Vol.
187,
September
No. 30,
BIOCHEMICAL
3, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1992
MULTIPLE
GROWTH
CHICKEN David W. Burt,
Jaquie M. Boswell,
Institute
mRNAS ARE EXPRESSED
ADIPOCYTE
Department AFRC
FACTOR
Edinburgh Roslin,
305
IN
CELLS
Ian R. Paton and Simon C. Butterwith
of Cellular
of Animal
PRECURSOR
1298-l
and Molecular
Physiology
Biology
and Genetics
Research
Research Station
Midlothian
EH25 9PS, UK
Received July 6, 1992 SUMMARY: We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-02, TGF-B3, TGF-I34 and bFGF genes in chicken adipocyte precursors. We also detect GH-independant, but differentiation-dependant IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation. 0 1992Academic Press,1°C.
Adipose tissue growth occurs through a combination of adipocyte hyperplasia and hypertrophy. Hyperplasia occurs via the proliferation and differentiation of adipocyte precursors. This process is under the control of hormones and growth factors, some of which may act in an autocrine and/or paracrine manner. Proliferation of primary and established adipogenic cell lines is stimulated by IGF-I, IGF-II, TGF-El, PDGF, aFGF and bFGF (l-2). Differentiation
of adipogenic cells is
stimulated by IGF-I (3-4) and inhibited by TGF-Dl (5-8). The effects of bFGF on adipocyte differentiation is cell-dependent, and can be inhibitory (9), stimulatory (10) or have no effect (8). The detection of IGF-I and TGF-Ol mRNAs in adipocyte precursors (11-13) suggests that these factors may act as autocrine and/or paracrine regulators of adipocyte cell proliferation differentiation.
and
Since we have shown that bFGF, TGF-Bl and IGF-I can influence chicken adipocyte precursor cell proliferation and differentiation in vitro (1, 2, 8), this study was undertaken to investigate the ability of these cells to express these growth factor genes. Our results indicate that mRNAs for bFGF, TGF-82, TGF-03, TGF-IJ4 and IGF-I are present in both proliferating and differentiated chicken adipocyte precursors. Taken together with the known action of these growth factors in Abbreviations: RT, reverse transcription; PCR, polymerase chain reaction; bp, base pairs; TGF-R, transforming growth factor-@ IGF, insulin-like growth factor; ; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; LPL, lipoprotein lipase; GH, Growth Hormone. 0006-291X/92
Copyright All rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1298
Vol.
187,
No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
vitro, our results support the hypothesis that these growth factors act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation. MATERIALS
AND METHODS
Preparation of Adipocyte Precursor Cell Cultures Chicken adipocyte precursors were prepared as described previously (14). They were cultured in Medium 199 supplemented with 4% (v/v) Ultroser G (Gibco, Paisley, Strathclyde, Scotland, U.K.) at 37OC in an atmosphere containing 5% CO;! with changes of medium every 2 days. Subconfluent monolayers of proliferating cells were harvested after 4 days in culture and differentiated adipocyte precursors 10 days after reaching confluence. Synthetic Oligonucleotides Synthetic oligonucleotides were synthesised by Oswel DNA services (Edinburgh University). The sequences of the primer pairs are shown in Table 1. An internal oligonucleotide specific for the IGF-I RT-PCR product (570-590 CTGTGCTCCAATAAAGCCACC; 15) was used as a probe for verification. Detection of mRNAs by RT-PCR Total RNA was isolated from whole tissues and cultured cells using the standard guanidine thiocyanate extraction procedure (16). First-strand cDNA was synthesised as described (17) using 1 ug of total RNA and random hexamers. For each RT reaction, a blank was prepared using all reagents except the RNA sample. PCR was carried out as described (17) using the primer pairs shown in Table 1. The reactions were denatured at 94OC (1 min), annealed at 50°C or 65OC (2 mins) and extended at 72OC (3 mins) for 20-35 cycles in a Biometra TRIO-BLOCK thermal cycler. Most primers were annealed at 5OOC,except bFGF at 65OC. A PCR blank was run as a control, using the RT blank. PCR products were resolved on 5% non-denaturing polyacrylamide gels along with molecular weight markers. In all experiments the RT-PCR reagent blank was negative.
