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Coordinate induction of fibronectin, fibronectin receptor, tropomyosin, and actin genes in serum-stimulated fibroblasts
Experimental
Cell Research 180 (1989) 537-545
Coordinate Induction of Fibronectin, Fibronectin Receptor, Tropomyosin, and Actin Genes in Serum-Stimulated Fi broblasts R.-P. RYSECK, H. MACDONALD-BRAVO, and R. BRAVO’ European
Molecular Biology Laboratory, 6900 Heidelberg, Federal
Postfach Republic
M. ZERIAL,
10.2209, Meyerhofstrasse
1,
of Germany
From a collection of more than 80 nonoverlapping clones, isolated by differential screening of a 1, cDNA library prepared from serum-stimulated cells in the presence of cycloheximide, we have identified four clones that encoded for components of the cytoskeleton and extracellular matrix. DNA sequencing of clones B2, V58, TTI, and P38 demonstrated that they corresponded to p-actin, a-tropomyosin, fibronectin, and the psubunit of fibronectin receptor. All four mRNA levels showed a detectable increase 30 min after stimulation and remained at high levels for at least 8 h. The half-lives of these mRNAs were found to be very long in contrast to those of other growth factor-inducible genes. An increase in transcription was observed for the four genes. Actin and fibronectin showed nearly maximal increase at 15 min, while fibronectin receptor and tropomyosin reached their maximum transcription at 1 h. These results demonstrated that four interacting components of the cytoskeleton and extracellular matrix are rapidly induced in stimulated quiescent cells, possibly reflecting part of the coordinate changes in gene expression that occur during embryogenesis and wound healing. @ 1989 Academic Press, Inc.
Several normal processes governed by growth factors, including embryonic development and wound repair, share key cellular events such as cell proliferation and cell movement. The basis of these phenomena lies in the response of the cells to growth factors, possibly through the regulation of specific gene expression. It is therefore expected that isolation of genes whose expression is controlled by growth factors in fibroblasts could lead to the identification of molecules that play an important role in cell proliferation, embryogenesis, or wound healing. The number of isolated genes that immediately respond to growth factors in tibroblasts has been rapidly increasing and better estimates suggest that the expression of at least 100 genes is controlled by growth factors [l-5]. Apart from C-$X and c-myc, only a few of these genes have been identified recently [6--l 11. Our laboratory has recently described the isolation of X0 independent clones whose expression is rapidly increased by growth factors as a direct consequence of the growth factor/receptor interaction demonstrated by their induction even in the presence of cycloheximide [5]. Here we report that four of these clones encode for interacting components of the cytoskeletal-extracellular matrix system, these being fibronectin, the P-subunit of tibronectin receptor, cx-tropomyosin, and actin. ’ To whom correspondence should be addressed. 537
Copynght 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827189 $03.00
538
yseck et al. MATERIALS
AN
Cell culture. NIH 3T3 cells were routinely grown in Dulbecco’s modeled Eagle‘s medium supplemented with 10% fetal calf serum (FCS) and antibiotics (100 units/ml penicillin, 50 &ml streptonaycm). Confluent cells were made quiescent by incubating them for 48 h in medium containing 1% FCS. Cultures were stimulated by refeeding with 20% FCS. When used, cycloheximide was added at 10 CLgimland actinomycin D at 1 ugiml. Northern blot analysis. Total RNA was prepared from celis using the guanidine hydrochloride procedure 1121.Poly(A)+ RNA was isolated by oligo(dT)-cellulose (Collaborative Research) column chromato~aphy 1131.The RNA was separated on agarose gels containing 6% formaldeb~de and blotted onto Gene Screen Plus (New England Nuclear) [14]. baited inserts [15] were 3’P-labeied by nick-transiation to a specific activity of 1-5X 10’ cpmipg 1161.~yb~d~zatio~ was carried alit in 50% formamide, 0.5% SDS, 5x SSC (1 x SSC = 150mMNaC1, 15 m&f sodium citrate) and 5x Denhardt’s solution at 42°C for 40 h. Filters were extensively washed in 0.1 x SSC containing c SDS at S6”C. Rot blot analysis. Poly(A)* RNA for these studies was purified from total A prepared as described above and dotted onto messenger-activated paper (mAP, Grgenics Ltd.), washed twice for I5 min in buffer, washed in 70% ethanol for 10 min, and dried. The poly(A)’ RNA was released by a 5-min ~nc~bation in HZ0 at 70°C. [“P]cDNA probes were made by priming with oIigo~dT) and synthesis was carried out by AMV reverse transcriptase. The purified radioactive samples were hybridized as before against recombinant pUC 19 plasmids containing the cDNA insert spotted onto a Gene Screen Plus membrane (New England Nuclear) [17]. Filters were washed in 0.1 x SSC containing 0.5% SDS at 60°C. Nuclear run-on transcription assay. Nuclei were isolated from NIH 3T3 cells as described by Greenberg and Ziff [lS] with some modi~cat~ons. One 500-cm’ dish (Nunc, Denmark) of quiescent or seem-stimulated cells was rinsed three times with 20 ml of ice-cold p~osp~ate-buffered saline for the indicated periods of time. Cells were scrapped in the same buffer and pelleted at XOOgfor 1 min. The cell pellet (approximately 1-2x 10’ cells) was resuspended in 400 ul of NP-40 lysis buffer (10 rr# Tris-I-ICI, pH 7.4, 10 mM NaCl, 3 mM MgC12, 0.5 % NP-40), incubated for 5 min on ice. and sheered through a Gilson tip (about 10 times). The suspension was gently loaded on top of a 4-m! solution containing 0.7 M sucrose, 60 m&# KCl, 15 mM NaCl, IS mM Tris-HCi, pH 7.5. 