherosclerosis. CNP has been recognized as a neuropeptide, being localized in brain and spinal cord and functioning in the regulation of body fluid volume and blood pressure in the central nervous system. By the present identification of CNP in the phorbol ester-stimulated THP-1 cell line, however, CNP may be thought to be synthesized in the peripheral tissue and to have a potential physiological function in the blood cell-vascular system through the paracrine mechanism, in addition to its function as a neuropeptide. We have surveyed for unidentified peptides in mammalian tissue using a bioassay for stimulant or relaxant effect on smooth muscle preparations (20,21). Since tissue concentrations of bioactive peptides are generally low, a large quantity of tissue is required for isolation of new peptides.

Especially in the case of human peptides, it is practically impossible to collect

enough tissue for isolation of unknown peptides. On the other hand, many cell lines established from human tissue or carcinomas have recently become available, which have already been transformed but still retain normal cell function to some extent. Thus, we began a search for new peptides in a series of human cell lines and isolated CNP-29 from culture medium of THP-1 cell line.

This evidence reveals that human cell lines are useful sources to identify

unknown bioactive peptides of human origin. Acknowledgments: This work was supported in part by research grants from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare of Japan. 703

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REFERENCES 1. Sudoh, T., Minamino, N., Kangawa, K. & Matsuo, H. (1990) Biochem. Biophys. Res. Commun., m,863870. 2. Wilcox, J.N., Augustine, A., Goeddel, D.V. & Lowe, D.G. (1991) Mol. Cell. Biol., 11, 3454-3462. 3. Ohyama, Y., Miyamoto, K., Saito, Y., Minamino, N., Kangawa, K. & Matsuo, H. (1992) Biochem. Biophys. Res. Commun., =,743749. 4. Furuya, M., Yoshida, M., Hayashi, Y., Ohnuma, N., Minamino, N., Kangawa, K. & Matsuo, H. (1991) Biochem. Biophys. Res. Commun., m,927931. 5. Minamino, N., Makino, Y., Tateyama, H., Kangawa, K. & Matsuo, H. (1991) Biochem. Biophys. Res. Commun., m,535542. 6. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T. & Tada, K. (1980) Int. J. Cancer. 26, 171- 176. 7. Tawaragi, Y., Fuchimura, K., Tanaka, S., Minamino, N., Kangawa, K. & Matsuo, H. (1991) Biochem. Biophys. Res. Commun., m,645651. 8. Kangawa, K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun., m,131-139. 9. Ueda, S., Minamino, N., Aburaya, M., Kangawa, K., Matsukura, S. & Matsuo, H. (1991) Biochem. Biophys. Res. Commun., m,759767. 10. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (1985) Biochem. Biophys. Res. Commun., 129,248255. 11. Tateyama, H., Hino, J., Minamino, N., Kangawa, K., Ogihara, T. & Matsuo, H. (1990) Biochem. Biophys. Res. Commun., m,1080-1087. 12. Chomczynski, P. & Sacci, N. (1986) Anal. Biochem., 162,156159. 13. Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (1990) FEBS Lett., 276, 209- 213. 14. Minamino, N., Kangawa, K. & Matsuo, H. (1990) Biochem. Biophys. Res. Commun., 170, 973-979. 15. Tsuchiya, S., Kobayashi, Y., Goto, Y., Okumura, H., Nakae, S., Konnno, T. & Tada, K. (1982) Cancer. Res., 42, 1530-1536. 16. Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y. & Shibai, H. (1987) Biochem. Biophys. Res. Commun., w,10951103. 17. Abe, T., Murakami, M. Sato, T., Kajiki, M., Ohno, M. & Kodaira, R. (1988) J. Biol. Chem., =,8941-8945. 18. Matsumoto, A., Naito, M., Itakura, H., Ikemoto, S., Asaoka, H., Hayakawa, I., Kanamori, H., Aburatani, H., Takaku, F., Suzuki, H., Kobari, Y., Miyai, T., Takahashi, K., Cohen, E.H., Wydro, R., Housman, D.E. & Kodama, T. (1990) Proc. Natl. Acad. Sci. USA, 87, 9133-9137. 19. Hasson, G.K., Holm, J., Holm, S., Fotev, Z., Hedrich, H.J. & Fingerle, J. (1991) Proc. Natl. Acad. Sci. USA, a, 10530-10534. 20. Kozawa, H., Hino, J., Minamino, N., Kangawa, K. & Matsuo, H. (1991) Biochem. Biophys. Res. Commun., m,588595. 21. Minamino, N., Kangawa, K. & Matsuo, H. (1988) Ann. N. Y. Acad. Sci., x,373-390.

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