Southern Blotting and Hybridisation Following electrophoresis, gels were treated in 0.5 M NaOWl.5 M NaCl for 30 mins, then in 1 M NH40Ac/0.02 M NaOH for 15 mins, transferred to Zetaprobe (Bio-Rad, Richmond, CA, U.S.A) membranes in 1 M NHdOAc/0.02 M NaOH by capillary action, rinsed in 2x SSC and baked. Oligonucleotides were labelled with 32P to a specific activity of 2x108 c.p.m. / ug using T4 polynucleotide kinase (16) and filters were hybridised as recommended by the manufacturer (BioRad). RESULTS Design and Validation
of Gene-Specific
Primers
for RT-PCR
Northern blot analysis lacks sufficient sensitivity to detect transcripts in small amounts of tissues, especially when those transcripts have short half-lives, as is the case for most growth factor mRNAs. We used the technique of RT-PCR, a technique that allows the detection of multiple mRNAs when cell numbers are small and/or mRNA transcripts are rare (17). We designed oligonucleotide primer pairs that bracket target sequences specific for each of the mRNAs listed in Table 1. We selected targets for amplification 150 to 300-bp in length containing a unique restriction enzyme site. The primer pairs were selected to cross intron-exon junctions, consequently genomic DNA would produce a larger product than the cDNA, allowing us to distinguish between signals due to mRNA or contaminating genomic DNA. The location of intronexon junctions was known in the case of some chicken genes (B-actin, 18; TGF-82, 19; LPL, 20; IGF-I, 21) or was inferred from homologous or duplicated genes (TGF-84, 22; TGF-83, 23; bFGF, 24). PCR products larger than the size expected for cDNA were not detected following RT1299
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BIOCHEMICAL
1992
Table Gene
AND
1. Growth
factor
PCR Primer
Product Size (bp)
BIOPHYSICAL
primer
RESEARCH
COMMUNICATIONS
pairs
Restriction Site
Restriction Products
5’-R-actin 3’-l3-actin
280
TGTGGTATCCATGAAACTA ATTCATCGTACTCCTGCTT
Kpd
185+95
(1)
5’-TGF-l32 3’.TGF-I32
269
AGGAATGTGCAGGATAATT ATTTTGGGTGTTTTGCCAA
Hi&II
192+77
(2)
5’-TGF-I33 3’-TGF-I33
200
GAGCAGAGTTCCGGGTGCT GTGCAGAAGCCACTCACGC
SSfI
137+35+28
(3)
5’.TGF-134 3’.TGF-I34
160
CACCGACTACTGCTTCGGC GTCGGCGCTCCAGATGTAC
PSfI
1 I o+so
(4)
5’-IGF-I 3’=IGF-I
200
GTATGTGGAGACAGAGGCTTC TTTGGCATATCAGTGTGGCGC
BUmHI
150+50
(5)
5’-LPL 3’.LPL
298
TAGACCAGCCATTCCTGAT TCTCCTTTACCAAGACGTG
P.UI
151+146
(6)
5’-bFGF 3’-bFGF
270
GATCCGCACATCAAACTGC GATACGTTTCTGTCCAGGTCC
Hitlfl
I%+112
(7)
All primer sequences given in 5’ to 3’ orientation. ‘Position: 3506-4140 (18): 2Position: 64529311 (19); 3Position: 482-681 (31); 4Position: X17-979 (32); 5Position: 439-638 (15); Qosition: 1203-1500 (33); 7Position: 432-701. PCR of any of the RNA sequences.
Since
kactin
samples, mRNA
indicating
that they were indeed
free of contaminating
DNA
is present in all tissues, primers for this gene were used to
monitor for efficient cDNA synthesis between different RNA samples. Growth
Factor
Expression
in Proliferating
and Differentiated
Chicken
Adipocyte
Precursor Cells Total RNA was prepared from either proliferating or differentiated cells from three independent primary cultures of chicken adipocyte precursors. First strand cDNA was synthesised and analysed for the mRNAs listed in Table 1. The growth factor mRNAs for TGF-02, TGF-l33, TGF-04 and bFGF were detected in both proliferating and differentiated adipocyte precursors (Fig. 1). The identity of PCR products was established by restriction enzyme mapping (Fig. 2). The PCR fragment amplified with the TGF-132 primers cut with Hi&III
to give two fragments 192-bp and
77-bp. The TGF-I33 products were digested with SstI to give 137-bp, 35-bp and 28-bp. The TGF-134 products were cleaved by PstI, to give lOO-bp and 50-bp. The PCR products for bFGF were confirmed by digestion with Hi@ to give fragments of 158-bp and 112-bp. IGF-I mRNA was absent or very low in proliferating adipocyte precursors (absent in 2/3 cell cultures), but was more abundant in differentiated cells (present in 3/3 cell cultures, an example is shown in Fig. 2). The identity of the IGF-I transcripts was confirmed by the size of the PCR product (200-bp), by hybridisation with an internal oligonucleotide probe and by cleavage with BumHI to generate a 150-bp band, that also hybridised to the same probe. 1300
Vol.
187,
No.