0.5 rn& spermidine, 0.15mV spermine,2 mM EDTA, 0.5 m&f EGTA, 14 a ~-mer~a~toe~ha~ol, and 0.1% Triton X-100 ~e~~~ug~d at 13OOgfor IO min. The nuclear pellet was resuspended in 400 ~1of storage buffer (40% glycerol, 5 m?& MgCL, 50 M Tris-HCl, pH 8.0, 0.1 mS EDTA) and immediately frozen. For the run-on transcription assay, the nuclei were thawed, pelleted at 1300x for 3 min, and resuspended is 80 ~1of storage buffer. Then 30 u1of 10x reaction buffer (700 ti KCl, 50 m&f MgClz. 50 miW’P?is-HCl. pH 8.0, 25 m DTT, 1 n&f EDTA), 15 ~1of IOX ATP, CTP. and GTP (4 miM), 1 ~1 of RNasin (53 units/$), 25 pI of distilled H@, and 250 ul of lyophilized [a-32P]WTP(3000 Ciimmol; Amersham) were added and incubated at 30°C for 30 min. The ‘?-labeled RNA was isolated as previously described [I9]. Approximately 2x lo6 cpm/ml was used for the hybridizations. These were performed in SO% formamide, 0.5% SDS containing 5x SSC and SX Denhardt’s solution at 42°C for 72 h, and filters were extensively washed in 0.1 x SSC containing 0.5 % SDS at 56°C. Filters were then treated for 20 min with RNase A (10 ugiml) in 2x SSC and washed for 30 min in 0.1 x SSC containing 0.1% SDS at room temperature. cDNA sequence determination and analysis. Several convenient restriction sites were used and the fragments generated were subsequently transferred into M13-derived vectors. §~~g~~~st~a~d~d DNA was prepared [20] and the nucleotide sequence was determined by the chain ~e~~~a~~o~method of Sanger el al. [Zl] using the Sequenase (USS) kit. Nucleotide and amino acid computer analyses were carried out using the UWGCG programs.
We have previously reported the isolation of over 80 ~onoverla~~~~~ clones by differential screening of a h cDNA library prepared with RNA from NI cells seam-stimulated in the presence of cyc~Qhexi~~idefor 4 h [9r]. As an att~rn~t to identify these cDNAs, double-stranded se~~e~ci~g of the ends of ail the c!ones
Expression
of cytoskeletal
genes during GOIGI
539
Fig. 1. Poly(A)+ RNAs from quiescent cells (Q) and quiescent cells stimulated with 20 % FCS in the presence of cycloheximide for 4 h (FCS) blotted and hybridized against purified nick-translated inserts of the indicated clones. In all cases 0.4 pg of RNA was applied per lane. Ribosomal size markers are indicated.
previously recloned in pUC19 was performed [22]. This preliminary information was used to search for homology through the DNA (EMBL, Genbank, NBR) and protein (Swissprot) data bases. Of the 70 cDNA sequences analyzed four clones B2, P38, TTl, and V58 were intially found to present significant homology with components of the cytoskeleton and extracellular matrix. The mRNAs corresponding to these clones were induced several-fold after serum stimulation of quiescent cells in the presence of cycloheximide (Fig. 1) demonstrating that they belonged to the group of genes whose expression is directly controlled by growth factors. The homology initially observed was confirmed by completing the sequence of each of these clones. The sequence of clone V58 (1.7 kbp) revealed an open reading frame (nucleotides l-846) which encoded an amino acid sequence of 282 residues corresponding to the nearly complete sequence described for rabbit a-tropomyosin [23] (Fig. 2A]. These sequences were found to have an overall similarity of 89%. The first 255 amino acids of V58 present 100% similary with rabbit a-tropomyosin, the rest of the sequence (27 residues) is only 15% similar. It is possible that these tropomyosins are alternative spliced products of the same gene [24]. Comparison of V58 amino acid sequence with other tropomyosin also revealed a significant similarity, although not as high as with rabbit a-tropomyosin (not shown). The nucleotide sequence of rabbit a-tropomyosin has not been described. The nucleotide sequence of V58 showed similarity with several tropomyosins in the coding sequence or 3’ untranslated region, but never in the overall sequence. Thus we believe that V58 could represent a new undescribed type of tropomyosin. The nucleotide sequence of clone P38 (1.1 kbp) showed an 83 % homology with the #I-subunit of human fibronectin receptor (HFNR) [25]) and 81% similarity with chicken and amphibian integrin [26-281 (not shown). This similarity included an open reading frame corresponding to the 204 carboxy-terminal amino acids of the P-subunit of HFNR and nearly 400 nucleotides of 3’ untranslated sequence
540 Wyseck et al. AIKKKMQMLiLDKENALDRiEQAEADKKAA~DRSKQLEDELVSLQKKLK~
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Fig. 2. Similarity between the amino acid sequence of (A) V58 and rabbit a-tropomyosin (RBTM); (B) P38 and P-subunit of human fibronectin receptor (HFNR); and (C) TTl and iat fibronectin (RFN).
