0 1992 Hamood Academic Publishers GmbH
Growth Factors, 1992, Vol. 6, pp. 209-218 Reprints available directly from the publisher Photocopying permitted by license only
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Molecular Cloning of CSF-1 Receptor from Rat Myoblasts. Sequence Analysis and Regulation During Myogenesis ANNE-GMLLE BORYCKI, MARTINE GUILLIER, MARIE-PIERRE LEIBOVITCH and SERGE ALEXANDRE LEIEOVITCH*
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Luboratoire d’oncologie Moliculaire UA 1158-URA 126 du CNRS, Institut Gustave Roussy 39, rue C . Desrnoulins 94800 Villquif, France
(Received August 8 1991, Accepted October 17 1991)
We have isolated and sequenced a cDNA (mrfms) encoding rat c-fms gene (CSF-1 receptor) from proliferating L6al myoblasts. The predicted amino acid sequence was highly identical with the c-fms protein found in monocytes and macrophages (98,76 and 84% identity from mouse, cat and human c-fms proteins, respectively). The mechanisms responsible for the regulation of mrfms gene expression during myogenesis were examined. Mrfms products were observed during proliferation of L6al myoblasts and were downregulated during differentiation. Run-on transcription assays demonstrated that the mrfms gene was transcriptionally active only in undifferentiated myoblasts. These findings suggested that mrfms levels in L6al myoblasts are controlled by transcriptional mechanisms. The half-life of mrfms transcripts was found to be at least 5 hr while inhibition of protein synthesis with cycloheximide (CHX) decreased this halflife to 30min without changes in the rate of m r f m s gene transcription. In addition oncogenic transformation of L6al myoblasts by the v-fms induced constitutive upregulation of m r f m s mRNAs, and nuclear run-on assays demonstrated that mrfms transcription was not growth-factor dependent. Furthermore, these findings with others previously published indicate that mrfms gene products may play a role in the normal and neoplastic growth of niuscular cells. KEYWORDS: CSF-1 receptor, myogenesis, regulation
of the v-fms oncogene of a feline sarcoma virus (Donner et al., 1982). Besides, the c-fms protoFor several years our laboratory has investigated oncogene was found to be constitutively active in the possible role of proto-oncogenes (c-onc. a variety of L6al-derived neoplastic transgenes) in differentiation versus neoplastic trans- formants (Leibovitch et al., 1986,1989). These finformation of skeletal muscle, using as main dings were intriguing in view of previous data model a clonal subline of rat myogenic cells showing that the CSF-1 receptor is closely related termed L6al and various neoplastic derivatives. or identical to the product of the c-ftns proto(Leibovitch et al., 1986). Among 15 proto-onco- oncogene (Sherr et al., 19851, and c-fms trangenes analyzed for mRNA accumulation, all of scripts were only found at consistent levels in them expressed more or less elevated levels of placenta tissue (Muller et al., 1983a), Choriotranscripts during L6al cell proliferation carcinoma cells and cells differentiated along the (Leibovitch et al., 1986; Hare1 et al., 1989) and in monocyte lineage (Muller et al., 1983b). Furtherparticular relatively high amounts of two tran- more, the c-fms product exhibits tyrosine kinase scripts 3.7kb and 2.0 kb long from the c-fms activity (Rettenmier et al., 1986; Woolford et al., proto-oncogene which is the cellular counterpart 1985) and specifically binds CSF-1 (Rettenmier et al., 1986). These and other data have demonstrated that the c-fms gene is related to a family of genes coding for growth factor receptors with *To whom correspondence should be addressed.
INTRODUCTION
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intrinsic tyrosine kinase activity (Barbacid and phage line J774.2 was maintained in culture Louver, 1981). Other data have shown that the essentiplly as described (Sacca et al., 1986). c-fms gene is expressed when human HL60 myeloid leukemic cells are induced to differentiate cDNA Library Screening, Subcloning and along the monocytic lineage with phorbol esters Sequencing (Sariban et al., 1985), that it is constitutively expressed in mature human peripheral blood A Agtl0 cDNA library prepared from rat L6al monocyte (Nienhuis et al., 1985; Sariban et al., myoblasts in our laboratory was screened (5x105 1985) and the level of c-fms transcripts is down- plaques) with a murine c-fms probe (i1755, regulated following activation of these cells Rothwell and Rohrschneider, 1987) as described previously (Leibovitch et al., 1990). Six positive (Horiguchi et al., 1988). Our further studies demonstrated that the clones were isolated from this library. Only a 3.7 kb long c-fms related transcripts from L6al single clone contained the entire coding region. myoblasts codes for a glycosylated protein with Nucleic acid sequence determination was perforassociated tyrosine kinase activity apparently med by the dideoxy method (Sanger et al., 1977) identical to the CSF-1 receptor of macrophages. It using the sequenase kit (US Biochemical). Both is abundant at the surface of proliferating myo- strands of the cDNA inserts were sequenced by blasts and disappears after the formation of using a combination of bidirectional Bal 31 produced nested deletions and restriction enzyme myotubes (Leibovitch et al., 1989). In the present studies we have cloned and fragments which were subcloned in the pUC18 sequenced a complete cDNA clone encoding a and pUC19 vectors. CSF-1 receptor from L6al rat myoblasts. We demonstrated that this cDNA clone, termed RNA Blot Analysis mrfms, is practically identical to the mouse macrophage CSF-1 receptor. We also show that regu- Total cellular' RNA was prepared by lysis in lation of its expression occurs at the transcrip- guanidium isothiocyanate buffer and cesium tional level and that the synthesis of a labile gradient centrifugation (Leibovitch et al., 1986). protein is responsible for stabilization of its RNA Twenty-five pg of RNA per lane were separated during the growth of L6al myoblasts. Further- electrophoretically on 1% agarose-formaldehyde more, we observe that the transformation of the gels and transferred to Hybond (Amersham, L6al myoblasts with the v-fms oncogene is corre- UK). Hybridization assays were made as lated by the loss of myogenic differentiation and described previously (Leibovitch et al., 1990). constitutive transcription of the mrfms indepen- Final washing was O.lxSSC, 0.1% SDS at 55-65 "C dently of the conditions of culture. depending on the probe. Nuclear Run-on Assays MATERIALS AND METHODS Cell Culture and DNA Transfection In the present study myogenic cell samples were collected at two different stages. First, as proliferating myoblasts after two days in growth medium containing 10% FCS. At this stage no muscle-specific gene is expressed. Second, the L6a1 cell line was maintained and induced to differentiate under previously described conditions (Hillion et al., 1984). L6al myoblasts were cotransfected with the pSM-FeSV vector (Donner et al., 1982) and the pSV2neo vector under previously described conditions (Leibovitch et al., 1987). The CSF-1 independent mouse macro-
Cells were washed once with ice-cold phosphate buffered saline then lysed in lysis buffer (10 mM Tris-HC1, pH=7.4, 10 mM NaC1, 3 mM MgC12, 0.5% Nonidet P40). Nuclei were collected and resuspended in nuclei freezing buffer (50 mM Tris-HC1, pH=8, 5 mM MgC12, 40% glycerol, 0.5 mM dithiothreitol) and stored at -80 "C until used. Nuclear run-on assays were performed as essentially described by Greenberg and Ziff (1984), using d 2 P UTP (>3000Ci/mM, NEN). Run-on transcript product's were purified through G-50 Column and hybridized to 5 p g of the denatured plasmids. Plasmids were denatured in 0.3 M NaOH for 30 min at 65 "C neutralized with 2 M ammonium acetate and
CSF-1 RECEPTOR IN MYOGENIC CELLS
then slot blotted onto Hybond. Following hybridization for 48 hr at 65 "C in 0.3 M NaCl, 10 mM Hepes, pH=7.4, 10 mM EDTA 0.2% SDS, filters were processed as described previously (Greenberg and Ziff, 1984).
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SDS PAGE Analysis L6al cells were incubated in methionine-free medium for 30 min before labeling for 1 hr with 250 pCi/ml 35S-methionine (1200 Ci/mMole, NEN). After labeling, cells were lyzed in RIPA buffer and immunoprecipitated as described previously (Leibovitch et al., 1989) using a rabbit polyclonal antiserum against the cytoplasmic domain of the v-fms protein. Recombinant DNA Probes The random primed DNA insert probes used in hybridization were: Glyceraldhyde 3 phosphate deshydrogenase (GAPDH): a 1.3 kb PstI fragment of the rat GAPDH cDNA (pRGAPDHl3) (Fort et al., 1985). Murine CSF-1 receptor (c-fms): a 3.7 kb EcoRI fragment containing the complete cDNA sequence of the murine c-fms protein (L755) (Rothwell et al., 1987). Rat myogenic fms protein: a 3.6 kb EcoRI fragment containing the complete cDNA sequence at the rat CSF-1 receptor (mrfms). Myogenin, a 1.5 kb EcoRI fragment of the rat myogenin cDNA clone (Wright et al., 1989). Myosin light chain two (MLCZ), a 0.5 kb PstI fragment of pLC2-18 (Garfinkel et al., 1982).