3, 1992
BIOCHEMICAL
I
Proliferating cells
AND
BIOPHYSICAL
Non-proliferating cells I
RESEARCH
COMMUNICATIONS
Blank I
pactin
TGF-p3
=
TGF-p
bFGF
Fig. 1. Expression of B-actin. TGF-82. TGF-03. TGF-D4. bFGF and LPL in proliferating and differentiated adipocyte precursors. RNA samples were reverse transcribed and amplified by PCR with gene-specific primers. Products were resolved on 5% non-denaturing polyacrylamide gels along with molecular weight markers (not shown). All the PCR products were of the expected size. PCR conditions: I&actin. TGF-132. TGF-134, LPL. 20 cycles; TGF-B3, 25 cycles; bFGF, 35 cycles.
pactin
TGF -
bFGF ml
TGF-p m leP+I
/+I
IGF-I
TGF - 82 .NP+I-P+I
LPL eNP+I
-NP+I
200
Fig. 2. Verification of TGF-f32, TGF-03, TGF-04, bFGF, IGF-I and LPL PCR amplification products by restriction enzyme mapping. cDNA from proliferating and differentiated adipocyte precursors was amplified with gene-specific primers. The amplified products were cleaved with the indicated restriction enzymes and the fragments separated on 5% non-denaturing polyacrylamide gels along with molecular weight markers (m). The PCR products from the amplification of cDNA with the IGF-I primers were processed as above, transferred to a nylon membrane and hybridised at high stringency to an internal oligonucleotide probe (see Materials & Methods). -, uncut PCR product; +, digest of PCR product; P, proliferating adipocyte precursors; NP, non-proliferating, differentiated adipocyte precursors.
1301
Vol.
187,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
paciin
TGF
- p2
TGF
- p3
TGF
- p4 bFGF
Fig. 3. Expression of TGF-82, TGF-83, TGF-1.54, bFGF and &actin genes in various chicken tissues. RNA samples were extracted from heart, fat pad, brain, ovary, kidney, leg muscle, liver and breast muscle from a 6 week old broiler hen, reverse transcribed and amplified by PCR with gene-specific primers. Products were resolved on 57~ non-denaturing polyacrylamide gels along with molecular weight markers. All the PCR products were of the expected size. B, RT-PCR blank. PCR conditions: all 30 cycles except bFGF: 3.5 cycles.
Expression
of Growth
Factor
mRNAs
in Adipose
Tissue
and
Various
Other
Tissues Total cellular growth
factor
RNA isolated mRNAs,
in vivo. For comparison, leg muscle,
mapping
TGF-IJ3
mRNA
lower levels were found
detected
giving
in vitro were also expressed
(Figs.
Verification
(data not shown),
samples. The growth
with BamHI,
nucleotide
transcripts
fat pads) was assayed for the same
from a range of tissues (heart, brain, ovary, kidney,
was also assayed by RT-PCR. enzyme
cell RNA
by digestion
TGF-I33 mRNA
whether isolated
in all the tissues examined
from the reported variable.
total RNA
by restriction
precursor
were detected confirmed
to determine
liver, breast muscle)
was established adipocyte
from adipose tissue (abdominal
factor mRNAs 3-4). The identity
rise to products
of RT-PCR
as described TGF-62,
of 150-bp
in Fig. 2, for
TGF-R4
of the IGF-I
was reproducibly
in liver. However,
detected
in fat pad, brain
and IGF-I
products
was
and 50-bp as predicted
sequence (15). The assays for TGF-I33 and bFGF mRNAs
with kidney,
products
and ovary
were more (Fig. 3), and
heart and skeletal muscle tissues. We were unable to detect
the B-actin product
was found at equivalent
levels in all tissues,
Fig. 4. Detection of IGF-I mRNA in various chicken tissues by RT-PCR. cDNA from the same tissues listed in Fig. 3 were amplified with IGF-I specific primers. The amplified products were cleaved with BumHI and the fragments separated on 5% non-denaturing polyacrylamide gels along with molecular weight markers (rn) and stained with ethidium bromide. In each case, a 200-bp band resolves into two bands of the expected size (15) and 50-bp). -, uncut PCR product; +, digest of PCR product; Blank, RT-PCR blank. PCR conditions: 35 cycles, 1302
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1992
BIOCHEMICAL
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BIOPHYSICAL
RESEARCH
COMMUNICATIONS
confirming that intact, amplifiable mRNA was present in these samples. Basic FGF was detected in fat pad, brain, ovary, kidney, and muscle from leg and breast, and was barely detectable in the heart or liver samples (Fig. 3).