(not shown). The comparison of the amino acid sequence encode reading frame of P38 with /?-subunit of HFNR showed a 92% similarity as illustrated in Fig. 2B. The nucleotide sequence of 0.9 kbp of clone TTE presented more than 90% homology to the 3’ end of human [29] and rat fibronectin [30] mRNA. This region included 103 amino acids of the carboxy-terminal and 593 nucleotides of the 3’ untranslated region of fibronectin mRNA. Figure 2 C shows the ammo acid sequence comparison between TTI and rat fibronectin. These seque
Expression
of cytoskeletal
genes during WIG1
541
FCS + CHX Q '30'lh
2h 4h 8h'
Fig. 3. Dot blot analysis of mRNA levels in serum-stimulated cells. 32P-labeled cDNA from quiescent cells stimulated with 20 % FCS for different times in the absence (A) or in the presence (B) of cycloheximide hybridized against recombinant pUC19 containing inserts of the indicated cDNA clones.
found to be 99% similar. Comparison with the human fibronectin revealed a slightly smaller similarity of 93 %. The nucleotide sequence of B2 (not shown) proved to be identical to ,!I-actin, previously described to be induced by growth factors in quiescent cells [18]. Kinetics
of Induction
To determine the time course of appearance and decay of actin, P-subunit of fibronectin receptor (FNR), fibronectin, and tropomyosin mRNAs, quiescent cells were stimulated with 20 % FCS and the poly(A)+ RNA extracted at different times was analyzed by a dot blot procedure. Recombinant pUC19 plasmid carrying each insert was spotted onto a Gene Screen Plus membrane and hybridized to [32P]cDNA probes derived from the stimulated cells mRNAs. Actin and fibronectin mRNA levels showed a significant increase 30 min after stimulation, reaching a maximum at 2 h and remaining high at least till 8 h (Fig. 3A). Induction of tropomyosin and ,&subunit of FNR mRNA levels was slower, reaching a maximum between 2 and 4 h but also remaining high till 8 h (Fig. 3A). This was in clear contrast to that of a well-known, transiently induced gene such as c-fos, whose mRNA reached undetectable levels after 2 h (Fig. 3A). The presence of cycloheximide during the induction period did not significantly affect the levels of the mRNAs of actin, tropomyosin, fibronectin, and P-subunit of FNR during the period of time analyzed (Fig. 3B). The effect of protein synthesis inhibition on the mRNA levels of genes transiently expressed is illustrated by c-fos. This superinduction observed in the presence of cycloheximide is in part due to the stabilization of mRNAs with very short half-lives such as c-fos. The fact that no effect was observed in actin, tropomyosin, P-subunit of FNR, and fibronectin mRNA levels could be an indication that these mRNAs are stable molecules. To determine this, quiescent cells stimulated with 20% FCS in the presence of cycloheximide for 4 h were carefully washed and incubated for different periods of time in 10% FCS containing actinomycin D in the presence or absence of cycloheximide. RNA was extracted at different times and the [32P]cDNA probes 35-898332
542
yseck et al.
i
KS + Act.D o’ 30’ 60' 90' 2h 4h
I
Fig. 4. Stability of mRNAs. [‘*P]cDNA was obtained from poly(A)’ RNA extracted fron quiescent ceils stimulated with 20% FCS in the presence of cycloheximide for 4 h (0’) or from equivalent cultures incubated for the indicated times in FCS plus 2~~~0~~~~~ in the absence (& or in the presence (B) of cyclohe~imide and hybridized as described under &hods to Gene Screen Plus membranes containing cDNA inserts in pUC19. In (A) cells were carefully washed three times with medium containing serum to eliminate the cycloheximide before adding FCS plus actinomycia C.
~erjved from these samples were a~a~y~e~by a dot blot proce NA levels 5f actin, clearly demo~straied that the subunit of FNR, and fibronectin did not decrease . 4A), suggesting that their half-lives were at least determined for c-f&s mRNA (10-15 mm). This was confirmed by the that in the presence of ~yclohexim~de ~~e~t~~alresults were obtai s of the four studied clones (Pig. , the stability of c-fos mRNA was would explain in part the previous observation that e on the level of induction of the four e synthesis of two of the proteins, p n and ~-trop~rny~si~, was mined after stimulation. Qnies~ent cells abeled fsr 1 h with ~~5~~~~~~~~~~~~~ rad~Qact~v~proteins were a~~a~~~e~ at different times after serum addition a by two-dimensional gel electrophoresis. synthesis of actin and tropomyosin rapi ftes s~imnlati5~ in 8 fashion similar to that observed for their mRN ~~a~$~~~~t~~~a~A~t~~Qti~~ by ~~r~~
The effect of serum on the transcription of the ~~~~~~b~~ genes was isolated nuclei from NIH 3T3 cells. Nuclei were is&ted at serum addition to quiescent cells and run-on tra~§~r~~t~on assay and ~ro~om~osin genes formed, As shown in Fig. SA, traus~~i~t~~~ sf the n, Actin remained ex~u~reased dramatizable within 15 min of serum a pressed at high levels for at least 8 h, in contrast to tr~~~myos~~ w sion reached basal levels after 4 h. The fibronectin and ~~s~b~~i~ of F owed a small increase in expression after 15 min rea6: , ~ibro~ect~n expression decrease ver spLowly7remaining high after 8 h. T expression of the receptor gene at nearly a maximum kvd up to 8 IL
Expression of cytoskeletal genes during GOIGl 543 Transcriptional FCS I I Q 15' 30' lh 2h 4h 8h
activation FCS + CHX I Q 15' 30' lh 2h 4h 8h
Fig. 5. Transcriptional activation induced by serum. Nuclei were isolated from quiescent cells (Q) or cells stimulated with 20 % FCS in the absence (A) or in the presence of cycloheximide (B) for the indicated times and run-on transcription assays were performed. The “P-labeled transcripts were hybridized against recombinant pUC19 containing the respective cDNAs immobilized on Gene Screen Plus membrane.
