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Transforming growth factor-β-induced gene expression of monocyte chemoattractant JE in mouse osteoblastic cells, MC3T3-E1
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1130-1136
October 31, 1991
TRANSFORMING GROWTH FACTOI%~-INDUCED GENE EXPRESSION OF MONOCYTE CIIEMOATTRACTANT J E IN MOUSE OSTEOBLASTIC CELLS, MC3T3-E1
Shigemasa Hanazawa, Akira Takeshita, Yoshio Tsukamoto*, Yas, hiro Kawata, Kazuzo Ohta Iwao Takara, and Shigeo Kitano
Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, J a p a n *Department of Phnrmacology, Osaka Dental College, 1-47, Kyobashi, Higashi-ku, Osaka 540, J a p a n Received September 18, 1991
A recent study demonstrated thnt PDGF-inducible J E is an inflammatory cytokine that directs chemotactic activity of monocytes. Accumulation of m o n o c y t e / m a c r o p h a g e lineage cells at site of b o n e tissue sites is v e r y important for formation of multinucleate osteoclasts, which mediate bone r e s o r p t i o n . Since t r a n s f o r m i n g g r o w t h factor-~(TGF-~) is a p o t e n t r e g u l a t o r in bone remodeling, we e x a m i n e d w h e t h e r TGF-~ induces J E gene expression in mouse osteoblastic cells, MC3T3-E1. TGF-~ induced a m a x i m u m J E mRNA expression at 3 h r a f t e r initiation of t h e cytokine t r e a t m e n t . This m a x i m a l expression was o b s e r v e d in w h e n TGF-~ was u s e d at a c o n c e n t r a t i o n of I ng/ml. The c h e m o t a c t i c activity for h u m a n monocytes was detected in conditioned medium of TGF-~treated cells, and the chemotactic activity was neutralized by a n t i 4 E serum treatment. © 1991
Academic
Press,
Inc.
JE and KC genes were cloned from mouse fibroblasts as plateletderived growth factor(PDGF)-inducible genes(l).
Therefore, many
investigators had assumed that these genes may play a functional role as intracellular mediators of the mitogenic response to PDGF and
*To whom correspondence should be addressed. Abbreviations used: PDGF, platelet-derived growth factor; TGF-~, t r a n s f o r m i n g growth factor-[~; a-MEM, a-modification of Eagle's m i n i m u m essential medium; CINC, cytokine-induced neutrophil chemoattractant; f-Met, formyl-methyonyl-leucine-phenylalanine. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1130
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
other growth factors. However, Rollins et a/.(2-4) recently indicated that the JE gene product has cytokine-like properties and, further, demonstrated that the human homolog of the murine JE is identical to a monocyte chemoattractant. Thus, the murine JE also may be a monocyte chemoattractant. On the other hand, Watanabe et a/.(5) demonstrated that KC is the mouse counterpart of CINC produced by a rat kidney epithelioid cell line. Osteoclasts are multinucleate cells that mediate bone resorption. A very recent study(6) has shown t h a t monocytes/macrophages are capable of differentiating into osteoclasts in cocultures with bone marrow-derived stromal cells. Therefore, accumulation of monocytes at sites of bone tissue may be very important for differentiation of multinucleate
osteoclasts. Although it has been reported t h a t
osteoblasts express gene of many kinds of cytokines in response to osteotropic factors, gene expression of monocyte chemoattractant JE in osteoblasts has not yet been demonstrated. It is very important for understanding
the functional role of osteoblasts in osteoclast
differentiation
to demonstrate
whether
the
cells express the
chemoattractant JE. TGF-[~ is a potent regulatory cytokine in bone remodeling(7,8). For example, this cytokine plays a functional role in the proliferation and differentiation of osteoblasts, and also regulates
differentiation of
osteoclastic cells. Therefore, in this study, we examined inducing effect of TGF-[~ on JE gene expression in mouse osteoblastic cells, MC3T3-E1. Our present study shows that TGF-I~ is a potent inducer of JE gene expression in these cells.