DISCUSSION
In this study we have used a method of mRNA phenotyping to show, for the first time, constitutive expression of TGF-82, TGF-133, TGF-84 and bFGF genes in chicken adipocyte precursors. We also detect differentiation-dependant IGF-I gene expression. The ability of TGF-81 to inhibit adipocyte differentiation
(5, 6, 8) and the constitutive
expression of TGF-8 mRNAs in chicken adipocyte precursors and 3T3-Ll cells (13) suggests that one or more TGF-1.3 isoforms may act as autocrine and/or paracrine negative regulators of adipogenesis. The response of chicken adipocyte precursors to TGF-02, TGF-83 or TGF-84 has not been tested yet, however, the activities of TGF-81, TGF-82 and TGF-03 are similar in most biological assays (25) and so we would expect that all the TGF-B isoforms would exert inhibitory effects on adipogenesis. Basic FGF has a widespread distribution
in adult tissues as determined by biochemical,
biological and immunological analyses of various tissues (26). Due to the short half-life of bFGF mRNA and the insensitivity of conventional mRNA assays, bFGF mRNA has only been detected in the hypothalamus (27), however, Koos & Olson (28) demonstrated the presence of bFGF mRNA in the ovary using the RT-PCR technique. Using this same approach we have detected bFGF mRNA in a range of tissues, including adipose tissue. We found bFGF mRNA in both proliferating and differentiated chicken adipocyte precursors. Since we have shown that exogenous bFGF stimulates the proliferation of chicken adipocyte precursors in vitro (2) but has no effect on differentiation (8) the detection of endogenous bFGF mRNA suggests it may act as an autocrine and/or paracrine regulator of cell proliferation. In an earlier paper we have shown that the proliferation and differentiation of chicken adipocyte precursors in vitro is stimulated by exogenous IGF-I (1). Taken together with the observations presented in this paper that IGF-I mRNA levels increase in response to adipocyte differentiation, we
suggest
that IGF-I may play an important role in adipogenesis. The induction of IGF-I gene
expression in confluent chicken adipocyte precursors is consistent with the results in the Ob1771 cell line (11). in which IGF-I mRNA is an early marker of adipocyte differentiation. In these cells IGF-I is required for the expression of late markers of adipocyte differentiation
(29), therefore,
IGF-I may act as an autocrine and/or paracrine regulator of adipocyte cell proliferation
and
differentiation. In contrast, to the work on other adipocyte precursors (1 l-12) we are able to detect IGF-I gene expression in GH-free medium (14). In addition, these low levels of IGF-I mRNA are not stimulated by exogenous GH in the chicken adipocyte cultures (data not shown). These observations may reflect a species difference and/or a difference in cell culture. For example, selection of cells in GH-rich culture medium (11-12) may select GH-dependent cells. Before extrapolating these in vitro results to the whole animal, we must remember that growth factor genes may be induced by placing cells in culture (30). However, the detection of the same growth factor mRNAs in freshly isolated adipose tissue supports a role for these growth factors in 1303
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3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
vivo. Adipose tissue is quite heterogeneous and contains fibroblasts, endothelial cells and connective tissues, in addition to adipocyte precursors at various stages of differentiation. To further characterise the cellular sites of growth factor expression in adipose tissue will require a more detailed study by in situ hybridisation and immunocytochemistry. In conclusion, we suggest that these growth factors may have a role to play in the regulation of adipocyte precursor cell proliferation and maturation, although a cause-and-effect relationship still remains to be demonstrated.
We wish to thank R. Zeller for providing the sequence of the chicken bFGF mRNA, E. Armstrong, R.K. Field and N. Russell for graphic work, A. Law and H. Griffin for helpful comments. ACKNOWLEDGMENTS:
REFERENCES
:. 3: 4. 2: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 2