21 1
755) as described in Materials and Methods. Six positive clones were identified under conditions of high stringency. The longest cDNA clone referred to here as mrfms (muscle rat fms) was subcloned for further analysis. The restriction enzyme map of the mrfms clone is indicated in Fig. 1. It is very similar to mouse, cat and human c-fms map especially in the 3' region of the cDNA. The nucleotide sequence of mrfms and its derived amino acid sequence are shown in Fig. 2. It contains 3667 bp which span the entire coding region, 75bp of 5' untranslated sequence and 661 bp 3' untranslated sequence. Its open reading frame of 2931 nucleotides from the 5' end start at position 76 and it codes for a polypeptide of 977aa. A polyadenylation signal ATTAAA is found at position 3645 and 16 nucleotides downstream from this poly A signal there is a tail of 12 A residues. K BC
B
X
mrfmS
HGURE 1 Restriction map of the m r f m s cDNA clone. The open box corresponds to the coding region. This clone was 100% sequenced from both strands B=BglZ, Bc=BclI, K=KpnI, H=HincII, Ps=PstI, Pv=PvuII, S=SacI, X=XbaI.
Comparison of the c-fms Proteins
The deduced amino acid sequences of mouse, cat and human c-fms gene products compared to the mrfms clone are shown in Fig. 3. This confirms RESULTS that c-fms proteins are highly conserved as are most other proto-oncogenes among vertebrate Cloning and Analysis of the c-fms cDNA species. The organisation of the mrfms protein is Clone from Rat Myoblasts (mrfms) very similar to the monocyte/macrophage CSF-I In previous reports using as a probe the v-fms receptor and can be divided into external, transclone we found that the c-fms proto-oncogene membrane and internal domains. The amino acid expressed two transcripts 2.0 kb and 3.7 kb long numbers and percent similarities between the in proliferating myoblasts and that both are pro- corresponding domains in mrfms, mouse c-fms, gressively eliminated during L6al cell differen- cat c-fms and human c-fms are given in Table 1. tiation. Furthermore, we showed that only the All four proteins are highly conserved in the 3.7kb mRNA spans the three domains of the internal kinase domain and less conserved in the CSF-1 receptor (Leibovitch et al., 1988). A lambda external glycosylated segment (Fig. 3). However, gtlO cDNA library was constructed using the overall amino acid homology of the external poly(A') RNA isolated from growing L 6 a l myo- domain between mrfms and mouse c-fms is 98%, blasts. 5x105 independent phage clones were between mrfms and cat c-fms it is 63% and mrfms screened with the murine c-fms probe (clone d and human c-fms it is 75%. As observed in the
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mouse c-fms product, mrfms protein contains nine potential N-linked glycosylation sites (Asn, X, Ser/thr) in the external domain (Fig. 3), six of the sites are also found in the same position in cat c-fms and human c-fms. Furthermore, the location of the ten cysteine residues in the external domain is conserved for all four c-fms proteins. The transmembrane domain formed by a stretch of 26 hydrophobic amino acids has 100, 77 and 85% homology with mouse, cat and human c-fms amino acid sequences, respectively. The intracellular domain is highly conserved between the 4 species with 90-98% amino acids identity (Table 1).This is also consistent with previous observations showing that like the c-fms proteins, the internal domain of mrfms proteins contains a region of homology with other tyrosine protein
kinases (Sherr, 1990). Furthermore the probable ATP binding site (residues 586-591) and lysine at 614 are identical between the four c-fms proteins. Control of mrfms Expression During Muscular Differentiation
Previous studies from our laboratory have demonstrated that consistent levels of c-fms related transcripts are expressed during proliferation of rat L6al myogenic cells as well as in all the neoplastic myoblasts studied. However, in non transformed cells these transcripts are eliminated during the myogenic process. (Leibovitch et al., 1986, 1988, 1989). These results have been extended to C2C12 mouse myogenic cells and we demonstrated that a glycoprotein of 170 kD (gp
CSF-1 RECEPTOR IN MYOGENIC CELLS
during myogenesis is shown in Fig. 4A and 4B. The expression pattern of the 3.7kb mrfms mRNAs paralleled the pattern of glycoproteins expression. No mrfms product is found after L6a 1 cells were switched to differentiation medium s nvsmsc RPKV CATPUSC LDPSI n Q H WIQGNA L for 4 days while, following their transfer to the HUKFUSC YSDGSS I s n Q F ’ G L C M RP V V F D 110 140 150 160 170 medium J774.2 macrophages continue to lIwlls Q E A W C L I ~ P A . L m S V S I F F ~ ~ € I I R I W ( V W S H T Y V Csame ~ msmsc P S express high level of c-fms transcripts. The supCATmSC D L L EAG VVR P Q N S P H T H FIENHV Q SAR wmsc D L L VEAG VVR PIMIINS P H T H FIQQDQSAL 190 200 210 220 230 pression of the expression at both the glycoproIIRFUWS V W G R E S T S T G I U ~ R V H P E P ~ I K ~ S K L V R I R G E U P I I L K R G MJSFUSC teins and mRNA levels in myotubes suggested D TV w QKDISG ATLT AE Q S I D N D S R H CATRISC SSVD N D € QHN nmmsc nc ~ v l IlS R QK I c ALT v AE that the regulation of mrfms gene did not occur 250 260 270 280 290 mms D T K L E I P L H S D W N Y Y - L S W W D A G I Y S C V I S W D V ~ A ~ F Q W ~ A Y at a translational level, but at the level of tranmsmsc TISQP H R LT n DH s N T T AW nns s v R CATFUSC APCQ n n L T~N D Q~ H n VQ KHSTS F R HLMRI$C N scription and/or mRNA accumulation in the 330 3 50 mms WLTSEQSWNSVGDSLIL~DAYPSIQHY~YLCPFFEWR. .KLEFITQMIY cytoplasm. Nuclear run-on assays (Fig. 5) msmsc L .. F CATFUSC T EKVD Q K V E GLESF SDY D . . D IKDT revealed that mrfms was transcriptionally active HWFUSC S N I T Mi N K W E GLGF SDH PEP ANA TKDT 370 380 390 400 I10 MRFIlS R Y T F K L F L H R V M S W G Q Y F L Q N ~ G ~ n L T F E L T ~ Y P P E L F C Din L6al proliferating myoblasts and switched off *oar*rc D . .._ CATFUSC ST S P L R R SFL R AG Q A R UTLI T L E in differentiated L6al myotubes, whereas no sigHWFUSC H T S P L P R S F L R F’G RA 1 TFI GT L A 430 440 450 460 470 nificant variation in transcriptional activity of lIRFUS V S G Y P Q P S V m E C R G H T D R C D E A Q ~ ~ ~ P ~ ~ Q K P F D K V I I Q S Q L P I G T ~ H n msmsc nL P GAPDH was observed in myotubes compared to V H E V H L A E VQ s S A G . L E S S CATFUSC A V DPY E H TV L T V E E LQ 5 HUUFUSC A N myoblasts. These results suggested that the 490 500 510 520 5?0 lIRRls I ( T Y F C K T H N S V G N S S Q Y F V S L M S I ( P L P D L S L ~ P ~ A ~ S ~ S L L V L L L L L L L Y K Y decrease in mrfms RNA level following differenmsmsc CATp?rSC R E I U F TYPIIAHT L LLT I A L nmmsc Q E IU SG UA IPI A AHTHP F I A L tiation occurred at the transcriptional level. 550 560 570 580 590 ..mRls K Q K P K Y Q V R Y I ( I I E R Y f f i N S ~ F I D ~ ~ Y ~ ~ E € P ~ ~ € G ~ L C A G A F G ~ ESubsequent AT studies were performed to deterIRISRlSC S CATMSC mine the stability of the mrfms mRNA. L6al cells nmmsc S 610 620 630 640 650 were treated with actinomycin D (Act.D) for various intervals. The levels of mrfms mFWA decreased about 40% after 4 hr of Act.D exposure (Fig. 6A). In contrast there was little if any effect on the level of GAPDH mRNA. These and other assays (not shown) suggest that the half life of NUSIWC R sn SEE c DG C A T W DG R HlMmSC .n SE mrfms RNA is 5-6 hr and is similar to that of 790 800 110 120 830 M R m ~ T ~ ~ ~ I G D P C ~ I ~ ~c-frrrs ~ ~ RNA I L in Y the ~ S ~ Human HL60 line (Weber et al., nvsIWc P D I CATPUSC R I €D 1989). In order to determine whether the stability mmsc N I €D 850 860 170 880 890 I(RR1S S Y C I W Y E I € S L C W P Y F ’ G I L ~ ~ K L ~ D G Y ~ Q W F A P ~ I Y S I of ~ Q Sthe ~ ~ mrfms transcripts is affected by inhibition nvsmsc n CATIWC S A A A of protein synthesis, or not, L6al myoblasts were HuLR(sC s A A A H 910 920 910 940 950 exposed to cycloheximide (CHX) for 30 min “S ~ r P Q I c € L I . Q B Q I R L e O ~ y ~ ~ ...GG(iS s ~ s D SsC % S S S W ~ S S E MUSRlSC ... before addition of Act.D for 90min. This treatCATJUSC S K QED V P Y T S S SSSSCRFG C M)IVIISC SFL am ~ l Tl ......_... RSGG s L ment caused a progressive decline in mrfms 970 971 mIW ~UCCEPCDIAQPLIQP”YQ€AC nvSIWC mRNA level (Fig. 6B) while no variation was CATIWC Q W M S C T Q observed for GAPDH mRNA. These results and FIGURE 3 Comparison of the deduced amino acid sequence those obtained with Act.D alone suggested that of mrfms and other c-fms proteins. The deduced amino acid inhibition of protein synthesis is associated with sequence of mrfms O W shown in Fig. 2 is compared with amino acid sequences from other vertebrate species given in destabilization of the mrfms mRNA. In another assay L6al myoblasts were exposed the literature. Changes for the murine, cat and human c-fms proteins are indicated in the corresponding lines. (.) indicates to CHX for various intervals. As shown in Fig. 6C that this amino acid is deleted. the level of mrfms decreased rapidly in presence of CHX, while no noticeable variations were 170) very similar to the CSF-1 receptor of mouse observed at the GAPDH mRNA level. Altogether macrophage is synthesized in L6al myoblasts via these findings supported the conclusion that a a short lived precursor of 115-116kD and an labile protein plays a role in the stabilization of immature gp130 (Leibovitch et al., 1989). The mrfms transcripts during proliferation of L6al expression of c-fms glycoproteins and mRNA myoblasts. Run-on assays were also performed Q
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TABLE 1 Amino acid number
Mrfms Mouse c-fms Cat c-fms Human c-fms Mrfms/mouse c-fms Mrfms/cat c-fms Mrfms/human c-fms
Total
External
Transmembrane
Internal
977 976 980 972 16/(98.4%) 240/(75.5%) 164/(83.1%)
510 510 514 512 7 (98.4%) 189 (63%) 127 (75.2%)
26 26 26 26 0 (100%) 6 (77%) 4 (84.6%)
441 440 440 434 9 (98%) 45 (89%) 33 (92.4%)
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Amino acid changes and percent similarity for mrfms, mouse c-fms, cat c-fms and human c-fms proteins are indicated respectively.