Materials a n d Methods
Reagents: H u m a n recombinant TGF-~ was obtained from King Brewer(Kakogawa, Japan). ~-MEM was obtained from Flow Lab. (McLean, VA ), fetal calf serum, from Hyclone(Logan,UT). nick 1131
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t r a n s l a t i o n kit, from Beringer M a n n h e i m (Beringer Yamanouchi, Tokyo, Japan). f-Met was purchased from Sigma Co.( St.Louis, MO). O s t e o b l a s t i c cell line a n d its culture: MC3T3-E1 cells, a mouse osteoblastic cell line( provided kindly by Dr. H. Kodama, Ohu University, Koriyama, Japan), were cultured to the subconfluent state in a - M E M with 10% fetal calf s e r u m at 3 7 o c and 5% CO 2 in air, washed, and then treated in serum-free a-MEM with test samples at various concentrations. At selected times, total RNA was extracted by the guanidine isothiocyanate procedure (9). C o n d i t i o n e d m e d i u m o f MC3T3-E1 cells: The confluent monolayer of the cells was treated with or without TGF-]~ at 1 ng/ml in serum-free ct-MEM. After incubation for 3 hr, the monolayer was washed five times to remove TGF-~ from the culture. Then the monolayer was further incubated in serum-free a-MEM. After a 24-hr incubation, the c u l t u r e s u p e r n a t a n t (conditioned m e d i u m ) w a s collected. The conditioned medium was assayed for monocyte chemotactic activity. N o r t h e r n b l o t t i n g analysis: Total RNA was separated on 1% agarose gel e l e c t r o p h o r e s i s and b l o t t e d onto a nylon m e m b r a n e ( M S I Magnagraph, Westboad, MA) and hybridized to nick-translated cDNA probes labeled with 5'-[a-32p]-dCTP for JE, KC and ~-actin. After hybridization, the m e m b r a n e s were washed sequentially, dried and then exposed to X-ray film (Eastman Kodak Co., Rochester,NY) at -700 C. ~-actin was used as an internal standard for quantification of total mRNA on each lane of the gel. M o n o c y t e c h e m o t a x i s assay: H u m a n peripheral blood mononuclear cells were isolated by the method utilizing Ficoll-Hypaque(Pharmacia LKB J a p a n , Tokyo, Japan). Monocyte chemotactic activity was m e a s u r e d in a 48-well m i c r o c h a m b e r a p p a r a t u s , as described previously(10), with h u m a n peripheral blood mononuclear cells used as the indicator cells. The conditioned medium t r e a t e d with or without anti-JE rabbit s e r u m ( p r o v i d e d kindly by Dr. B.J.Rollins, D a n a F a r b e r Cancer Institute, Boston, MA) or n o n i m m u n e rabbit s e r u m was diluted in Gey's balanced salt solution containing 1% bovine s e r u m albumin. After the test media had been incubated for 90 min with the indicator cells, the filter of the microchamber apparatus was removed, and the filter was fixed in methanol and then stained with Diff-Quik(Kokusai Shiyaku, Kobe, Japan). The data are expressed as the mean+SD of total n u m b e r of m i g r a t i n g monocytes/oil i m m e r s i o n field in triplicate assays. Results Firstly we examined the effect of TGF-~ on J E gene expression in MC3T3-E 1 cells. When the cells were cultured in the absence of TGF-~ in serum-free a-MEM, no J E mRNA was detected. However, in the presence of the cytokine, a high level of the JE mRNA was detected at 3 1132
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A JE
JE
[~-Actin
[3-Actin
0
1
3
6
12
24
0
Treatment time (hr) 20
(9
B
10
20 ~]
15
"4 15
1
ii 10
Treatmenttime (hr)
0.01 0.1 1 TGF-[~ (ng/ml)
o - .
®
o o.o, o., TGF-~
, l O
(ng]ml)
Fig.1. Time course o f J E mRNA level in MC3T3-E1 cells t r e a t e d with TGF-~. The cells were incubated with or without TGF-~(1 ng/ml) for indicated periods(hr). Then total RNA was isolated, and 5 pg/lane of the RNA applied to Northern blotting analysis(A). Quantification of the J E mRNA levels in panel A was done by d e n s i t o m e t r y and is expressed as a relative intensity(B). Fi~.2. Effect o f TGF-[~ dose o n induction o f J E mRNA expression in MC3T3-E1 cells. The cells were incubated with or without various concentrations of TGF-[3(0.01-10 ng/ml). After 3 hr, total RNA was isolated, and 5 pg/]ane of the RNA was applied to Northern blotting analysis(A). Quantification of the JE mRNA levels in panel A was done by densitometry and is expressed as a relative intensity(B).