dramatic contrast to these results was the expression of the c-fos gene as shown in Fig. 5A. Similar experiments with nuclei isolated from quiescent cells stimulated in the presence of cycloheximide were performed (Fig. 5B). A slight effect on the transcriptional activation of actin, fibronectin, and FNR genes could be observed. All three presented a small increase in their levels of expression compared to that in the absence of cycloheximide. Tropomyosin gene expression, the most transient of this group, was significantly prolonged, although not as much as that observed for the c-fos gene. These results demonstrated that the increase in actin, tibronectin, ,&subunit of FNR, and tropomyosin mRNAs in serum-stimulated cells results at least in part from the direct stimulation of their genes. DISCUSSION The changes in gene expression induced by growth factors in quiescent cells are generally considered only as part of the mitogenic response. However, it is possible that many of these gene products are necessary to integrate and coordinate complex biological processes in which cell proliferation is a common event. We have demonstrated that the expression of four interacting components of the cytoskeleton and matrix, i.e., actin, P-subunit of FNR, a-tropomyosin, and fibronectin is rapidly induced by serum in quiescent tibroblast cells. The effect of serum stimulation on their mRNA levels lasts for at least 8 h, in contrast to other mRNAs like c-fos that show a very rapid but transient induction. While this work was in preparation, the stimulation of the fibronectin gene by growth factors was reported [3 11. The nuclear run-on experiments showed that transcription of these genes increased rapidly after serum stimulation and that it remained high for several hours, with the exception of tropomyosin. However, due to the long half-life of
544
yseck et al.
NA, it was still present at high levels 8 h after stirn~~at~o~. As transcription activation also occurred in the presence of ~y~~obex~rn~d~we can conclude that it is a direct consequence of the growth factorlrece The results presented here are of interest especially if they are mtegrated in biological processes such as wound healing and embryogenesis in which several l-tans role growth factors act in concert. It is known that fibroblasts play in cutaneous wound repair. They rapidly migrate to the woun and their most important function is the remodelling of the extra~el~~~ar matrix. The fibroblasts that migrate to the wound region rapidly secrete an extensive fibronectin matrix possibly required for the organization of collagen [32--341. In addition, other matrix components are codeposited with ronectin suggesting that its presence is essential in the organization of the gra ation tissue matrix. Fibronectin is a high-molecular-weight protein that is able to interact with several ma~romolecuies using different structural domains of its subunits (reviewed Hn [35]). One of these is a binding domain that is able to interact wit receptor, a glycoprotein involved in the transmembra~e linkage between fibronectin and actin (for review see [28]). ~ibro~e~ti~ receptor, in its ca as a transmembrane linker between the extracellular matrix nd the cytoskefeton, is presumed to function in cell migration in embryos an probably in wound at the /?-subunit hng [36]. Moreover, it has been recently WA is expressed in gastrula and neurula sta embryos WI. A~~~~~~~~~Q~ of fibronectin receptor mRNA at the gastrula stage coincides with the onset of cell ion. Fibronectin levels also increase during this period [37] an en shown to occur in contact with an extracellular matrix ric tin [37-391. The increase in gene expression of actin can be c changes in cytoskeleton and cellular motility occurring rapi Several studies have shown that these initial. cytoskeletal changes are parried by the structural organization of actin filaments into stress fibers 1. Interestingly, we have observed t myosin, a ~orn~o~~~t of the stress fibers [43] and an important modu~at is also indua [44]. The multiplicity of tropomyosin isoforms in ~bro~lasts [45, 461 raises t possibility that specific associations of giv isoforms with actin le to different anges in relative Ieve of different actin structures. It is possible then that forms of tropomyosin in the cell may alter the organization of the actin microhlaments [44] ~ Altogether, these results would suggest that the immediate changes in actin, tropomyosin, tibronectin, and fibronectin ceptor expression after st~~~~~at~o~ of quiescent fibroblasts are correlated with e normal response of cebis in several complex biological processes. I. 2. 3. 4. 5.