B
A
M q 1
200.
92-
2
3
4 kD ,170 130
6 9,
3 0-
FIGURE 4 Expression of mrfms products in myoblasts L6al ceils. (A) Northern blotting analysis of mrfms (RNA); Aliquots of 2.5pg of poly(A+) RNA from tissue cultured cells were processed sequentially for Northern blot hybridization with the
32P
random priming labeled inserts of mrfms, myogenin and MLCZ clones. Autoradiograms were exposed overnight for mrfms probe and 5 hr for myogenin and M L U probes. (B)Lysates of % methionine labeled L6al myoblasts (lanes 1-2), J774.2 cells (lane 3) or L6al myotubes (lane 4) were immunoprecipitated with the commercially rabbit polyclonal antibody to fms oncoprotein (Cat. no: OA-11-815 C.R.B. Cambridge, UK) (lanes 2-4) or preimmune serum (lane 1).130 and 170 kD c-fms bands are indicated.
CSF-1 RECFPTOR IN WOGENIC CELLS
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under the same conditions to determine whether exposure to CHX affects the transcription of the mrfms gene. No detectable effect on the rate of the mrfms transcription in treated compared to untreated myoblasts was observed (data not shown). This confirmed that the effects of CHX are at the post-transcriptional level. In addition
f
- rnrfn s -
I
--Gapd I puc
-1
-1 -III
FIGURE 5 Analysis of the relative levels of m r f m s gene transcription in proliferating L6al myoblasts and fully differentiated myotubes. Nuclear run-on assays were performed on nuclei isolated from Ma1 myoblasts and myotubes. 32P labeled nuclear RNA was hybridized as described in Materials and Methods.
m
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we have never detected mrfms transcripts in differentiated myotubes similarly treated with CHX and/or Act.D.
Mrfms is Constitutively Expressed After Transformation of L6al Myoblasts by v-fms We previously found that c-fms-related gene products are consistently expressed in spontaneous neoplastic transformants derived from non fusing L6al cells clones maintained under proliferating as well as differentiating conditions (Leibovitch et al., 1986, 1988). We have also observed that the various L6al cell clones of transformants obtained by transfecting different oncogenes, including v-fos, displayed elevated levels of c-fms related transcripts (our unpublished data). On the other hand, we had previously found that the endogenous c-fos gene was inactivated in all v-fos induced transformants derived from L6al cells (Leibovitch et al., 1977). It was therefore interesting to analyze mrfms RNA in v-fms induced transformants derived from L6al cells. For that purpose L6al cell-cultures were cotransfected with a pSVneo gene and with a recombinant provirus v-fms cloned in pBR322 as described in Materials and Methods and selected for focus-forming cells in soft agar:
.-
FIGURE 6 (A) Half life of mrfms in L6al myoblasts. Actinomycin D (ActD, 5 p g / m l ) was added for the indicated times. Twenty-five pg of total cellular RNA was analyzed as described in Materials and Methods and hybridized to the %P labeled mrfms or Gapdh probes. The levels of mrfms mRNA were measured by scanning densitometry and expressed as a percent of the control level obtained in non treated cells and plotted against time. A half-life of 5 hr was calculated from the slope. (B)Effects of actinomycin (Act.D) and cycloheximide (CHX)on m r f m s mRNA levels in L6al myoblasts. L6al myoblasts were treated with CHX (10pg/ml) 30 min before addition of Act.D (5 pg/ml) at indicated times. Total cellular RNA was hybridized sequentially to mrfms and Gapdh probes as indicated in Fig. 6A. The calculated half-life of the mrfms RNA was 35-40min. (C) Effects of cycloheximide (CHX) on mrfms mRNA levels in L6al myoblasts. CHX (10pg/ml) was added for the indicated times. Total cellular RNA was hybridized to the 3? mrfms and Gapdh probes. The calculated half-life on the mrfms RNA was 30 min.
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several hundred foci were obtained in separate assays. Among those, 15 cloned sublines were established and characterized as non differentiating neoplastic transformants (as in the case of other L6al derived transformants). When analyzed for integration all 15 lines were shown to contain from 3 to 25 v-fms copies in their genome. All of them expressed relatively high levels of a 8.5 kb v-fms transcript long, as well as the 3.7 kb mrfms mRNA independently of the serum concentration in the culture medium. Run-on assays were performed in order to determine whether transformation by the v-fms oncogene affects transcription of the endogenous mrfms gene. Experiments made with 3 clonal transformants gave results identical to those shown in Fig. 7A for the L6K5 fms clone. In the latter clone relatively high transcriptional rates of mrfms RNA were observed in the differentiation (2% FCS) as well as in the proliferation medium. However the mrfms probe cross-hybridizises with the v-fms mRNA. Northern blot allowed us to discriminate the two gene products. In Fig. 7B, a strong hybridization with the v-fms transcripts is visible, and much higher levels of endogenous mrfms mRNA are observed in transformed compared to non transformed myoblasts.
DISCUSSION In previous studies we used v-fms probes to demonstrate that the rat L6al and C2C12 lines of myogenic cells express an abundant amount of c-fms related transcript 3.7 kb long during their growth phase (the s a y size as in the J774.2 line of murine macrophages). The transcript became undetectable during the formation of myotubes following the transfer of growth-arrested cells to the low-serum differentiation medium (Leibovitch et al., 1986,1988,1989).High levels of this 3.7 kb transcript were also observed in a variety of neoplastic transformants derived from myogenic cells even when these transformants were maintained in the low serum differentiation medium (Leibovitch et al., 1986, 1989). Besides, low reproducible amounts of c-fms RNA were detected in fetal but not in adult rat skeletal muscles (Leibovitch et al., 1988). These findings were at variance with previous reports according to which expression of the c-fms gene appeared to be restricted to hematopoietic monocytes, placenta and macrophage containing tissues (Arceci et al., 1989; Stanley et al., 1983; Regenstief and Rossant, 1989). In the present study we have isolated from proliferating L6al Eat cells and
1
2
1
2
v-f m s mrfms
L6KSfms
L6d
LbKSfms
FIGURE 7 Constitutive expression of mrfms gene in v-fms transformant myoblasts. (A) Nuclear run-on assays were performed 1 on nuclei isolated from L 6 a l myoblasts and L6K5 fms transformants cultured in proliferation medium (10% FCS) or differentiation medium (2%FCS)32p labeled nuclear RNA was hybridized as described in Materials and Methods. (B)Aliquots of 2 5 p g of total cellular RNA were analyzed by Northern blotting experiments as described in Materials and Methods and hybridized to 32Pmrfms insert probe. Exposure time was overnight (lane 1, proliferation medium; lane 2, differentiation medium).