h r a f t e r i n i t i a t i o n of t h e cytokine t r e a t m e n t , a n d t h e r e a f t e r t h e m R N A level d e c r e a s e d p r o g r e s s i v e l y u n t i l t h e e x p e r i m e n t w a s t e r m i n a t e d a t 24 hr(Fig.1). Fig.2 s h o w s a d o s e - r e s p o n s e of J E m R N A levels in w h e n t h e cells w e r e t r e a t e d for 3 h r w i t h TGF-~. T G F - ~ a t 1 n g / m l i n d u c e d t h e m a x i m a l J E m R N A level in the cells. O n t h e o t h e r h a n d , t h e m R N A of t h e n e u t r o p h i l c h e m o a t t r a c t t a n t K C w a s n o t d e t e c t e d in t h e cells u n d e r
the same
experimental
conditions as u s e d for J E m R N A e x p r e s s i o n ( d a t a n o t shown). N e x t , we e x a m i n e d t h e p r e s e n c e of t h e f u n c t i o n a l J E g e n e p r o d u c t in c o n d i t i o n e d m e d i u m of T G F - ~ - t r e a t e d cells. T h e c o n d i t i o n e d m e d i u m of TGF-~-treated
cells w a s
diluted,
and
c h e m o t a c t i c a c t i v i t y . Fig.3 s h o w s t h a t a c t i v i t y in t h e c o n d i t i o n e d m e d i u m 1133
assayed
for m o n o c y t e
the monocyte chemotactic
was dilution dependent.
The
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
70 "0
on--
60
~o C E'~ • ~
50
8E mE
8O A
40
!;,-- 30
o_~ 0
0
6~
40
oo
~__= I
1/2
®
60
~E
20
0
~
I
I
1/4
1/8
20 o
Q
Dilution
oo
Fig.3, Monocyte chemotactic activity in conditioned medium of TGF~-treated MC3T3-E1 cells. The cells were incubated with(0) or without ( 0 ) TGF-~(1 ng/ml). After 24 hr, the conditioned medium was prepared and applied to the monocyte chemotactic assay. The results are expressed as mean+SD of triplicates. The number of migrating cells obtained with f-Met(10-8M), as positive control, was 72±2. Fig.4. Neutralizing effect of anti-JE serum on monocyte chemotactic activity in conditioned m e d i u m of TGF-~-treated MC3T3-E 1 cells. The cells were incubated with TGF-~(1 ng/ml). After 24 hr, the conditioned medium was prepared and treated for 90 rain with anti-JE rabbit serum or nonimmunized rabbit serum at a final dilution of 1/500, and then applied to the monocyte chemotactic assay. Control was conditioned medium from cells without TGF-~. The results are expressed as mean+SD of triplicates.
c h e m o t a c t i c a c t i v i t y was m a r k e d l y n e u t r a l i z e d b y t r e a t m e n t of the conditioned m e d i u m with a n t i - J E r a b b i t serum(Fig.4).
Dis~l~on M a n y r e c e n t studies(6,11) h a v e d e m o n s t r a t e d t h a t osteoclasts are d e r i v e d f r o m cells of t h e c i r c u l a t i n g m o n o c y t e / m a c r o p h a g e lineage. T h e r e f o r e , a c c u m u l a t i o n of such cells a t site of bone t i s s u e sites is v e r y i m p o r t a n t for f o r m a t i o n of m u l t i n u c l e a t e osteoclasts. T h e p r e s e n t s t u d y shows t h a t TGF-~ s t r o n g l y i n d u c e d J E m R N A e x p r e s s i o n in m o u s e osteoblasts, MC3T3-E1. To our knowledge, this is t h e first s t u d y to p r e s e n t evidence t h a t osteoblasts express t h e J E gene, whose p r o d u c t is a c h e m o a t t r a c t a n t for circulating monocytes. The J E m R N A was also i n d u c e d b y i n t e r l e u k i n - l - ~ a n d t u m o r necrosis factora, p o t e n t osteotropic cytokine ( u n p u b l i s h e d data). O n t h e o t h e r h a n d , 1134
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the mRNA of the neutrophil chemoattractant KC was not expressed in the cells treated with TGF-~, tumor necrosis factor-a, interleukin-1, lipopolysaccharide, or TPA, a potent activator of protein kinase C (unpublished data). These results are very interesting to us, because it is known that both JE and KC genes were coexpressed in fibroblasts and
peritoneal
macrophages
treated
with
interleukin-1
or
lipopolysaccharide(12-14). This selectivity of osteoblasts for gene expression of monocyte chemoattractant JE suggests the possibility that
these
cells
can
cause
the
selective
accumulation
of
monocyte/macrophage lineage cells, which cells subsequently give rise to osteoclasts in bone tissues. Quite recently Udagawa et a/.(6) d e m o n s t r a t e d t h a t m a t u r e monocytes and macrophages differentiate into osteoclasts in cooperation with stromal cells. Also some recent studies(15-17) have suggested that macrophage-colony stimulating factor produced by stromal cells plays an important role in the proliferation and differentiation of osteoclast progenitors. Therefore, in future studies, it will be very important to demonstrate w h e t h e r JE acts as a c h e m o a t t r a c t a n t for osteoclast progenitor, and cooperates with macrophage-colony stimulating
factor
in
proliferation
and
differentiation of osteoclast progenitors. Acknowledgments We thank Dr. C.D. Stiles of Harvard University for providing mouse JE and KC cDNA probes. And we also express our gratitude to Dr. B.J. Rollins of Dana-Faber Cancer Institute for providing anti-JE serum.