Butterwith, S.C., and Goddard, C. (1991) .I. Endocrinol. 131, 203-209. Butterwith, S.C., Peddie, C.D., and Goddard, C. (1992) J. Endocrinol. 132 562. Smith P.J., Wise, L.S., Berkowitz, R., Nan, C., and Rubin, C.S. (1988) J. Biol. Chem. 263, 9402-9408. Gaillard, D., Negrel, R., Lagarde, M., and Ailhaud, G. (1989) Biochem. J. 257, 389-397. Ignotz, R.A., and Massague, J. (1985) Proc. Natl. Acad. Sci. U.S.A . 82, 8.530-8534. Torti, F.M., Torti, S.V., Larrick., J.W., and Ringold, G.M. (1989) J. Cell Biol. 108, 1105-1113. Dani, C., Amri,E.-Z., Bertrand, B., Enerback, S., Bjursell, G., Grimaldi, P., and Ailhaud, G. (1990) J. Cell. Biochem. 43, 103-l 10. Butterwith, S.C., and Gilroy, M. (1991) Comp. Biochem. Physiol. lOOA, 473-476. Navre, M., and Ringold, G.M. (1989) J. Cell. Biol. 109, 1857-1863. Broad, T.E., and Ham, R.G. (1983) European J. Biochem. 135, 33-39. Doglio, A., Dani, C., Fredrikson, G., Grimaldi, P., and Ailhaud, G. (1987) EMBO J. 6, 401 l-4016. Gaskins, H.R., Kim, J.-W., Wright, J.T., Rund, L.A., and Hausman, G.J. (1990) Endocrinol. 126, 622-630. Weiner, F.R., Shah, A., Smith, P.J., and Rubin, C.S. (1989) Biochem. 28, 4094-4099. Butterwith, S.C., and Griffin, H.D. (1989) Comp. Biochem. Physiol. 94A, 721-724. Kajimoto, Y., and Rotwein, P. (1989) Mol. Endocrinol. 3, 1907-1913. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory. Rappolee, D.A., Wang, A., Mark, D., and Werb, Z. (1989) J. Cell. Biochem. 39, l-l 1. Kost, T.A., Theodoralkis, N., and Hughes, S.H. (1983) Nucleic Acids Res. 11, 82878301. Burt, D.W., and Paton, I.R. (1991) DNA & Cell Biol. 10, 723-734. Cooper, D.A., Lu, S.C., Viswanath, R., Freiman, R.N., and Bensadoun, A. (1992) B&him. Biophys. Acta 1129, 166-171. Kajimoto, Y., and Rotwein, P. (1991) J. Biol. Chem. 266, 9724-9731. Derynck, R., Rhee, L., Chen, E.Y., and Tilburg, A.V. (1987) Nucleic Acids Res. 15, 3188-3189. Derynck, R., Lindquist, P.B., Lee, A., Wen, D., Tamm, J., Gracar, J.L., Rhee, L., Mason, A.J., Miller, D.A., Coffey, R.J., Moses, H.L., and Chen, E.Y. (1988) EMBO J. 7, 3737-3743. Abraham, J.A., Whang, J.L., Tumolo, A., Mergia, A., and Fiddes, J.C. (1986) Cold Spring Harbor Symposia on Quantitative Biology 51,657-668. Gracar, J.L., Miller, D.A., Arrick, B.A., Lyons, R.M., Moses, H.L., and Derynck, R. (1989) Mol. Endocrinol. 3, 1977-1986. Klagsburn, M. (1989) Progress in Growth Factor Res. 1, 207-235. Shimasaki, S., Emoto, N., Koba, A., Mercado, M., Shibata, F., Cooksey, K., Baird, A., and Ling, N. (1988) Biochem. Biophys. Res. Commun. 157, 256-263. 1304
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1992
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AND
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Koos, R.D., and Olson, C.E. (1989) Mol. Endocrinol. 3, 2041-2048. Doglio, A., Dani, C., Grimaldi, P., and Ailhaud, G. (1986) Biochem. J. 238. 123-129. Boswell, J.M., Yui, M.A., Endres, S., Burt, D.W., and Kelley, V.E. (1988) J. Immunol. 141, 118-124. Jakowlew, S.B., Dillard, P.J., Kondaiah, P., Sporn, M.B., and Roberts, A.B. (1988a) Mol. Endocrinol. 2, 747-755. Jakowlew, S.B., Dillard, P.J., Sporn, M.B., and Roberts, A.B. (1988b) Mol. Endocrinol. 2, 1186-1195. Cooper, D.A., Stein, J.C., Strieleman, P.J., and Bensadoun, A. (1989) Biochim. Biophys. Acta 1008,92-101.
1305
187,
September
No. 30,
BIOCHEMICAL
3, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1992
MULTIPLE
GROWTH
CHICKEN David W. Burt,
Jaquie M. Boswell,
Institute
mRNAS ARE EXPRESSED
ADIPOCYTE
Department AFRC
FACTOR
Edinburgh Roslin,
305
IN
CELLS
Ian R. Paton and Simon C. Butterwith
of Cellular
of Animal
PRECURSOR
1298-l
and Molecular
Physiology
Biology
and Genetics
Research
Research Station
Midlothian
EH25 9PS, UK
Received July 6, 1992 SUMMARY: We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-02, TGF-B3, TGF-I34 and bFGF genes in chicken adipocyte precursors. We also detect GH-independant, but differentiation-dependant IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation. 0 1992Academic Press,1°C.
Adipose tissue growth occurs through a combination of adipocyte hyperplasia and hypertrophy. Hyperplasia occurs via the proliferation and differentiation of adipocyte precursors. This process is under the control of hormones and growth factors, some of which may act in an autocrine and/or paracrine manner. Proliferation of primary and established adipogenic cell lines is stimulated by IGF-I, IGF-II, TGF-El, PDGF, aFGF and bFGF (l-2). Differentiation
of adipogenic cells is
stimulated by IGF-I (3-4) and inhibited by TGF-Dl (5-8). The effects of bFGF on adipocyte differentiation is cell-dependent, and can be inhibitory (9), stimulatory (10) or have no effect (8). The detection of IGF-I and TGF-Ol mRNAs in adipocyte precursors (11-13) suggests that these factors may act as autocrine and/or paracrine regulators of adipocyte cell proliferation differentiation.
and
Since we have shown that bFGF, TGF-Bl and IGF-I can influence chicken adipocyte precursor cell proliferation and differentiation in vitro (1, 2, 8), this study was undertaken to investigate the ability of these cells to express these growth factor genes. Our results indicate that mRNAs for bFGF, TGF-82, TGF-03, TGF-IJ4 and IGF-I are present in both proliferating and differentiated chicken adipocyte precursors. Taken together with the known action of these growth factors in Abbreviations: RT, reverse transcription; PCR, polymerase chain reaction; bp, base pairs; TGF-R, transforming growth factor-@ IGF, insulin-like growth factor; ; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; LPL, lipoprotein lipase; GH, Growth Hormone. 0006-291X/92
Copyright All rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1298
Vol.