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CSF-1 RECEF'T€)R IN MYOGENIC CELLS
sequenced a complete cDNA clone termed mrfms. Using it as a probe, we confirm here that c-fms transcripts are abundant during the growth phase of rat myogenic cells and are eliminated after completion of the myogenic process. We also confirm our previous observation (Leibovitch et al., 1989) that the 3.7 kb long RNA spans the three domains of the CSF-1 receptor. Moreover, in contrast to previous data showing that expression of the c-fms gene is regulated at the translational level in human blood monocytes and HL60 cells induced to differentiate along the monocytic lineage (Weber et al., 1989), we demonstrate here that this gene is transcriptionally downregulated during the process of myogenesis. However, in the myogenic as well as in the monocytic lineages, a post translational mechanism involving the synthesis of a labile protein appears to stabilize the c-fms transcripts. We show in addition that endogenous c-fms transcripts are overexpressed in all the 15 sublines of non fusing transformants derived from L6al cells and remain overexpressed when maintained in a low serum medium. This suggests that the expression of v-fms activates or maintains the expression of endogenous c-fms. Recently, using two distinct anti-c-fms and anti-v-fms antisera, we found that c-fms related proteins located at the surface of proliferating L6al myoblasts undergo the same two-step glycosylation processing as the CSF-1 receptor of macrophages and contain a similar intracellular tyrosine kinase domain, whereas no c-fms related protein was detected in myotubes (Leibovitch et al., 1989). This finding raised the question of whether this protein is encoded by the CSF-1 receptor gene itself or by another closely related gene. The present study brings forth two sorts of data that strongly argue in favour of the former possibility. First, expression of c-fms related proteins in proliferating and not in differentiated myotubes is confirmed with a third specific antibody. Second, by our sequencing of the mrfms clone which confirms the amino acid homologies between the rat and the mouse (Rothwell and Rohrschneider, 19871, cat (Woolford et al., 1988) and human (Coussens et al., 1986) CSF-1 receptor genes. In particular this similarity exceeds 98% with the mouse gene and most of the deduced amino acid changes are conservative. Our demonstration that the c-fms gene is expressed in proliferating myoblasts and not in myotubes, and appears to
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be constitutively activated in our various neoplastic myoblasts studied strongly suggests that in muscle lineages this gene codes for the receptor of a CSF-1 like growth factor (if not CSF-1 itself) as it does in the monocytic lineages. Moreover, this hypothesis is consistent with a recent report showing that myogenically determined fibroblastic cells express the CSF-1 gene in the same manner as do hematopoietic lineages in response to activation. Moreover, conditioned media from these myogenically determined cells were shown to support in vitro colony formation of bone marrow monocytes (Harrington et al., 1990). We recently obtained preliminary results showing that L6al myogenic cells expressed CSF-1 gene products during the growth phase as well as during the myogenic process. However, accumulation of CSF-1 transcripts was markedly lower in differentiated myotubes than in proliferating myoblats. These results suggest that an autocrine mechanism involving CSF-1 and its receptor may play an important part in the growth of normal muscle stem cells and in their neoplastic proliferation. This mechanism could be itself regulated by inhibition of c-fms transcription during myogenesis. This may have interesting implications regarding the mechanism of muscle regeneration and malignant transformation. ACKNOWLEDGMENTS We thank Dr V. Rothwell and L. Rohrschneider for plasmids A755, A. Harel-Bellan for helpful discussion, Dr J. Hare1 for his continued encouragement and support and Marie-France Maman for preparing this manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS), INSERM and Association pour la Recherche contre le Cancer, and Association Francaise contre les Myopathies. The sequences data in the publication have been assigned to the following EMBL/Genbank Data accession number: X61479.
REFERENCES Arceci, R. J., Shanahan, F., Stanley, E. R. and Pollard, J. W. (1989) Temporal expression and location of colony stimulating factor 1 (CSF-1) and its receptor in the female reproductive tract are consistent with CSF-1 regulated placenta development. Proc. Nutl. Acad. Sci. USA 86,8818-8822. Barbacid, M. and Louver, A. V. (1981) Gene products of McDonough feline sarcoma virus have an in vitro-associ-
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ated protein kinase that phorphorylates tyrosine residues: lack of detection of this enzymatic activity in vivo. ]. Virol. 28,551-566. Coussens, L., Van Beveren, C. Smith, D., Chen, E., Mitchell, R. L., Isake C. M., Verma, I. M. and Ullrich, A. (1986) Structural alteration of viral homologue of receptor protooncogene fms at carboxy terminus. Nature 320,277-280. Dormer, L., Fedele, L. A., Garan, C. F., Anderson, S. J. and Sherr, C. J. (1982) McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (0-fms).1. Virol. 41,489-500. Fort, P., Marty, L., Piechaczyk, M.,El. Sabrouty, S., Dani, C., Jeanteur, P. and Blanchard, J. M. (1985) Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate deshydrogenase multigenic family. Nucl. Acid Res. 13,1431-1442. Garfinkel, L. F. Periasamy, M. and Nadal-Ginard, B. (1982). Cloning, identification and characterization of a actin, myosin light 1 , 2 and 3, hopomyosin and troponin C and T. ]. Biol Chem. 257,11078-11086. Greenberg, M. E. and Ziff, E. B. (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311,433-438. Harel, J., Leibovitch, M. P., Guillier, M., Borycki, A. G. and Leibovitch, S. A. (1989) Protooncogenes and differentiation versus transformation of striated muscle cells. Annals of the N . Y . Acad. Sci. 567,187-207. Harrington, M. A., Falkenburg, J. H., Daub, R. and Broxmeyer, H. E. (1990) Effect of myogenic and adipogenic differentiation on expression of colony stimulating factors genes. Mol. Cell. Biol. 10,4948-4952. Hillion, J., Leibovitch, S. A., Leibovitch, M. P., Raymondjean, M., Kruh, J. and Harel, J. (1984) Changes in gene expression following neoplastic transformation of rat myogenic cells. Cancer Res. 44,2959-2965. Horiguchi, J., Sariban, E. and Kufe, D. (1988) Transcriptional and posttranscriptional regulation of colony-stimulating factorl gene expression in human monocytes. Mol. Cell. Biol. 8,3951-3954. Leibovitch, S. A., Leibovitch, M. P., Guillier, M., Hillion, J. and Harel, J. (1986) Differential expression of proto-oncogenes related to transformation and cancer progression in rat myoblasts. Cancer Res. 46,4097-4103. Leibovitch, S . A., Leibovitch, M. P., Guillier, M. and Harel, J. (1988) Expression of two c-fms related gene products in rat muscular stem cells. Oncogene Res. 2,293-298. Leibovitch, S . A., Leibovitch, M. P., Borycki, A. G. and Harel, J. (1989) Expression of CSF-1 receptor-related proteins in muscular stem cells. Oncogene Res. 4,157-162. Leibovitch S . A., Lenormand, J. L., Leibovitch, M. P., Guillier, M., Mallard, L. and Harel, J. (1990) Rat myogenic c-mos cDNA: cloning sequence analysis and regulation during muscle development. Oncogene 5,1149-1157.
Muller, R., Slamon, D. J., Adamson, E. D., Tremblay, J. M., Muller, D., Cline, M. J. and Verma, I. M. (1983a) Transcriptional of c-onc genes, ki-rus and c-fms during mouse development. Ml. Cell. Biol. 3, 1062-1069. Muller, R., Tremblay, E., Adamson, E. and Verma I. M. (1983b) Tissue and cell type specific expression of two human c-onc genes. Nature 304,45434546. Nienhuis, A. W., BUM, H. F., Turner, P. H., Gopal Venkat, T., Nash, W. G., OBrien, S. J. and Sherr, C. J. (1985) Expression of the human c-fms proto-oncogene in hematopoietic cells and its deletion in the 5q-syndrome. Cell 42,421-428. Regenstreif, L. and Rossant, J. (1989) Expression of the c-fms proto-oncogene and of the cytokine, CSF-1 during mouse embryogenesis. Develop. Biol. 133,284-294. Rettenmier, C. W., Sacca, R., Furman, W. L., Roussel, M. L., Holt, J. T., Nienhuis, A. W., Stanley, E. R. and Sherr, C. J. (1986) Expression of the human c-fms proto-oncogenes product (colony-stimulating factor 1 receptor) on periph. eral blood mononuclear cells and choriocarcinoma cell lines. ]. Clin. Invest. 77,1740-1746. Rothwell, V. M. and Rohrschneider L. R. (1987) Murine c-fms cDNA: cloning, sequence analysis and retroviral expression. Oncogene Res. 1,311-324. Sanger, F., Nicken, S. and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sd. (USA) 74,5463-5467. Sariban, E., Mitchell, T and Kufe, D. (1985) Expression of the c-fms proto-oncogene during human monocyte differentiation. Nature 316,64-66. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F. Look, A. T. and Stanley E. R. (1985) The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor CSF-1. Cell 41,665-675. Sherr, C. J. (1988) The c-fms proto-oncogene. Bioch. Bioph. Act. 948,225-243. Sherr, C. J. (1990) Colony stimulating factorl receptor. Blood 75,l-12. Stanley, E. R., Guilbert, L. J., Tushinski, R. S. and Bortelmez, S. M. (1983) CSF-I: a mononuclear phagocyte lineagespecific hematopoietic growth factor. ]. Cell. Biol. 21, 151-1 59. Weber, B., Horigushi, J., Luebbers, R., Sherman, M. and Kufe, D. (1989) Posttranscriptional stabilization of c-fms mRNA by a labile protein during Human monocytic differentiation. Mol. Cell. Biol. 9,769-775. Woolford, J . , McAuliffe, A. and Rohrschneider, L. R. (1988) Activation of the feline c-fms proto-oncogene: Multiple alterations are required to generate a fully transformed phenotype. Cell 55,965-977. Wright, W. E., Sasson, D. and Lin, V. K. (1989) Myogenin, a factor regulating myogenesis has a domain homologous to MyoDl . Cell 56,777-783.