References 1. Cochran,B.H., Reffel,A.C., and .Stiles,C.D. (1983) Cell 33, 939-947. 2. Rollins,B.J., Morrison,E.D., and Stiles,C.D. (1988) Proc. Natl.Acad. Sci. USA. 85, 3738-3742. 3. Rollins,B.J., Stier,P. Ernst,T.E., and Wong,G.G. (1989) Mol. Cell. Biol. 9, 4687-4695. 4. Rollins,B.J., Yoshimura, T., Leonard,E.J., and Pober,J.S. (1990) Amer. J. Pathol. 136, 1229-1233. 1135
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Watanabe.K., Konishi,K., Fujioka, M., Kinoshita,S., and Nakagawa,H. (1989) J. Biol. Chem. 264, 19559-19563. 6. Udagawa,N., Takahashi, N., Akatsu,T., Tanaka,H., Sakaki,T., Nishihara,T., Koga,T., Martin,T.J., and Suda,T. (1990) Proc. Natl. Acad. Sci. USA. 87, 7260-7264. 7. Tashijian,A. Voekel,H.E.F., Lazzaro,M., Singer,F.R., Roberts,A.B., Derynck,K.R., Winkler,M.E., and Levine,L. (1985) Proc. Natl Acad. Sci. USA. 82, 4535-4538. . Robey,P.G., Young,M.F., Franders,K.C., Roche,N.S., Kondaiah,P., Reddi,A.H., Termine,J.D., Sporn,M.B., and Robert,A.B. (1987) J. Cell Biol. 105, 457-463. 9. Chirgwin,J.M.A., Przbyla,A.E., Macdonard,R.J., and Rutter,W.J. (1979) Biochemistry 18, 5295-5299. 10. Falk,W., Goodwin,R.H., and Leonard,E.J. (1980) J. Immunol. Methods 33,239-247. 11. Mundy,G.R., and Roodman,G.D. (1987) Osteoclast ontology and function. In Bone and Mineral Res.(Peck,W.A. ed.) Elsevier, Amsterdam, 5,209-279. 12. Koerner,T.J., Hamilton,T.A., Introna,M., Tannenbaum,C., Bast,R.C.,Tannenbaum,C.S., and Hamilton,T.A. (1987) BiChem. Biophys. Res. Commun. 149, 969-974. 13. Tannenbaum,C.S>Major,J.A., Poptic,E.J., DiCorleto,P.Eo, and Hamilton,T.A.J. Cell Physiol. 144. 77-83. 14. Hall,D.J., Brownlee,C., and Stiles,C.D. (1989) J. Cell. Physiol. 141, 154-159. 15. Jedzejcczak,W.W., Bartocci,A., Ferrante,A.W., Ansari,A.A., Sell,K.W., P18)ollard,J.W., and Stanley,C.D. (1990) Proc. Natl. Acad. Sci. USA. 87, 4858-4832. 16. Yoshida,H., Hayashi,T., Kunisada,T., Ogawa,M., Nishikawa,S., Okumura, H., Suda,T., Shultz,L.D., and Nishikawa,S. (1990) Nature 345, 442-444. 17. Kodama,H., Nose,M., Nishida,S., and Yamazaki,A. (1991) J. Exp. Med. 173, 1291-1294.