187,
No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
vitro, our results support the hypothesis that these growth factors act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation. MATERIALS
AND METHODS
Preparation of Adipocyte Precursor Cell Cultures Chicken adipocyte precursors were prepared as described previously (14). They were cultured in Medium 199 supplemented with 4% (v/v) Ultroser G (Gibco, Paisley, Strathclyde, Scotland, U.K.) at 37OC in an atmosphere containing 5% CO;! with changes of medium every 2 days. Subconfluent monolayers of proliferating cells were harvested after 4 days in culture and differentiated adipocyte precursors 10 days after reaching confluence. Synthetic Oligonucleotides Synthetic oligonucleotides were synthesised by Oswel DNA services (Edinburgh University). The sequences of the primer pairs are shown in Table 1. An internal oligonucleotide specific for the IGF-I RT-PCR product (570-590 CTGTGCTCCAATAAAGCCACC; 15) was used as a probe for verification. Detection of mRNAs by RT-PCR Total RNA was isolated from whole tissues and cultured cells using the standard guanidine thiocyanate extraction procedure (16). First-strand cDNA was synthesised as described (17) using 1 ug of total RNA and random hexamers. For each RT reaction, a blank was prepared using all reagents except the RNA sample. PCR was carried out as described (17) using the primer pairs shown in Table 1. The reactions were denatured at 94OC (1 min), annealed at 50°C or 65OC (2 mins) and extended at 72OC (3 mins) for 20-35 cycles in a Biometra TRIO-BLOCK thermal cycler. Most primers were annealed at 5OOC,except bFGF at 65OC. A PCR blank was run as a control, using the RT blank. PCR products were resolved on 5% non-denaturing polyacrylamide gels along with molecular weight markers. In all experiments the RT-PCR reagent blank was negative.
Southern Blotting and Hybridisation Following electrophoresis, gels were treated in 0.5 M NaOWl.5 M NaCl for 30 mins, then in 1 M NH40Ac/0.02 M NaOH for 15 mins, transferred to Zetaprobe (Bio-Rad, Richmond, CA, U.S.A) membranes in 1 M NHdOAc/0.02 M NaOH by capillary action, rinsed in 2x SSC and baked. Oligonucleotides were labelled with 32P to a specific activity of 2x108 c.p.m. / ug using T4 polynucleotide kinase (16) and filters were hybridised as recommended by the manufacturer (BioRad). RESULTS Design and Validation
of Gene-Specific
Primers
for RT-PCR
Northern blot analysis lacks sufficient sensitivity to detect transcripts in small amounts of tissues, especially when those transcripts have short half-lives, as is the case for most growth factor mRNAs. We used the technique of RT-PCR, a technique that allows the detection of multiple mRNAs when cell numbers are small and/or mRNA transcripts are rare (17). We designed oligonucleotide primer pairs that bracket target sequences specific for each of the mRNAs listed in Table 1. We selected targets for amplification 150 to 300-bp in length containing a unique restriction enzyme site. The primer pairs were selected to cross intron-exon junctions, consequently genomic DNA would produce a larger product than the cDNA, allowing us to distinguish between signals due to mRNA or contaminating genomic DNA. The location of intronexon junctions was known in the case of some chicken genes (B-actin, 18; TGF-82, 19; LPL, 20; IGF-I, 21) or was inferred from homologous or duplicated genes (TGF-84, 22; TGF-83, 23; bFGF, 24). PCR products larger than the size expected for cDNA were not detected following RT1299
Vol.
187,
No.
3,
BIOCHEMICAL
1992
Table Gene
AND
1. Growth
factor
PCR Primer
Product Size (bp)
BIOPHYSICAL
primer
RESEARCH
COMMUNICATIONS
pairs
Restriction Site
Restriction Products
5’-R-actin 3’-l3-actin
280
TGTGGTATCCATGAAACTA ATTCATCGTACTCCTGCTT
Kpd
185+95
(1)
5’-TGF-l32 3’.TGF-I32
269
AGGAATGTGCAGGATAATT ATTTTGGGTGTTTTGCCAA
Hi&II
192+77
(2)
5’-TGF-I33 3’-TGF-I33
200
GAGCAGAGTTCCGGGTGCT GTGCAGAAGCCACTCACGC
SSfI
137+35+28
(3)
5’.TGF-134 3’.TGF-I34
160
CACCGACTACTGCTTCGGC GTCGGCGCTCCAGATGTAC
PSfI
1 I o+so
(4)
5’-IGF-I 3’=IGF-I
200
GTATGTGGAGACAGAGGCTTC TTTGGCATATCAGTGTGGCGC
BUmHI
150+50
(5)
5’-LPL 3’.LPL
298
TAGACCAGCCATTCCTGAT TCTCCTTTACCAAGACGTG
P.UI
151+146
(6)
5’-bFGF 3’-bFGF
270
GATCCGCACATCAAACTGC GATACGTTTCTGTCCAGGTCC
Hitlfl
I%+112
(7)
All primer sequences given in 5’ to 3’ orientation. ‘Position: 3506-4140 (18): 2Position: 64529311 (19); 3Position: 482-681 (31); 4Position: X17-979 (32); 5Position: 439-638 (15); Qosition: 1203-1500 (33); 7Position: 432-701. PCR of any of the RNA sequences.