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41. Goldman, R. D., Lazarides, E., Pollack, R., and Wever, K. (1975) Exp. Cell Res. 90, 333-344. 42. Goldman, R. D., Schloss, J. A., and Starger, J. M. (1976) in Cell Motility (Goldman, R. D., Pollard, T., and Rosenbaum, J., Eds.), Vol. A, pp. 217-245, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 43. Lazarides, E. {1975) .I. Cell Biol. 65, 549-561. 44. Payne, M. R., and Rudnick, S. E. (1984) Trends Biochem. Sci. 9, 361-363. 45. Matsumura, F., Lin, J. J. C., Yamashiro-Matsumura, S., Thomas, G. P., and Topp, W. C. (1983) J. Biol. Chem. 258, 13,954-13,964. 46. Matsumura, F., Yamashiro-Matsumura, S., and Lin, J. J. C. (1983) J. Biol. Chem, 258, 6636-6644. Received July 15, 1988
Printed
in Sweden
Cell Research 180 (1989) 537-545
Coordinate Induction of Fibronectin, Fibronectin Receptor, Tropomyosin, and Actin Genes in Serum-Stimulated Fi broblasts R.-P. RYSECK, H. MACDONALD-BRAVO, and R. BRAVO’ European
Molecular Biology Laboratory, 6900 Heidelberg, Federal
Postfach Republic
M. ZERIAL,
10.2209, Meyerhofstrasse
1,
of Germany
From a collection of more than 80 nonoverlapping clones, isolated by differential screening of a 1, cDNA library prepared from serum-stimulated cells in the presence of cycloheximide, we have identified four clones that encoded for components of the cytoskeleton and extracellular matrix. DNA sequencing of clones B2, V58, TTI, and P38 demonstrated that they corresponded to p-actin, a-tropomyosin, fibronectin, and the psubunit of fibronectin receptor. All four mRNA levels showed a detectable increase 30 min after stimulation and remained at high levels for at least 8 h. The half-lives of these mRNAs were found to be very long in contrast to those of other growth factor-inducible genes. An increase in transcription was observed for the four genes. Actin and fibronectin showed nearly maximal increase at 15 min, while fibronectin receptor and tropomyosin reached their maximum transcription at 1 h. These results demonstrated that four interacting components of the cytoskeleton and extracellular matrix are rapidly induced in stimulated quiescent cells, possibly reflecting part of the coordinate changes in gene expression that occur during embryogenesis and wound healing. @ 1989 Academic Press, Inc.
Several normal processes governed by growth factors, including embryonic development and wound repair, share key cellular events such as cell proliferation and cell movement. The basis of these phenomena lies in the response of the cells to growth factors, possibly through the regulation of specific gene expression. It is therefore expected that isolation of genes whose expression is controlled by growth factors in fibroblasts could lead to the identification of molecules that play an important role in cell proliferation, embryogenesis, or wound healing. The number of isolated genes that immediately respond to growth factors in tibroblasts has been rapidly increasing and better estimates suggest that the expression of at least 100 genes is controlled by growth factors [l-5]. Apart from C-$X and c-myc, only a few of these genes have been identified recently [6--l 11. Our laboratory has recently described the isolation of X0 independent clones whose expression is rapidly increased by growth factors as a direct consequence of the growth factor/receptor interaction demonstrated by their induction even in the presence of cycloheximide [5]. Here we report that four of these clones encode for interacting components of the cytoskeletal-extracellular matrix system, these being fibronectin, the P-subunit of tibronectin receptor, cx-tropomyosin, and actin. ’ To whom correspondence should be addressed. 537
Copynght 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827189 $03.00
538
yseck et al. MATERIALS
AN
Cell culture. NIH 3T3 cells were routinely grown in Dulbecco’s modeled Eagle‘s medium supplemented with 10% fetal calf serum (FCS) and antibiotics (100 units/ml penicillin, 50 &ml streptonaycm). Confluent cells were made quiescent by incubating them for 48 h in medium containing 1% FCS. Cultures were stimulated by refeeding with 20% FCS. When used, cycloheximide was added at 10 CLgimland actinomycin D at 1 ugiml. Northern blot analysis. Total RNA was prepared from celis using the guanidine hydrochloride procedure 1121.Poly(A)+ RNA was isolated by oligo(dT)-cellulose (Collaborative Research) column chromato~aphy 1131.The RNA was separated on agarose gels containing 6% formaldeb~de and blotted onto Gene Screen Plus (New England Nuclear) [14]. baited inserts [15] were 3’P-labeied by nick-transiation to a specific activity of 1-5X 10’ cpmipg 1161.