1136
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1130-1136
October 31, 1991
TRANSFORMING GROWTH FACTOI%~-INDUCED GENE EXPRESSION OF MONOCYTE CIIEMOATTRACTANT J E IN MOUSE OSTEOBLASTIC CELLS, MC3T3-E1
Shigemasa Hanazawa, Akira Takeshita, Yoshio Tsukamoto*, Yas, hiro Kawata, Kazuzo Ohta Iwao Takara, and Shigeo Kitano
Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, J a p a n *Department of Phnrmacology, Osaka Dental College, 1-47, Kyobashi, Higashi-ku, Osaka 540, J a p a n Received September 18, 1991
A recent study demonstrated thnt PDGF-inducible J E is an inflammatory cytokine that directs chemotactic activity of monocytes. Accumulation of m o n o c y t e / m a c r o p h a g e lineage cells at site of b o n e tissue sites is v e r y important for formation of multinucleate osteoclasts, which mediate bone r e s o r p t i o n . Since t r a n s f o r m i n g g r o w t h factor-~(TGF-~) is a p o t e n t r e g u l a t o r in bone remodeling, we e x a m i n e d w h e t h e r TGF-~ induces J E gene expression in mouse osteoblastic cells, MC3T3-E1. TGF-~ induced a m a x i m u m J E mRNA expression at 3 h r a f t e r initiation of t h e cytokine t r e a t m e n t . This m a x i m a l expression was o b s e r v e d in w h e n TGF-~ was u s e d at a c o n c e n t r a t i o n of I ng/ml. The c h e m o t a c t i c activity for h u m a n monocytes was detected in conditioned medium of TGF-~treated cells, and the chemotactic activity was neutralized by a n t i 4 E serum treatment. © 1991
Academic
Press,
Inc.
JE and KC genes were cloned from mouse fibroblasts as plateletderived growth factor(PDGF)-inducible genes(l).
Therefore, many
investigators had assumed that these genes may play a functional role as intracellular mediators of the mitogenic response to PDGF and
*To whom correspondence should be addressed. Abbreviations used: PDGF, platelet-derived growth factor; TGF-~, t r a n s f o r m i n g growth factor-[~; a-MEM, a-modification of Eagle's m i n i m u m essential medium; CINC, cytokine-induced neutrophil chemoattractant; f-Met, formyl-methyonyl-leucine-phenylalanine. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1130
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
other growth factors. However, Rollins et a/.(2-4) recently indicated that the JE gene product has cytokine-like properties and, further, demonstrated that the human homolog of the murine JE is identical to a monocyte chemoattractant. Thus, the murine JE also may be a monocyte chemoattractant. On the other hand, Watanabe et a/.(5) demonstrated that KC is the mouse counterpart of CINC produced by a rat kidney epithelioid cell line. Osteoclasts are multinucleate cells that mediate bone resorption. A very recent study(6) has shown t h a t monocytes/macrophages are capable of differentiating into osteoclasts in cocultures with bone marrow-derived stromal cells. Therefore, accumulation of monocytes at sites of bone tissue may be very important for differentiation of multinucleate
osteoclasts. Although it has been reported t h a t
osteoblasts express gene of many kinds of cytokines in response to osteotropic factors, gene expression of monocyte chemoattractant JE in osteoblasts has not yet been demonstrated. It is very important for understanding
the functional role of osteoblasts in osteoclast
differentiation
to demonstrate
whether
the
cells express the
chemoattractant JE. TGF-[~ is a potent regulatory cytokine in bone remodeling(7,8). For example, this cytokine plays a functional role in the proliferation and differentiation of osteoblasts, and also regulates
differentiation of
osteoclastic cells. Therefore, in this study, we examined inducing effect of TGF-[~ on JE gene expression in mouse osteoblastic cells, MC3T3-E1. Our present study shows that TGF-I~ is a potent inducer of JE gene expression in these cells.