Since
kactin
samples, mRNA
indicating
that they were indeed
free of contaminating
DNA
is present in all tissues, primers for this gene were used to
monitor for efficient cDNA synthesis between different RNA samples. Growth
Factor
Expression
in Proliferating
and Differentiated
Chicken
Adipocyte
Precursor Cells Total RNA was prepared from either proliferating or differentiated cells from three independent primary cultures of chicken adipocyte precursors. First strand cDNA was synthesised and analysed for the mRNAs listed in Table 1. The growth factor mRNAs for TGF-02, TGF-l33, TGF-04 and bFGF were detected in both proliferating and differentiated adipocyte precursors (Fig. 1). The identity of PCR products was established by restriction enzyme mapping (Fig. 2). The PCR fragment amplified with the TGF-132 primers cut with Hi&III
to give two fragments 192-bp and
77-bp. The TGF-I33 products were digested with SstI to give 137-bp, 35-bp and 28-bp. The TGF-134 products were cleaved by PstI, to give lOO-bp and 50-bp. The PCR products for bFGF were confirmed by digestion with Hi@ to give fragments of 158-bp and 112-bp. IGF-I mRNA was absent or very low in proliferating adipocyte precursors (absent in 2/3 cell cultures), but was more abundant in differentiated cells (present in 3/3 cell cultures, an example is shown in Fig. 2). The identity of the IGF-I transcripts was confirmed by the size of the PCR product (200-bp), by hybridisation with an internal oligonucleotide probe and by cleavage with BumHI to generate a 150-bp band, that also hybridised to the same probe. 1300
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Blank I
pactin
TGF-p3
=
TGF-p
bFGF
Fig. 1. Expression of B-actin. TGF-82. TGF-03. TGF-D4. bFGF and LPL in proliferating and differentiated adipocyte precursors. RNA samples were reverse transcribed and amplified by PCR with gene-specific primers. Products were resolved on 5% non-denaturing polyacrylamide gels along with molecular weight markers (not shown). All the PCR products were of the expected size. PCR conditions: I&actin. TGF-132. TGF-134, LPL. 20 cycles; TGF-B3, 25 cycles; bFGF, 35 cycles.
pactin
TGF -
bFGF ml
TGF-p m leP+I
/+I
IGF-I
TGF - 82 .NP+I-P+I
LPL eNP+I
-NP+I
200
Fig. 2. Verification of TGF-f32, TGF-03, TGF-04, bFGF, IGF-I and LPL PCR amplification products by restriction enzyme mapping. cDNA from proliferating and differentiated adipocyte precursors was amplified with gene-specific primers. The amplified products were cleaved with the indicated restriction enzymes and the fragments separated on 5% non-denaturing polyacrylamide gels along with molecular weight markers (m). The PCR products from the amplification of cDNA with the IGF-I primers were processed as above, transferred to a nylon membrane and hybridised at high stringency to an internal oligonucleotide probe (see Materials & Methods). -, uncut PCR product; +, digest of PCR product; P, proliferating adipocyte precursors; NP, non-proliferating, differentiated adipocyte precursors.
1301
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paciin
TGF
- p2
TGF
- p3
TGF
- p4 bFGF
Fig. 3. Expression of TGF-82, TGF-83, TGF-1.54, bFGF and &actin genes in various chicken tissues. RNA samples were extracted from heart, fat pad, brain, ovary, kidney, leg muscle, liver and breast muscle from a 6 week old broiler hen, reverse transcribed and amplified by PCR with gene-specific primers. Products were resolved on 57~ non-denaturing polyacrylamide gels along with molecular weight markers. All the PCR products were of the expected size. B, RT-PCR blank. PCR conditions: all 30 cycles except bFGF: 3.5 cycles.
Expression
of Growth
Factor
mRNAs
in Adipose
Tissue
and
Various
Other
Tissues Total cellular growth
factor
RNA isolated mRNAs,
in vivo. For comparison, leg muscle,
mapping
TGF-IJ3
mRNA
lower levels were found
detected
giving
in vitro were also expressed
(Figs.