~yb~d~zatio~ was carried alit in 50% formamide, 0.5% SDS, 5x SSC (1 x SSC = 150mMNaC1, 15 m&f sodium citrate) and 5x Denhardt’s solution at 42°C for 40 h. Filters were extensively washed in 0.1 x SSC containing c SDS at S6”C. Rot blot analysis. Poly(A)* RNA for these studies was purified from total A prepared as described above and dotted onto messenger-activated paper (mAP, Grgenics Ltd.), washed twice for I5 min in buffer, washed in 70% ethanol for 10 min, and dried. The poly(A)’ RNA was released by a 5-min ~nc~bation in HZ0 at 70°C. [“P]cDNA probes were made by priming with oIigo~dT) and synthesis was carried out by AMV reverse transcriptase. The purified radioactive samples were hybridized as before against recombinant pUC 19 plasmids containing the cDNA insert spotted onto a Gene Screen Plus membrane (New England Nuclear) [17]. Filters were washed in 0.1 x SSC containing 0.5% SDS at 60°C. Nuclear run-on transcription assay. Nuclei were isolated from NIH 3T3 cells as described by Greenberg and Ziff [lS] with some modi~cat~ons. One 500-cm’ dish (Nunc, Denmark) of quiescent or seem-stimulated cells was rinsed three times with 20 ml of ice-cold p~osp~ate-buffered saline for the indicated periods of time. Cells were scrapped in the same buffer and pelleted at XOOgfor 1 min. The cell pellet (approximately 1-2x 10’ cells) was resuspended in 400 ul of NP-40 lysis buffer (10 rr# Tris-I-ICI, pH 7.4, 10 mM NaCl, 3 mM MgC12, 0.5 % NP-40), incubated for 5 min on ice. and sheered through a Gilson tip (about 10 times). The suspension was gently loaded on top of a 4-m! solution containing 0.7 M sucrose, 60 m&# KCl, 15 mM NaCl, IS mM Tris-HCi, pH 7.5. 0.5 rn& spermidine, 0.15mV spermine,2 mM EDTA, 0.5 m&f EGTA, 14 a ~-mer~a~toe~ha~ol, and 0.1% Triton X-100 ~e~~~ug~d at 13OOgfor IO min. The nuclear pellet was resuspended in 400 ~1of storage buffer (40% glycerol, 5 m?& MgCL, 50 M Tris-HCl, pH 8.0, 0.1 mS EDTA) and immediately frozen. For the run-on transcription assay, the nuclei were thawed, pelleted at 1300x for 3 min, and resuspended is 80 ~1of storage buffer. Then 30 u1of 10x reaction buffer (700 ti KCl, 50 m&f MgClz. 50 miW’P?is-HCl. pH 8.0, 25 m DTT, 1 n&f EDTA), 15 ~1of IOX ATP, CTP. and GTP (4 miM), 1 ~1 of RNasin (53 units/$), 25 pI of distilled H@, and 250 ul of lyophilized [a-32P]WTP(3000 Ciimmol; Amersham) were added and incubated at 30°C for 30 min. The ‘?-labeled RNA was isolated as previously described [I9]. Approximately 2x lo6 cpm/ml was used for the hybridizations. These were performed in SO% formamide, 0.5% SDS containing 5x SSC and SX Denhardt’s solution at 42°C for 72 h, and filters were extensively washed in 0.1 x SSC containing 0.5 % SDS at 56°C. Filters were then treated for 20 min with RNase A (10 ugiml) in 2x SSC and washed for 30 min in 0.1 x SSC containing 0.1% SDS at room temperature. cDNA sequence determination and analysis. Several convenient restriction sites were used and the fragments generated were subsequently transferred into M13-derived vectors. §~~g~~~st~a~d~d DNA was prepared [20] and the nucleotide sequence was determined by the chain ~e~~~a~~o~method of Sanger el al. [Zl] using the Sequenase (USS) kit. Nucleotide and amino acid computer analyses were carried out using the UWGCG programs.
We have previously reported the isolation of over 80 ~onoverla~~~~~ clones by differential screening of a h cDNA library prepared with RNA from NI cells seam-stimulated in the presence of cyc~Qhexi~~idefor 4 h [9r]. As an att~rn~t to identify these cDNAs, double-stranded se~~e~ci~g of the ends of ail the c!ones
Expression
of cytoskeletal
genes during GOIGI
539
Fig. 1. Poly(A)+ RNAs from quiescent cells (Q) and quiescent cells stimulated with 20 % FCS in the presence of cycloheximide for 4 h (FCS) blotted and hybridized against purified nick-translated inserts of the indicated clones. In all cases 0.4 pg of RNA was applied per lane. Ribosomal size markers are indicated.
previously recloned in pUC19 was performed [22]. This preliminary information was used to search for homology through the DNA (EMBL, Genbank, NBR) and protein (Swissprot) data bases. Of the 70 cDNA sequences analyzed four clones B2, P38, TTl, and V58 were intially found to present significant homology with components of the cytoskeleton and extracellular matrix. The mRNAs corresponding to these clones were induced several-fold after serum stimulation of quiescent cells in the presence of cycloheximide (Fig. 1) demonstrating that they belonged to the group of genes whose expression is directly controlled by growth factors. The homology initially observed was confirmed by completing the sequence of each of these clones. The sequence of clone V58 (1.7 kbp) revealed an open reading frame (nucleotides l-846) which encoded an amino acid sequence of 282 residues corresponding to the nearly complete sequence described for rabbit a-tropomyosin [23] (Fig. 2A]. These sequences were found to have an overall similarity of 89%. The first 255 amino acids of V58 present 100% similary with rabbit a-tropomyosin, the rest of the sequence (27 residues) is only 15% similar. It is possible that these tropomyosins are alternative spliced products of the same gene [24]. Comparison of V58 amino acid sequence with other tropomyosin also revealed a significant similarity, although not as high as with rabbit a-tropomyosin (not shown). The nucleotide sequence of rabbit a-tropomyosin has not been described. The nucleotide sequence of V58 showed similarity with several tropomyosins in the coding sequence or 3’ untranslated region, but never in the overall sequence. Thus we believe that V58 could represent a new undescribed type of tropomyosin. The nucleotide sequence of clone P38 (1.1 kbp) showed an 83 % homology with the #I-subunit of human fibronectin receptor (HFNR) [25]) and 81% similarity with chicken and amphibian integrin [26-281 (not shown). This similarity included an open reading frame corresponding to the 204 carboxy-terminal amino acids of the P-subunit of HFNR and nearly 400 nucleotides of 3’ untranslated sequence
540 Wyseck et al. AIKKKMQMLiLDKENALDRiEQAEADKKAA~DRSKQLEDELVSLQKKLK~