Materials a n d Methods
Reagents: H u m a n recombinant TGF-~ was obtained from King Brewer(Kakogawa, Japan). ~-MEM was obtained from Flow Lab. (McLean, VA ), fetal calf serum, from Hyclone(Logan,UT). nick 1131
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t r a n s l a t i o n kit, from Beringer M a n n h e i m (Beringer Yamanouchi, Tokyo, Japan). f-Met was purchased from Sigma Co.( St.Louis, MO). O s t e o b l a s t i c cell line a n d its culture: MC3T3-E1 cells, a mouse osteoblastic cell line( provided kindly by Dr. H. Kodama, Ohu University, Koriyama, Japan), were cultured to the subconfluent state in a - M E M with 10% fetal calf s e r u m at 3 7 o c and 5% CO 2 in air, washed, and then treated in serum-free a-MEM with test samples at various concentrations. At selected times, total RNA was extracted by the guanidine isothiocyanate procedure (9). C o n d i t i o n e d m e d i u m o f MC3T3-E1 cells: The confluent monolayer of the cells was treated with or without TGF-]~ at 1 ng/ml in serum-free ct-MEM. After incubation for 3 hr, the monolayer was washed five times to remove TGF-~ from the culture. Then the monolayer was further incubated in serum-free a-MEM. After a 24-hr incubation, the c u l t u r e s u p e r n a t a n t (conditioned m e d i u m ) w a s collected. The conditioned medium was assayed for monocyte chemotactic activity. N o r t h e r n b l o t t i n g analysis: Total RNA was separated on 1% agarose gel e l e c t r o p h o r e s i s and b l o t t e d onto a nylon m e m b r a n e ( M S I Magnagraph, Westboad, MA) and hybridized to nick-translated cDNA probes labeled with 5'-[a-32p]-dCTP for JE, KC and ~-actin. After hybridization, the m e m b r a n e s were washed sequentially, dried and then exposed to X-ray film (Eastman Kodak Co., Rochester,NY) at -700 C. ~-actin was used as an internal standard for quantification of total mRNA on each lane of the gel. M o n o c y t e c h e m o t a x i s assay: H u m a n peripheral blood mononuclear cells were isolated by the method utilizing Ficoll-Hypaque(Pharmacia LKB J a p a n , Tokyo, Japan). Monocyte chemotactic activity was m e a s u r e d in a 48-well m i c r o c h a m b e r a p p a r a t u s , as described previously(10), with h u m a n peripheral blood mononuclear cells used as the indicator cells. The conditioned medium t r e a t e d with or without anti-JE rabbit s e r u m ( p r o v i d e d kindly by Dr. B.J.Rollins, D a n a F a r b e r Cancer Institute, Boston, MA) or n o n i m m u n e rabbit s e r u m was diluted in Gey's balanced salt solution containing 1% bovine s e r u m albumin. After the test media had been incubated for 90 min with the indicator cells, the filter of the microchamber apparatus was removed, and the filter was fixed in methanol and then stained with Diff-Quik(Kokusai Shiyaku, Kobe, Japan). The data are expressed as the mean+SD of total n u m b e r of m i g r a t i n g monocytes/oil i m m e r s i o n field in triplicate assays. Results Firstly we examined the effect of TGF-~ on J E gene expression in MC3T3-E 1 cells. When the cells were cultured in the absence of TGF-~ in serum-free a-MEM, no J E mRNA was detected. However, in the presence of the cytokine, a high level of the JE mRNA was detected at 3 1132
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A JE
JE
[~-Actin
[3-Actin
0
1
3
6
12
24
0
Treatment time (hr) 20
(9
B
10
20 ~]
15
"4 15
1
ii 10
Treatmenttime (hr)
0.01 0.1 1 TGF-[~ (ng/ml)
o - .
®
o o.o, o., TGF-~
, l O
(ng]ml)
Fig.1. Time course o f J E mRNA level in MC3T3-E1 cells t r e a t e d with TGF-~. The cells were incubated with or without TGF-~(1 ng/ml) for indicated periods(hr). Then total RNA was isolated, and 5 pg/lane of the RNA applied to Northern blotting analysis(A). Quantification of the J E mRNA levels in panel A was done by d e n s i t o m e t r y and is expressed as a relative intensity(B). Fi~.2. Effect o f TGF-[~ dose o n induction o f J E mRNA expression in MC3T3-E1 cells. The cells were incubated with or without various concentrations of TGF-[3(0.01-10 ng/ml). After 3 hr, total RNA was isolated, and 5 pg/]ane of the RNA was applied to Northern blotting analysis(A). Quantification of the JE mRNA levels in panel A was done by densitometry and is expressed as a relative intensity(B).