Growth Factors, 1992, Vol. 6, pp. 209-218 Reprints available directly from the publisher Photocopying permitted by license only
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Molecular Cloning of CSF-1 Receptor from Rat Myoblasts. Sequence Analysis and Regulation During Myogenesis ANNE-GMLLE BORYCKI, MARTINE GUILLIER, MARIE-PIERRE LEIBOVITCH and SERGE ALEXANDRE LEIEOVITCH*
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Luboratoire d’oncologie Moliculaire UA 1158-URA 126 du CNRS, Institut Gustave Roussy 39, rue C . Desrnoulins 94800 Villquif, France
(Received August 8 1991, Accepted October 17 1991)
We have isolated and sequenced a cDNA (mrfms) encoding rat c-fms gene (CSF-1 receptor) from proliferating L6al myoblasts. The predicted amino acid sequence was highly identical with the c-fms protein found in monocytes and macrophages (98,76 and 84% identity from mouse, cat and human c-fms proteins, respectively). The mechanisms responsible for the regulation of mrfms gene expression during myogenesis were examined. Mrfms products were observed during proliferation of L6al myoblasts and were downregulated during differentiation. Run-on transcription assays demonstrated that the mrfms gene was transcriptionally active only in undifferentiated myoblasts. These findings suggested that mrfms levels in L6al myoblasts are controlled by transcriptional mechanisms. The half-life of mrfms transcripts was found to be at least 5 hr while inhibition of protein synthesis with cycloheximide (CHX) decreased this halflife to 30min without changes in the rate of m r f m s gene transcription. In addition oncogenic transformation of L6al myoblasts by the v-fms induced constitutive upregulation of m r f m s mRNAs, and nuclear run-on assays demonstrated that mrfms transcription was not growth-factor dependent. Furthermore, these findings with others previously published indicate that mrfms gene products may play a role in the normal and neoplastic growth of niuscular cells. KEYWORDS: CSF-1 receptor, myogenesis, regulation
of the v-fms oncogene of a feline sarcoma virus (Donner et al., 1982). Besides, the c-fms protoFor several years our laboratory has investigated oncogene was found to be constitutively active in the possible role of proto-oncogenes (c-onc. a variety of L6al-derived neoplastic transgenes) in differentiation versus neoplastic trans- formants (Leibovitch et al., 1986,1989). These finformation of skeletal muscle, using as main dings were intriguing in view of previous data model a clonal subline of rat myogenic cells showing that the CSF-1 receptor is closely related termed L6al and various neoplastic derivatives. or identical to the product of the c-ftns proto(Leibovitch et al., 1986). Among 15 proto-onco- oncogene (Sherr et al., 19851, and c-fms trangenes analyzed for mRNA accumulation, all of scripts were only found at consistent levels in them expressed more or less elevated levels of placenta tissue (Muller et al., 1983a), Choriotranscripts during L6al cell proliferation carcinoma cells and cells differentiated along the (Leibovitch et al., 1986; Hare1 et al., 1989) and in monocyte lineage (Muller et al., 1983b). Furtherparticular relatively high amounts of two tran- more, the c-fms product exhibits tyrosine kinase scripts 3.7kb and 2.0 kb long from the c-fms activity (Rettenmier et al., 1986; Woolford et al., proto-oncogene which is the cellular counterpart 1985) and specifically binds CSF-1 (Rettenmier et al., 1986). These and other data have demonstrated that the c-fms gene is related to a family of genes coding for growth factor receptors with *To whom correspondence should be addressed.
INTRODUCTION
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intrinsic tyrosine kinase activity (Barbacid and phage line J774.2 was maintained in culture Louver, 1981). Other data have shown that the essentiplly as described (Sacca et al., 1986). c-fms gene is expressed when human HL60 myeloid leukemic cells are induced to differentiate cDNA Library Screening, Subcloning and along the monocytic lineage with phorbol esters Sequencing (Sariban et al., 1985), that it is constitutively expressed in mature human peripheral blood A Agtl0 cDNA library prepared from rat L6al monocyte (Nienhuis et al., 1985; Sariban et al., myoblasts in our laboratory was screened (5x105 1985) and the level of c-fms transcripts is down- plaques) with a murine c-fms probe (i1755, regulated following activation of these cells Rothwell and Rohrschneider, 1987) as described previously (Leibovitch et al., 1990). Six positive (Horiguchi et al., 1988). Our further studies demonstrated that the clones were isolated from this library. Only a 3.7 kb long c-fms related transcripts from L6al single clone contained the entire coding region. myoblasts codes for a glycosylated protein with Nucleic acid sequence determination was perforassociated tyrosine kinase activity apparently med by the dideoxy method (Sanger et al., 1977) identical to the CSF-1 receptor of macrophages. It using the sequenase kit (US Biochemical). Both is abundant at the surface of proliferating myo- strands of the cDNA inserts were sequenced by blasts and disappears after the formation of using a combination of bidirectional Bal 31 produced nested deletions and restriction enzyme myotubes (Leibovitch et al., 1989). In the present studies we have cloned and fragments which were subcloned in the pUC18 sequenced a complete cDNA clone encoding a and pUC19 vectors. CSF-1 receptor from L6al rat myoblasts. We demonstrated that this cDNA clone, termed RNA Blot Analysis mrfms, is practically identical to the mouse macrophage CSF-1 receptor. We also show that regu- Total cellular' RNA was prepared by lysis in lation of its expression occurs at the transcrip- guanidium isothiocyanate buffer and cesium tional level and that the synthesis of a labile gradient centrifugation (Leibovitch et al., 1986). protein is responsible for stabilization of its RNA Twenty-five pg of RNA per lane were separated during the growth of L6al myoblasts. Further- electrophoretically on 1% agarose-formaldehyde more, we observe that the transformation of the gels and transferred to Hybond (Amersham, L6al myoblasts with the v-fms oncogene is corre- UK). Hybridization assays were made as lated by the loss of myogenic differentiation and described previously (Leibovitch et al., 1990). constitutive transcription of the mrfms indepen- Final washing was O.lxSSC, 0.1% SDS at 55-65 "C dently of the conditions of culture. depending on the probe. Nuclear Run-on Assays MATERIALS AND METHODS Cell Culture and DNA Transfection In the present study myogenic cell samples were collected at two different stages. First, as proliferating myoblasts after two days in growth medium containing 10% FCS. At this stage no muscle-specific gene is expressed. Second, the L6a1 cell line was maintained and induced to differentiate under previously described conditions (Hillion et al., 1984). L6al myoblasts were cotransfected with the pSM-FeSV vector (Donner et al., 1982) and the pSV2neo vector under previously described conditions (Leibovitch et al., 1987). The CSF-1 independent mouse macro-
Cells were washed once with ice-cold phosphate buffered saline then lysed in lysis buffer (10 mM Tris-HC1, pH=7.4, 10 mM NaC1, 3 mM MgC12, 0.5% Nonidet P40). Nuclei were collected and resuspended in nuclei freezing buffer (50 mM Tris-HC1, pH=8, 5 mM MgC12, 40% glycerol, 0.5 mM dithiothreitol) and stored at -80 "C until used. Nuclear run-on assays were performed as essentially described by Greenberg and Ziff (1984), using d 2 P UTP (>3000Ci/mM, NEN). Run-on transcript product's were purified through G-50 Column and hybridized to 5 p g of the denatured plasmids. Plasmids were denatured in 0.3 M NaOH for 30 min at 65 "C neutralized with 2 M ammonium acetate and
CSF-1 RECEPTOR IN MYOGENIC CELLS
then slot blotted onto Hybond. Following hybridization for 48 hr at 65 "C in 0.3 M NaCl, 10 mM Hepes, pH=7.4, 10 mM EDTA 0.2% SDS, filters were processed as described previously (Greenberg and Ziff, 1984).
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SDS PAGE Analysis L6al cells were incubated in methionine-free medium for 30 min before labeling for 1 hr with 250 pCi/ml 35S-methionine (1200 Ci/mMole, NEN). After labeling, cells were lyzed in RIPA buffer and immunoprecipitated as described previously (Leibovitch et al., 1989) using a rabbit polyclonal antiserum against the cytoplasmic domain of the v-fms protein. Recombinant DNA Probes The random primed DNA insert probes used in hybridization were: Glyceraldhyde 3 phosphate deshydrogenase (GAPDH): a 1.3 kb PstI fragment of the rat GAPDH cDNA (pRGAPDHl3) (Fort et al., 1985). Murine CSF-1 receptor (c-fms): a 3.7 kb EcoRI fragment containing the complete cDNA sequence of the murine c-fms protein (L755) (Rothwell et al., 1987). Rat myogenic fms protein: a 3.6 kb EcoRI fragment containing the complete cDNA sequence at the rat CSF-1 receptor (mrfms). Myogenin, a 1.5 kb EcoRI fragment of the rat myogenin cDNA clone (Wright et al., 1989). Myosin light chain two (MLCZ), a 0.5 kb PstI fragment of pLC2-18 (Garfinkel et al., 1982).