I/ / I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ! Ill
V58
1I i I I I I I I I I I I I
AIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEDELVSLQKKLKG
RBTM
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I I I I I I I I I I I I I I I I I I I I I I I / I//l
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! I I I I I I I I I I I I I I I ! ! II/I
TEDELDKYSEALKDAQEKLELAEKKATDFEADVASLNRRIQLVEEELDRA
RBTM
QERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKMFXQEIQLKEAK
V58
lIIIlIIlIIII/lII//IlI/lII/Il!ll/Il////lI///II/II/I QERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKM~IQEIQLKEAK
RBTM
HIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTVTN
V58
IIIIII//IIIIIIII/I/II/lIilI/!Il////l/iII//III/~//I HIAEDADRKYEEVARKLVIIESDLERAEERAF,LSEGKCALEEELKTVTN
RBTM
NLKSLEAQAEKYSQKEDKYEEEIKVLSDKLKEAET~FAERSVTKLEKS
V58
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIi NLKSLEAQAEKYSQKEDKYEEEIKVLSDKLKEAETRAEFAERSVTKLEKS
A
1DDLEEKVAhKEENLSMH;MLDQTLLELNNM
IIIII
I
II
RBTM
V58
I
IDDLEDELYAQKLKYKAISEELDHALNDMTSI
RBTM
DTGPCLASNGQICNGRGICECGACKCTDPKFQGPTCETCQTCLGVCAE.YK
II
I I/Il//lI/II//lI/
liI/lllIi/
l/I
P38
I///l//II/l/
DTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCE~CQTCLGVCAEHK
HFNR
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II/ll/l/iIII/l/II
III/
I/lI//li/
/I/I//l
P38
III
/III/
ECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEK
BFNR
DIDDCWFYFTYSVNGNNEAIVHVVETPDCPTGPDIIPIVAGVVAGIVLIG
P38
I lIlI/lI/IIIlIlII
II/l/
I !iIii/I!IIlIII//III//I
DVDDCWFYFTYSVNGNNEVHVVENPECPTGPDIIPIVAGWAGIVLIG LALLLIWXLiMIIHDRREFiKFEKEKMNAkDTGENPIYiSAVTTVVNPi
HFNR P38
IlI/lI//III/I/l/I/III/!II///l/;llll/ll/lll;!l//li~ LALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTWNPK
B
YEGK
HFNR
P38
IIII YEGK
HFNR
EFKCDPHEA&'DDGKTYH;GEQWQKEYL~AICSCTCFG~QRGWRCDN&
TTl
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiIIIIIII/IiIIIIIIII EFKCDPHEATCYDDGKTYHVGEQWQKEYLGAICSCTCFGGQRGWRCDNCR
RFN
RPGAAEPSPDGTTGHTYNQYTQRYNQRTNTNVNCPIECFMPLDVQADRDD
TT:
//IIIIIIIII/III/IlliIIlI
Il~!I/I/l/Ill//lIl!lIIIII
RPGAAEPSPDGTTGHTYNQYTQRYHQRTNT~CPIECFhf?LDVQADRDD SRE
RFN
TTl
III SFCE WN
Fig. 2. Similarity between the amino acid sequence of (A) V58 and rabbit a-tropomyosin (RBTM); (B) P38 and P-subunit of human fibronectin receptor (HFNR); and (C) TTl and iat fibronectin (RFN).
(not shown). The comparison of the amino acid sequence encode reading frame of P38 with /?-subunit of HFNR showed a 92% similarity as illustrated in Fig. 2B. The nucleotide sequence of 0.9 kbp of clone TTE presented more than 90% homology to the 3’ end of human [29] and rat fibronectin [30] mRNA. This region included 103 amino acids of the carboxy-terminal and 593 nucleotides of the 3’ untranslated region of fibronectin mRNA. Figure 2 C shows the ammo acid sequence comparison between TTI and rat fibronectin. These seque
Expression
of cytoskeletal
genes during WIG1
541
FCS + CHX Q '30'lh
2h 4h 8h'
Fig. 3. Dot blot analysis of mRNA levels in serum-stimulated cells. 32P-labeled cDNA from quiescent cells stimulated with 20 % FCS for different times in the absence (A) or in the presence (B) of cycloheximide hybridized against recombinant pUC19 containing inserts of the indicated cDNA clones.
found to be 99% similar. Comparison with the human fibronectin revealed a slightly smaller similarity of 93 %. The nucleotide sequence of B2 (not shown) proved to be identical to ,!I-actin, previously described to be induced by growth factors in quiescent cells [18]. Kinetics
of Induction
To determine the time course of appearance and decay of actin, P-subunit of fibronectin receptor (FNR), fibronectin, and tropomyosin mRNAs, quiescent cells were stimulated with 20 % FCS and the poly(A)+ RNA extracted at different times was analyzed by a dot blot procedure. Recombinant pUC19 plasmid carrying each insert was spotted onto a Gene Screen Plus membrane and hybridized to [32P]cDNA probes derived from the stimulated cells mRNAs. Actin and fibronectin mRNA levels showed a significant increase 30 min after stimulation, reaching a maximum at 2 h and remaining high at least till 8 h (Fig. 3A). Induction of tropomyosin and ,&subunit of FNR mRNA levels was slower, reaching a maximum between 2 and 4 h but also remaining high till 8 h (Fig. 3A). This was in clear contrast to that of a well-known, transiently induced gene such as c-fos, whose mRNA reached undetectable levels after 2 h (Fig. 3A). The presence of cycloheximide during the induction period did not significantly affect the levels of the mRNAs of actin, tropomyosin, fibronectin, and P-subunit of FNR during the period of time analyzed (Fig. 3B). The effect of protein synthesis inhibition on the mRNA levels of genes transiently expressed is illustrated by c-fos. This superinduction observed in the presence of cycloheximide is in part due to the stabilization of mRNAs with very short half-lives such as c-fos. The fact that no effect was observed in actin, tropomyosin, P-subunit of FNR, and fibronectin mRNA levels could be an indication that these mRNAs are stable molecules. To determine this, quiescent cells stimulated with 20% FCS in the presence of cycloheximide for 4 h were carefully washed and incubated for different periods of time in 10% FCS containing actinomycin D in the presence or absence of cycloheximide. RNA was extracted at different times and the [32P]cDNA probes 35-898332
542
yseck et al.