h r a f t e r i n i t i a t i o n of t h e cytokine t r e a t m e n t , a n d t h e r e a f t e r t h e m R N A level d e c r e a s e d p r o g r e s s i v e l y u n t i l t h e e x p e r i m e n t w a s t e r m i n a t e d a t 24 hr(Fig.1). Fig.2 s h o w s a d o s e - r e s p o n s e of J E m R N A levels in w h e n t h e cells w e r e t r e a t e d for 3 h r w i t h TGF-~. T G F - ~ a t 1 n g / m l i n d u c e d t h e m a x i m a l J E m R N A level in the cells. O n t h e o t h e r h a n d , t h e m R N A of t h e n e u t r o p h i l c h e m o a t t r a c t t a n t K C w a s n o t d e t e c t e d in t h e cells u n d e r
the same
experimental
conditions as u s e d for J E m R N A e x p r e s s i o n ( d a t a n o t shown). N e x t , we e x a m i n e d t h e p r e s e n c e of t h e f u n c t i o n a l J E g e n e p r o d u c t in c o n d i t i o n e d m e d i u m of T G F - ~ - t r e a t e d cells. T h e c o n d i t i o n e d m e d i u m of TGF-~-treated
cells w a s
diluted,
and
c h e m o t a c t i c a c t i v i t y . Fig.3 s h o w s t h a t a c t i v i t y in t h e c o n d i t i o n e d m e d i u m 1133
assayed
for m o n o c y t e
the monocyte chemotactic
was dilution dependent.
The
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
70 "0
on--
60
~o C E'~ • ~
50
8E mE
8O A
40
!;,-- 30
o_~ 0
0
6~
40
oo
~__= I
1/2
®
60
~E
20
0
~
I
I
1/4
1/8
20 o
Q
Dilution
oo
Fig.3, Monocyte chemotactic activity in conditioned medium of TGF~-treated MC3T3-E1 cells. The cells were incubated with(0) or without ( 0 ) TGF-~(1 ng/ml). After 24 hr, the conditioned medium was prepared and applied to the monocyte chemotactic assay. The results are expressed as mean+SD of triplicates. The number of migrating cells obtained with f-Met(10-8M), as positive control, was 72±2. Fig.4. Neutralizing effect of anti-JE serum on monocyte chemotactic activity in conditioned m e d i u m of TGF-~-treated MC3T3-E 1 cells. The cells were incubated with TGF-~(1 ng/ml). After 24 hr, the conditioned medium was prepared and treated for 90 rain with anti-JE rabbit serum or nonimmunized rabbit serum at a final dilution of 1/500, and then applied to the monocyte chemotactic assay. Control was conditioned medium from cells without TGF-~. The results are expressed as mean+SD of triplicates.
c h e m o t a c t i c a c t i v i t y was m a r k e d l y n e u t r a l i z e d b y t r e a t m e n t of the conditioned m e d i u m with a n t i - J E r a b b i t serum(Fig.4).
Dis~l~on M a n y r e c e n t studies(6,11) h a v e d e m o n s t r a t e d t h a t osteoclasts are d e r i v e d f r o m cells of t h e c i r c u l a t i n g m o n o c y t e / m a c r o p h a g e lineage. T h e r e f o r e , a c c u m u l a t i o n of such cells a t site of bone t i s s u e sites is v e r y i m p o r t a n t for f o r m a t i o n of m u l t i n u c l e a t e osteoclasts. T h e p r e s e n t s t u d y shows t h a t TGF-~ s t r o n g l y i n d u c e d J E m R N A e x p r e s s i o n in m o u s e osteoblasts, MC3T3-E1. To our knowledge, this is t h e first s t u d y to p r e s e n t evidence t h a t osteoblasts express t h e J E gene, whose p r o d u c t is a c h e m o a t t r a c t a n t for circulating monocytes. The J E m R N A was also i n d u c e d b y i n t e r l e u k i n - l - ~ a n d t u m o r necrosis factora, p o t e n t osteotropic cytokine ( u n p u b l i s h e d data). O n t h e o t h e r h a n d , 1134
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the mRNA of the neutrophil chemoattractant KC was not expressed in the cells treated with TGF-~, tumor necrosis factor-a, interleukin-1, lipopolysaccharide, or TPA, a potent activator of protein kinase C (unpublished data). These results are very interesting to us, because it is known that both JE and KC genes were coexpressed in fibroblasts and
peritoneal
macrophages
treated
with
interleukin-1
or
lipopolysaccharide(12-14). This selectivity of osteoblasts for gene expression of monocyte chemoattractant JE suggests the possibility that
these
cells
can
cause
the
selective
accumulation
of
monocyte/macrophage lineage cells, which cells subsequently give rise to osteoclasts in bone tissues. Quite recently Udagawa et a/.(6) d e m o n s t r a t e d t h a t m a t u r e monocytes and macrophages differentiate into osteoclasts in cooperation with stromal cells. Also some recent studies(15-17) have suggested that macrophage-colony stimulating factor produced by stromal cells plays an important role in the proliferation and differentiation of osteoclast progenitors. Therefore, in future studies, it will be very important to demonstrate w h e t h e r JE acts as a c h e m o a t t r a c t a n t for osteoclast progenitor, and cooperates with macrophage-colony stimulating
factor
in
proliferation
and
differentiation of osteoclast progenitors. Acknowledgments We thank Dr. C.D. Stiles of Harvard University for providing mouse JE and KC cDNA probes. And we also express our gratitude to Dr. B.J. Rollins of Dana-Faber Cancer Institute for providing anti-JE serum.