21 1
755) as described in Materials and Methods. Six positive clones were identified under conditions of high stringency. The longest cDNA clone referred to here as mrfms (muscle rat fms) was subcloned for further analysis. The restriction enzyme map of the mrfms clone is indicated in Fig. 1. It is very similar to mouse, cat and human c-fms map especially in the 3' region of the cDNA. The nucleotide sequence of mrfms and its derived amino acid sequence are shown in Fig. 2. It contains 3667 bp which span the entire coding region, 75bp of 5' untranslated sequence and 661 bp 3' untranslated sequence. Its open reading frame of 2931 nucleotides from the 5' end start at position 76 and it codes for a polypeptide of 977aa. A polyadenylation signal ATTAAA is found at position 3645 and 16 nucleotides downstream from this poly A signal there is a tail of 12 A residues. K BC
B
X
mrfmS
HGURE 1 Restriction map of the m r f m s cDNA clone. The open box corresponds to the coding region. This clone was 100% sequenced from both strands B=BglZ, Bc=BclI, K=KpnI, H=HincII, Ps=PstI, Pv=PvuII, S=SacI, X=XbaI.
Comparison of the c-fms Proteins
The deduced amino acid sequences of mouse, cat and human c-fms gene products compared to the mrfms clone are shown in Fig. 3. This confirms RESULTS that c-fms proteins are highly conserved as are most other proto-oncogenes among vertebrate Cloning and Analysis of the c-fms cDNA species. The organisation of the mrfms protein is Clone from Rat Myoblasts (mrfms) very similar to the monocyte/macrophage CSF-I In previous reports using as a probe the v-fms receptor and can be divided into external, transclone we found that the c-fms proto-oncogene membrane and internal domains. The amino acid expressed two transcripts 2.0 kb and 3.7 kb long numbers and percent similarities between the in proliferating myoblasts and that both are pro- corresponding domains in mrfms, mouse c-fms, gressively eliminated during L6al cell differen- cat c-fms and human c-fms are given in Table 1. tiation. Furthermore, we showed that only the All four proteins are highly conserved in the 3.7kb mRNA spans the three domains of the internal kinase domain and less conserved in the CSF-1 receptor (Leibovitch et al., 1988). A lambda external glycosylated segment (Fig. 3). However, gtlO cDNA library was constructed using the overall amino acid homology of the external poly(A') RNA isolated from growing L 6 a l myo- domain between mrfms and mouse c-fms is 98%, blasts. 5x105 independent phage clones were between mrfms and cat c-fms it is 63% and mrfms screened with the murine c-fms probe (clone d and human c-fms it is 75%. As observed in the
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mouse c-fms product, mrfms protein contains nine potential N-linked glycosylation sites (Asn, X, Ser/thr) in the external domain (Fig. 3), six of the sites are also found in the same position in cat c-fms and human c-fms. Furthermore, the location of the ten cysteine residues in the external domain is conserved for all four c-fms proteins. The transmembrane domain formed by a stretch of 26 hydrophobic amino acids has 100, 77 and 85% homology with mouse, cat and human c-fms amino acid sequences, respectively. The intracellular domain is highly conserved between the 4 species with 90-98% amino acids identity (Table 1).This is also consistent with previous observations showing that like the c-fms proteins, the internal domain of mrfms proteins contains a region of homology with other tyrosine protein
kinases (Sherr, 1990). Furthermore the probable ATP binding site (residues 586-591) and lysine at 614 are identical between the four c-fms proteins. Control of mrfms Expression During Muscular Differentiation
Previous studies from our laboratory have demonstrated that consistent levels of c-fms related transcripts are expressed during proliferation of rat L6al myogenic cells as well as in all the neoplastic myoblasts studied. However, in non transformed cells these transcripts are eliminated during the myogenic process. (Leibovitch et al., 1986, 1988, 1989). These results have been extended to C2C12 mouse myogenic cells and we demonstrated that a glycoprotein of 170 kD (gp
CSF-1 RECEPTOR IN MYOGENIC CELLS
during myogenesis is shown in Fig. 4A and 4B. The expression pattern of the 3.7kb mrfms mRNAs paralleled the pattern of glycoproteins expression. No mrfms product is found after L6a 1 cells were switched to differentiation medium s nvsmsc RPKV CATPUSC LDPSI n Q H WIQGNA L for 4 days while, following their transfer to the HUKFUSC YSDGSS I s n Q F ’ G L C M RP V V F D 110 140 150 160 170 medium J774.2 macrophages continue to lIwlls Q E A W C L I ~ P A . L m S V S I F F ~ ~ € I I R I W ( V W S H T Y V Csame ~ msmsc P S express high level of c-fms transcripts. The supCATmSC D L L EAG VVR P Q N S P H T H FIENHV Q SAR wmsc D L L VEAG VVR PIMIINS P H T H FIQQDQSAL 190 200 210 220 230 pression of the expression at both the glycoproIIRFUWS V W G R E S T S T G I U ~ R V H P E P ~ I K ~ S K L V R I R G E U P I I L K R G MJSFUSC teins and mRNA levels in myotubes suggested D TV w QKDISG ATLT AE Q S I D N D S R H CATRISC SSVD N D € QHN nmmsc nc ~ v l IlS R QK I c ALT v AE that the regulation of mrfms gene did not occur 250 260 270 280 290 mms D T K L E I P L H S D W N Y Y - L S W W D A G I Y S C V I S W D V ~ A ~ F Q W ~ A Y at a translational level, but at the level of tranmsmsc TISQP H R LT n DH s N T T AW nns s v R CATFUSC APCQ n n L T~N D Q~ H n VQ KHSTS F R HLMRI$C N scription and/or mRNA accumulation in the 330 3 50 mms WLTSEQSWNSVGDSLIL~DAYPSIQHY~YLCPFFEWR. .KLEFITQMIY cytoplasm. Nuclear run-on assays (Fig. 5) msmsc L .. F CATFUSC T EKVD Q K V E GLESF SDY D . . D IKDT revealed that mrfms was transcriptionally active HWFUSC S N I T Mi N K W E GLGF SDH PEP ANA TKDT 370 380 390 400 I10 MRFIlS R Y T F K L F L H R V M S W G Q Y F L Q N ~ G ~ n L T F E L T ~ Y P P E L F C Din L6al proliferating myoblasts and switched off *oar*rc D . .._ CATFUSC ST S P L R R SFL R AG Q A R UTLI T L E in differentiated L6al myotubes, whereas no sigHWFUSC H T S P L P R S F L R F’G RA 1 TFI GT L A 430 440 450 460 470 nificant variation in transcriptional activity of lIRFUS V S G Y P Q P S V m E C R G H T D R C D E A Q ~ ~ ~ P ~ ~ Q K P F D K V I I Q S Q L P I G T ~ H n msmsc nL P GAPDH was observed in myotubes compared to V H E V H L A E VQ s S A G . L E S S CATFUSC A V DPY E H TV L T V E E LQ 5 HUUFUSC A N myoblasts. These results suggested that the 490 500 510 520 5?0 lIRRls I ( T Y F C K T H N S V G N S S Q Y F V S L M S I ( P L P D L S L ~ P ~ A ~ S ~ S L L V L L L L L L L Y K Y decrease in mrfms RNA level following differenmsmsc CATp?rSC R E I U F TYPIIAHT L LLT I A L nmmsc Q E IU SG UA IPI A AHTHP F I A L tiation occurred at the transcriptional level. 550 560 570 580 590 ..mRls K Q K P K Y Q V R Y I ( I I E R Y f f i N S ~ F I D ~ ~ Y ~ ~ E € P ~ ~ € G ~ L C A G A F G ~ ESubsequent AT studies were performed to deterIRISRlSC S CATMSC mine the stability of the mrfms mRNA. L6al cells nmmsc S 610 620 630 640 650 were treated with actinomycin D (Act.D) for various intervals. The levels of mrfms mFWA decreased about 40% after 4 hr of Act.D exposure (Fig. 6A). In contrast there was little if any effect on the level of GAPDH mRNA. These and other assays (not shown) suggest that the half life of NUSIWC R sn SEE c DG C A T W DG R HlMmSC .n SE mrfms RNA is 5-6 hr and is similar to that of 790 800 110 120 830 M R m ~ T ~ ~ ~ I G D P C ~ I ~ ~c-frrrs ~ ~ RNA I L in Y the ~ S ~ Human HL60 line (Weber et al., nvsIWc P D I CATPUSC R I €D 1989). In order to determine whether the stability mmsc N I €D 850 860 170 880 890 I(RR1S S Y C I W Y E I € S L C W P Y F ’ G I L ~ ~ K L ~ D G Y ~ Q W F A P ~ I Y S I of ~ Q Sthe ~ ~ mrfms transcripts is affected by inhibition nvsmsc n CATIWC S A A A of protein synthesis, or not, L6al myoblasts were HuLR(sC s A A A H 910 920 910 940 950 exposed to cycloheximide (CHX) for 30 min “S ~ r P Q I c € L I . Q B Q I R L e O ~ y ~ ~ ...GG(iS s ~ s D SsC % S S S W ~ S S E MUSRlSC ... before addition of Act.D for 90min. This treatCATJUSC S K QED V P Y T S S SSSSCRFG C M)IVIISC SFL am ~ l Tl ......_... RSGG s L ment caused a progressive decline in mrfms 970 971 mIW ~UCCEPCDIAQPLIQP”YQ€AC nvSIWC mRNA level (Fig. 6B) while no variation was CATIWC Q W M S C T Q observed for GAPDH mRNA. These results and FIGURE 3 Comparison of the deduced amino acid sequence those obtained with Act.D alone suggested that of mrfms and other c-fms proteins. The deduced amino acid inhibition of protein synthesis is associated with sequence of mrfms O W shown in Fig. 2 is compared with amino acid sequences from other vertebrate species given in destabilization of the mrfms mRNA. In another assay L6al myoblasts were exposed the literature. Changes for the murine, cat and human c-fms proteins are indicated in the corresponding lines. (.) indicates to CHX for various intervals. As shown in Fig. 6C that this amino acid is deleted. the level of mrfms decreased rapidly in presence of CHX, while no noticeable variations were 170) very similar to the CSF-1 receptor of mouse observed at the GAPDH mRNA level. Altogether macrophage is synthesized in L6al myoblasts via these findings supported the conclusion that a a short lived precursor of 115-116kD and an labile protein plays a role in the stabilization of immature gp130 (Leibovitch et al., 1989). The mrfms transcripts during proliferation of L6al expression of c-fms glycoproteins and mRNA myoblasts. Run-on assays were also performed Q
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TABLE 1 Amino acid number
Mrfms Mouse c-fms Cat c-fms Human c-fms Mrfms/mouse c-fms Mrfms/cat c-fms Mrfms/human c-fms
Total
External
Transmembrane
Internal
977 976 980 972 16/(98.4%) 240/(75.5%) 164/(83.1%)
510 510 514 512 7 (98.4%) 189 (63%) 127 (75.2%)
26 26 26 26 0 (100%) 6 (77%) 4 (84.6%)
441 440 440 434 9 (98%) 45 (89%) 33 (92.4%)
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Amino acid changes and percent similarity for mrfms, mouse c-fms, cat c-fms and human c-fms proteins are indicated respectively.