Verification
(data not shown),
samples. The growth
with BamHI,
nucleotide
transcripts
fat pads) was assayed for the same
from a range of tissues (heart, brain, ovary, kidney,
was also assayed by RT-PCR. enzyme
cell RNA
by digestion
TGF-I33 mRNA
whether isolated
in all the tissues examined
from the reported variable.
total RNA
by restriction
precursor
were detected confirmed
to determine
liver, breast muscle)
was established adipocyte
from adipose tissue (abdominal
factor mRNAs 3-4). The identity
rise to products
of RT-PCR
as described TGF-62,
of 150-bp
in Fig. 2, for
TGF-R4
of the IGF-I
was reproducibly
in liver. However,
detected
in fat pad, brain
and IGF-I
products
was
and 50-bp as predicted
sequence (15). The assays for TGF-I33 and bFGF mRNAs
with kidney,
products
and ovary
were more (Fig. 3), and
heart and skeletal muscle tissues. We were unable to detect
the B-actin product
was found at equivalent
levels in all tissues,
Fig. 4. Detection of IGF-I mRNA in various chicken tissues by RT-PCR. cDNA from the same tissues listed in Fig. 3 were amplified with IGF-I specific primers. The amplified products were cleaved with BumHI and the fragments separated on 5% non-denaturing polyacrylamide gels along with molecular weight markers (rn) and stained with ethidium bromide. In each case, a 200-bp band resolves into two bands of the expected size (15) and 50-bp). -, uncut PCR product; +, digest of PCR product; Blank, RT-PCR blank. PCR conditions: 35 cycles, 1302
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confirming that intact, amplifiable mRNA was present in these samples. Basic FGF was detected in fat pad, brain, ovary, kidney, and muscle from leg and breast, and was barely detectable in the heart or liver samples (Fig. 3).
DISCUSSION
In this study we have used a method of mRNA phenotyping to show, for the first time, constitutive expression of TGF-82, TGF-133, TGF-84 and bFGF genes in chicken adipocyte precursors. We also detect differentiation-dependant IGF-I gene expression. The ability of TGF-81 to inhibit adipocyte differentiation
(5, 6, 8) and the constitutive
expression of TGF-8 mRNAs in chicken adipocyte precursors and 3T3-Ll cells (13) suggests that one or more TGF-1.3 isoforms may act as autocrine and/or paracrine negative regulators of adipogenesis. The response of chicken adipocyte precursors to TGF-02, TGF-83 or TGF-84 has not been tested yet, however, the activities of TGF-81, TGF-82 and TGF-03 are similar in most biological assays (25) and so we would expect that all the TGF-B isoforms would exert inhibitory effects on adipogenesis. Basic FGF has a widespread distribution
in adult tissues as determined by biochemical,
biological and immunological analyses of various tissues (26). Due to the short half-life of bFGF mRNA and the insensitivity of conventional mRNA assays, bFGF mRNA has only been detected in the hypothalamus (27), however, Koos & Olson (28) demonstrated the presence of bFGF mRNA in the ovary using the RT-PCR technique. Using this same approach we have detected bFGF mRNA in a range of tissues, including adipose tissue. We found bFGF mRNA in both proliferating and differentiated chicken adipocyte precursors. Since we have shown that exogenous bFGF stimulates the proliferation of chicken adipocyte precursors in vitro (2) but has no effect on differentiation (8) the detection of endogenous bFGF mRNA suggests it may act as an autocrine and/or paracrine regulator of cell proliferation. In an earlier paper we have shown that the proliferation and differentiation of chicken adipocyte precursors in vitro is stimulated by exogenous IGF-I (1). Taken together with the observations presented in this paper that IGF-I mRNA levels increase in response to adipocyte differentiation, we
suggest
that IGF-I may play an important role in adipogenesis. The induction of IGF-I gene
expression in confluent chicken adipocyte precursors is consistent with the results in the Ob1771 cell line (11). in which IGF-I mRNA is an early marker of adipocyte differentiation. In these cells IGF-I is required for the expression of late markers of adipocyte differentiation
(29), therefore,
IGF-I may act as an autocrine and/or paracrine regulator of adipocyte cell proliferation
and
differentiation. In contrast, to the work on other adipocyte precursors (1 l-12) we are able to detect IGF-I gene expression in GH-free medium (14). In addition, these low levels of IGF-I mRNA are not stimulated by exogenous GH in the chicken adipocyte cultures (data not shown). These observations may reflect a species difference and/or a difference in cell culture. For example, selection of cells in GH-rich culture medium (11-12) may select GH-dependent cells. Before extrapolating these in vitro results to the whole animal, we must remember that growth factor genes may be induced by placing cells in culture (30). However, the detection of the same growth factor mRNAs in freshly isolated adipose tissue supports a role for these growth factors in 1303
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vivo. Adipose tissue is quite heterogeneous and contains fibroblasts, endothelial cells and connective tissues, in addition to adipocyte precursors at various stages of differentiation. To further characterise the cellular sites of growth factor expression in adipose tissue will require a more detailed study by in situ hybridisation and immunocytochemistry. In conclusion, we suggest that these growth factors may have a role to play in the regulation of adipocyte precursor cell proliferation and maturation, although a cause-and-effect relationship still remains to be demonstrated.
We wish to thank R. Zeller for providing the sequence of the chicken bFGF mRNA, E. Armstrong, R.K. Field and N. Russell for graphic work, A. Law and H. Griffin for helpful comments. ACKNOWLEDGMENTS:
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