References 1. Cochran,B.H., Reffel,A.C., and .Stiles,C.D. (1983) Cell 33, 939-947. 2. Rollins,B.J., Morrison,E.D., and Stiles,C.D. (1988) Proc. Natl.Acad. Sci. USA. 85, 3738-3742. 3. Rollins,B.J., Stier,P. Ernst,T.E., and Wong,G.G. (1989) Mol. Cell. Biol. 9, 4687-4695. 4. Rollins,B.J., Yoshimura, T., Leonard,E.J., and Pober,J.S. (1990) Amer. J. Pathol. 136, 1229-1233. 1135
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5. Watanabe.K., Konishi,K., Fujioka, M., Kinoshita,S., and Nakagawa,H. (1989) J. Biol. Chem. 264, 19559-19563. 6. Udagawa,N., Takahashi, N., Akatsu,T., Tanaka,H., Sakaki,T., Nishihara,T., Koga,T., Martin,T.J., and Suda,T. (1990) Proc. Natl. Acad. Sci. USA. 87, 7260-7264. 7. Tashijian,A. Voekel,H.E.F., Lazzaro,M., Singer,F.R., Roberts,A.B., Derynck,K.R., Winkler,M.E., and Levine,L. (1985) Proc. Natl Acad. Sci. USA. 82, 4535-4538. . Robey,P.G., Young,M.F., Franders,K.C., Roche,N.S., Kondaiah,P., Reddi,A.H., Termine,J.D., Sporn,M.B., and Robert,A.B. (1987) J. Cell Biol. 105, 457-463. 9. Chirgwin,J.M.A., Przbyla,A.E., Macdonard,R.J., and Rutter,W.J. (1979) Biochemistry 18, 5295-5299. 10. Falk,W., Goodwin,R.H., and Leonard,E.J. (1980) J. Immunol. Methods 33,239-247. 11. Mundy,G.R., and Roodman,G.D. (1987) Osteoclast ontology and function. In Bone and Mineral Res.(Peck,W.A. ed.) Elsevier, Amsterdam, 5,209-279. 12. Koerner,T.J., Hamilton,T.A., Introna,M., Tannenbaum,C., Bast,R.C.,Tannenbaum,C.S., and Hamilton,T.A. (1987) BiChem. Biophys. Res. Commun. 149, 969-974. 13. Tannenbaum,C.S>Major,J.A., Poptic,E.J., DiCorleto,P.Eo, and Hamilton,T.A.J. Cell Physiol. 144. 77-83. 14. Hall,D.J., Brownlee,C., and Stiles,C.D. (1989) J. Cell. Physiol. 141, 154-159. 15. Jedzejcczak,W.W., Bartocci,A., Ferrante,A.W., Ansari,A.A., Sell,K.W., P18)ollard,J.W., and Stanley,C.D. (1990) Proc. Natl. Acad. Sci. USA. 87, 4858-4832. 16. Yoshida,H., Hayashi,T., Kunisada,T., Ogawa,M., Nishikawa,S., Okumura, H., Suda,T., Shultz,L.D., and Nishikawa,S. (1990) Nature 345, 442-444. 17. Kodama,H., Nose,M., Nishida,S., and Yamazaki,A. (1991) J. Exp. Med. 173, 1291-1294.
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