B
A
M q 1
200.
92-
2
3
4 kD ,170 130
6 9,
3 0-
FIGURE 4 Expression of mrfms products in myoblasts L6al ceils. (A) Northern blotting analysis of mrfms (RNA); Aliquots of 2.5pg of poly(A+) RNA from tissue cultured cells were processed sequentially for Northern blot hybridization with the
32P
random priming labeled inserts of mrfms, myogenin and MLCZ clones. Autoradiograms were exposed overnight for mrfms probe and 5 hr for myogenin and M L U probes. (B)Lysates of % methionine labeled L6al myoblasts (lanes 1-2), J774.2 cells (lane 3) or L6al myotubes (lane 4) were immunoprecipitated with the commercially rabbit polyclonal antibody to fms oncoprotein (Cat. no: OA-11-815 C.R.B. Cambridge, UK) (lanes 2-4) or preimmune serum (lane 1).130 and 170 kD c-fms bands are indicated.
CSF-1 RECFPTOR IN WOGENIC CELLS
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under the same conditions to determine whether exposure to CHX affects the transcription of the mrfms gene. No detectable effect on the rate of the mrfms transcription in treated compared to untreated myoblasts was observed (data not shown). This confirmed that the effects of CHX are at the post-transcriptional level. In addition
f
- rnrfn s -
I
--Gapd I puc
-1
-1 -III
FIGURE 5 Analysis of the relative levels of m r f m s gene transcription in proliferating L6al myoblasts and fully differentiated myotubes. Nuclear run-on assays were performed on nuclei isolated from Ma1 myoblasts and myotubes. 32P labeled nuclear RNA was hybridized as described in Materials and Methods.
m
215
we have never detected mrfms transcripts in differentiated myotubes similarly treated with CHX and/or Act.D.
Mrfms is Constitutively Expressed After Transformation of L6al Myoblasts by v-fms We previously found that c-fms-related gene products are consistently expressed in spontaneous neoplastic transformants derived from non fusing L6al cells clones maintained under proliferating as well as differentiating conditions (Leibovitch et al., 1986, 1988). We have also observed that the various L6al cell clones of transformants obtained by transfecting different oncogenes, including v-fos, displayed elevated levels of c-fms related transcripts (our unpublished data). On the other hand, we had previously found that the endogenous c-fos gene was inactivated in all v-fos induced transformants derived from L6al cells (Leibovitch et al., 1977). It was therefore interesting to analyze mrfms RNA in v-fms induced transformants derived from L6al cells. For that purpose L6al cell-cultures were cotransfected with a pSVneo gene and with a recombinant provirus v-fms cloned in pBR322 as described in Materials and Methods and selected for focus-forming cells in soft agar:
.-
FIGURE 6 (A) Half life of mrfms in L6al myoblasts. Actinomycin D (ActD, 5 p g / m l ) was added for the indicated times. Twenty-five pg of total cellular RNA was analyzed as described in Materials and Methods and hybridized to the %P labeled mrfms or Gapdh probes. The levels of mrfms mRNA were measured by scanning densitometry and expressed as a percent of the control level obtained in non treated cells and plotted against time. A half-life of 5 hr was calculated from the slope. (B)Effects of actinomycin (Act.D) and cycloheximide (CHX)on m r f m s mRNA levels in L6al myoblasts. L6al myoblasts were treated with CHX (10pg/ml) 30 min before addition of Act.D (5 pg/ml) at indicated times. Total cellular RNA was hybridized sequentially to mrfms and Gapdh probes as indicated in Fig. 6A. The calculated half-life of the mrfms RNA was 35-40min. (C) Effects of cycloheximide (CHX) on mrfms mRNA levels in L6al myoblasts. CHX (10pg/ml) was added for the indicated times. Total cellular RNA was hybridized to the 3? mrfms and Gapdh probes. The calculated half-life on the mrfms RNA was 30 min.
BORYCKI et al.
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several hundred foci were obtained in separate assays. Among those, 15 cloned sublines were established and characterized as non differentiating neoplastic transformants (as in the case of other L6al derived transformants). When analyzed for integration all 15 lines were shown to contain from 3 to 25 v-fms copies in their genome. All of them expressed relatively high levels of a 8.5 kb v-fms transcript long, as well as the 3.7 kb mrfms mRNA independently of the serum concentration in the culture medium. Run-on assays were performed in order to determine whether transformation by the v-fms oncogene affects transcription of the endogenous mrfms gene. Experiments made with 3 clonal transformants gave results identical to those shown in Fig. 7A for the L6K5 fms clone. In the latter clone relatively high transcriptional rates of mrfms RNA were observed in the differentiation (2% FCS) as well as in the proliferation medium. However the mrfms probe cross-hybridizises with the v-fms mRNA. Northern blot allowed us to discriminate the two gene products. In Fig. 7B, a strong hybridization with the v-fms transcripts is visible, and much higher levels of endogenous mrfms mRNA are observed in transformed compared to non transformed myoblasts.
DISCUSSION In previous studies we used v-fms probes to demonstrate that the rat L6al and C2C12 lines of myogenic cells express an abundant amount of c-fms related transcript 3.7 kb long during their growth phase (the s a y size as in the J774.2 line of murine macrophages). The transcript became undetectable during the formation of myotubes following the transfer of growth-arrested cells to the low-serum differentiation medium (Leibovitch et al., 1986,1988,1989).High levels of this 3.7 kb transcript were also observed in a variety of neoplastic transformants derived from myogenic cells even when these transformants were maintained in the low serum differentiation medium (Leibovitch et al., 1986, 1989). Besides, low reproducible amounts of c-fms RNA were detected in fetal but not in adult rat skeletal muscles (Leibovitch et al., 1988). These findings were at variance with previous reports according to which expression of the c-fms gene appeared to be restricted to hematopoietic monocytes, placenta and macrophage containing tissues (Arceci et al., 1989; Stanley et al., 1983; Regenstief and Rossant, 1989). In the present study we have isolated from proliferating L6al Eat cells and
1
2
1
2
v-f m s mrfms
L6KSfms
L6d
LbKSfms
FIGURE 7 Constitutive expression of mrfms gene in v-fms transformant myoblasts. (A) Nuclear run-on assays were performed 1 on nuclei isolated from L 6 a l myoblasts and L6K5 fms transformants cultured in proliferation medium (10% FCS) or differentiation medium (2%FCS)32p labeled nuclear RNA was hybridized as described in Materials and Methods. (B)Aliquots of 2 5 p g of total cellular RNA were analyzed by Northern blotting experiments as described in Materials and Methods and hybridized to 32Pmrfms insert probe. Exposure time was overnight (lane 1, proliferation medium; lane 2, differentiation medium).
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CSF-1 RECEF'T€)R IN MYOGENIC CELLS
sequenced a complete cDNA clone termed mrfms. Using it as a probe, we confirm here that c-fms transcripts are abundant during the growth phase of rat myogenic cells and are eliminated after completion of the myogenic process. We also confirm our previous observation (Leibovitch et al., 1989) that the 3.7 kb long RNA spans the three domains of the CSF-1 receptor. Moreover, in contrast to previous data showing that expression of the c-fms gene is regulated at the translational level in human blood monocytes and HL60 cells induced to differentiate along the monocytic lineage (Weber et al., 1989), we demonstrate here that this gene is transcriptionally downregulated during the process of myogenesis. However, in the myogenic as well as in the monocytic lineages, a post translational mechanism involving the synthesis of a labile protein appears to stabilize the c-fms transcripts. We show in addition that endogenous c-fms transcripts are overexpressed in all the 15 sublines of non fusing transformants derived from L6al cells and remain overexpressed when maintained in a low serum medium. This suggests that the expression of v-fms activates or maintains the expression of endogenous c-fms. Recently, using two distinct anti-c-fms and anti-v-fms antisera, we found that c-fms related proteins located at the surface of proliferating L6al myoblasts undergo the same two-step glycosylation processing as the CSF-1 receptor of macrophages and contain a similar intracellular tyrosine kinase domain, whereas no c-fms related protein was detected in myotubes (Leibovitch et al., 1989). This finding raised the question of whether this protein is encoded by the CSF-1 receptor gene itself or by another closely related gene. The present study brings forth two sorts of data that strongly argue in favour of the former possibility. First, expression of c-fms related proteins in proliferating and not in differentiated myotubes is confirmed with a third specific antibody. Second, by our sequencing of the mrfms clone which confirms the amino acid homologies between the rat and the mouse (Rothwell and Rohrschneider, 19871, cat (Woolford et al., 1988) and human (Coussens et al., 1986) CSF-1 receptor genes. In particular this similarity exceeds 98% with the mouse gene and most of the deduced amino acid changes are conservative. Our demonstration that the c-fms gene is expressed in proliferating myoblasts and not in myotubes, and appears to
217
be constitutively activated in our various neoplastic myoblasts studied strongly suggests that in muscle lineages this gene codes for the receptor of a CSF-1 like growth factor (if not CSF-1 itself) as it does in the monocytic lineages. Moreover, this hypothesis is consistent with a recent report showing that myogenically determined fibroblastic cells express the CSF-1 gene in the same manner as do hematopoietic lineages in response to activation. Moreover, conditioned media from these myogenically determined cells were shown to support in vitro colony formation of bone marrow monocytes (Harrington et al., 1990). We recently obtained preliminary results showing that L6al myogenic cells expressed CSF-1 gene products during the growth phase as well as during the myogenic process. However, accumulation of CSF-1 transcripts was markedly lower in differentiated myotubes than in proliferating myoblats. These results suggest that an autocrine mechanism involving CSF-1 and its receptor may play an important part in the growth of normal muscle stem cells and in their neoplastic proliferation. This mechanism could be itself regulated by inhibition of c-fms transcription during myogenesis. This may have interesting implications regarding the mechanism of muscle regeneration and malignant transformation. ACKNOWLEDGMENTS We thank Dr V. Rothwell and L. Rohrschneider for plasmids A755, A. Harel-Bellan for helpful discussion, Dr J. Hare1 for his continued encouragement and support and Marie-France Maman for preparing this manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS), INSERM and Association pour la Recherche contre le Cancer, and Association Francaise contre les Myopathies. The sequences data in the publication have been assigned to the following EMBL/Genbank Data accession number: X61479.
REFERENCES Arceci, R. J., Shanahan, F., Stanley, E. R. and Pollard, J. W. (1989) Temporal expression and location of colony stimulating factor 1 (CSF-1) and its receptor in the female reproductive tract are consistent with CSF-1 regulated placenta development. Proc. Nutl. Acad. Sci. USA 86,8818-8822. Barbacid, M. and Louver, A. V. (1981) Gene products of McDonough feline sarcoma virus have an in vitro-associ-
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ated protein kinase that phorphorylates tyrosine residues: lack of detection of this enzymatic activity in vivo. ]. Virol. 28,551-566. Coussens, L., Van Beveren, C. Smith, D., Chen, E., Mitchell, R. L., Isake C. M., Verma, I. M. and Ullrich, A. (1986) Structural alteration of viral homologue of receptor protooncogene fms at carboxy terminus. Nature 320,277-280. Dormer, L., Fedele, L. A., Garan, C. F., Anderson, S. J. and Sherr, C. J. (1982) McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (0-fms).1. Virol. 41,489-500. Fort, P., Marty, L., Piechaczyk, M.,El. Sabrouty, S., Dani, C., Jeanteur, P. and Blanchard, J. M. (1985) Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate deshydrogenase multigenic family. Nucl. Acid Res. 13,1431-1442. Garfinkel, L. F. Periasamy, M. and Nadal-Ginard, B. (1982). Cloning, identification and characterization of a actin, myosin light 1 , 2 and 3, hopomyosin and troponin C and T. ]. Biol Chem. 257,11078-11086. Greenberg, M. E. and Ziff, E. B. (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311,433-438. Harel, J., Leibovitch, M. P., Guillier, M., Borycki, A. G. and Leibovitch, S. A. (1989) Protooncogenes and differentiation versus transformation of striated muscle cells. Annals of the N . Y . Acad. Sci. 567,187-207. Harrington, M. A., Falkenburg, J. H., Daub, R. and Broxmeyer, H. E. (1990) Effect of myogenic and adipogenic differentiation on expression of colony stimulating factors genes. Mol. Cell. Biol. 10,4948-4952. Hillion, J., Leibovitch, S. A., Leibovitch, M. P., Raymondjean, M., Kruh, J. and Harel, J. (1984) Changes in gene expression following neoplastic transformation of rat myogenic cells. Cancer Res. 44,2959-2965. Horiguchi, J., Sariban, E. and Kufe, D. (1988) Transcriptional and posttranscriptional regulation of colony-stimulating factorl gene expression in human monocytes. Mol. Cell. Biol. 8,3951-3954. Leibovitch, S. A., Leibovitch, M. P., Guillier, M., Hillion, J. and Harel, J. (1986) Differential expression of proto-oncogenes related to transformation and cancer progression in rat myoblasts. Cancer Res. 46,4097-4103. Leibovitch, S . A., Leibovitch, M. P., Guillier, M. and Harel, J. (1988) Expression of two c-fms related gene products in rat muscular stem cells. Oncogene Res. 2,293-298. Leibovitch, S . A., Leibovitch, M. P., Borycki, A. G. and Harel, J. (1989) Expression of CSF-1 receptor-related proteins in muscular stem cells. Oncogene Res. 4,157-162. Leibovitch S . A., Lenormand, J. L., Leibovitch, M. P., Guillier, M., Mallard, L. and Harel, J. (1990) Rat myogenic c-mos cDNA: cloning sequence analysis and regulation during muscle development. Oncogene 5,1149-1157.
Muller, R., Slamon, D. J., Adamson, E. D., Tremblay, J. M., Muller, D., Cline, M. J. and Verma, I. M. (1983a) Transcriptional of c-onc genes, ki-rus and c-fms during mouse development. Ml. Cell. Biol. 3, 1062-1069. Muller, R., Tremblay, E., Adamson, E. and Verma I. M. (1983b) Tissue and cell type specific expression of two human c-onc genes. Nature 304,45434546. Nienhuis, A. W., BUM, H. F., Turner, P. H., Gopal Venkat, T., Nash, W. G., OBrien, S. J. and Sherr, C. J. (1985) Expression of the human c-fms proto-oncogene in hematopoietic cells and its deletion in the 5q-syndrome. Cell 42,421-428. Regenstreif, L. and Rossant, J. (1989) Expression of the c-fms proto-oncogene and of the cytokine, CSF-1 during mouse embryogenesis. Develop. Biol. 133,284-294. Rettenmier, C. W., Sacca, R., Furman, W. L., Roussel, M. L., Holt, J. T., Nienhuis, A. W., Stanley, E. R. and Sherr, C. J. (1986) Expression of the human c-fms proto-oncogenes product (colony-stimulating factor 1 receptor) on periph. eral blood mononuclear cells and choriocarcinoma cell lines. ]. Clin. Invest. 77,1740-1746. Rothwell, V. M. and Rohrschneider L. R. (1987) Murine c-fms cDNA: cloning, sequence analysis and retroviral expression. Oncogene Res. 1,311-324. Sanger, F., Nicken, S. and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sd. (USA) 74,5463-5467. Sariban, E., Mitchell, T and Kufe, D. (1985) Expression of the c-fms proto-oncogene during human monocyte differentiation. Nature 316,64-66. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F. Look, A. T. and Stanley E. R. (1985) The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor CSF-1. Cell 41,665-675. Sherr, C. J. (1988) The c-fms proto-oncogene. Bioch. Bioph. Act. 948,225-243. Sherr, C. J. (1990) Colony stimulating factorl receptor. Blood 75,l-12. Stanley, E. R., Guilbert, L. J., Tushinski, R. S. and Bortelmez, S. M. (1983) CSF-I: a mononuclear phagocyte lineagespecific hematopoietic growth factor. ]. Cell. Biol. 21, 151-1 59. Weber, B., Horigushi, J., Luebbers, R., Sherman, M. and Kufe, D. (1989) Posttranscriptional stabilization of c-fms mRNA by a labile protein during Human monocytic differentiation. Mol. Cell. Biol. 9,769-775. Woolford, J . , McAuliffe, A. and Rohrschneider, L. R. (1988) Activation of the feline c-fms proto-oncogene: Multiple alterations are required to generate a fully transformed phenotype. Cell 55,965-977. Wright, W. E., Sasson, D. and Lin, V. K. (1989) Myogenin, a factor regulating myogenesis has a domain homologous to MyoDl . Cell 56,777-783.