i
KS + Act.D o’ 30’ 60' 90' 2h 4h
I
Fig. 4. Stability of mRNAs. [‘*P]cDNA was obtained from poly(A)’ RNA extracted fron quiescent ceils stimulated with 20% FCS in the presence of cycloheximide for 4 h (0’) or from equivalent cultures incubated for the indicated times in FCS plus 2~~~0~~~~~ in the absence (& or in the presence (B) of cyclohe~imide and hybridized as described under &hods to Gene Screen Plus membranes containing cDNA inserts in pUC19. In (A) cells were carefully washed three times with medium containing serum to eliminate the cycloheximide before adding FCS plus actinomycia C.
~erjved from these samples were a~a~y~e~by a dot blot proce NA levels 5f actin, clearly demo~straied that the subunit of FNR, and fibronectin did not decrease . 4A), suggesting that their half-lives were at least determined for c-f&s mRNA (10-15 mm). This was confirmed by the that in the presence of ~yclohexim~de ~~e~t~~alresults were obtai s of the four studied clones (Pig. , the stability of c-fos mRNA was would explain in part the previous observation that e on the level of induction of the four e synthesis of two of the proteins, p n and ~-trop~rny~si~, was mined after stimulation. Qnies~ent cells abeled fsr 1 h with ~~5~~~~~~~~~~~~~ rad~Qact~v~proteins were a~~a~~~e~ at different times after serum addition a by two-dimensional gel electrophoresis. synthesis of actin and tropomyosin rapi ftes s~imnlati5~ in 8 fashion similar to that observed for their mRN ~~a~$~~~~t~~~a~A~t~~Qti~~ by ~~r~~
The effect of serum on the transcription of the ~~~~~~b~~ genes was isolated nuclei from NIH 3T3 cells. Nuclei were is&ted at serum addition to quiescent cells and run-on tra~§~r~~t~on assay and ~ro~om~osin genes formed, As shown in Fig. SA, traus~~i~t~~~ sf the n, Actin remained ex~u~reased dramatizable within 15 min of serum a pressed at high levels for at least 8 h, in contrast to tr~~~myos~~ w sion reached basal levels after 4 h. The fibronectin and ~~s~b~~i~ of F owed a small increase in expression after 15 min rea6: , ~ibro~ect~n expression decrease ver spLowly7remaining high after 8 h. T expression of the receptor gene at nearly a maximum kvd up to 8 IL
Expression of cytoskeletal genes during GOIGl 543 Transcriptional FCS I I Q 15' 30' lh 2h 4h 8h
activation FCS + CHX I Q 15' 30' lh 2h 4h 8h
Fig. 5. Transcriptional activation induced by serum. Nuclei were isolated from quiescent cells (Q) or cells stimulated with 20 % FCS in the absence (A) or in the presence of cycloheximide (B) for the indicated times and run-on transcription assays were performed. The “P-labeled transcripts were hybridized against recombinant pUC19 containing the respective cDNAs immobilized on Gene Screen Plus membrane.
dramatic contrast to these results was the expression of the c-fos gene as shown in Fig. 5A. Similar experiments with nuclei isolated from quiescent cells stimulated in the presence of cycloheximide were performed (Fig. 5B). A slight effect on the transcriptional activation of actin, fibronectin, and FNR genes could be observed. All three presented a small increase in their levels of expression compared to that in the absence of cycloheximide. Tropomyosin gene expression, the most transient of this group, was significantly prolonged, although not as much as that observed for the c-fos gene. These results demonstrated that the increase in actin, tibronectin, ,&subunit of FNR, and tropomyosin mRNAs in serum-stimulated cells results at least in part from the direct stimulation of their genes. DISCUSSION The changes in gene expression induced by growth factors in quiescent cells are generally considered only as part of the mitogenic response. However, it is possible that many of these gene products are necessary to integrate and coordinate complex biological processes in which cell proliferation is a common event. We have demonstrated that the expression of four interacting components of the cytoskeleton and matrix, i.e., actin, P-subunit of FNR, a-tropomyosin, and fibronectin is rapidly induced by serum in quiescent tibroblast cells. The effect of serum stimulation on their mRNA levels lasts for at least 8 h, in contrast to other mRNAs like c-fos that show a very rapid but transient induction. While this work was in preparation, the stimulation of the fibronectin gene by growth factors was reported [3 11. The nuclear run-on experiments showed that transcription of these genes increased rapidly after serum stimulation and that it remained high for several hours, with the exception of tropomyosin. However, due to the long half-life of
544
yseck et al.
NA, it was still present at high levels 8 h after stirn~~at~o~. As transcription activation also occurred in the presence of ~y~~obex~rn~d~we can conclude that it is a direct consequence of the growth factorlrece The results presented here are of interest especially if they are mtegrated in biological processes such as wound healing and embryogenesis in which several l-tans role growth factors act in concert. It is known that fibroblasts play in cutaneous wound repair. They rapidly migrate to the woun and their most important function is the remodelling of the extra~el~~~ar matrix. The fibroblasts that migrate to the wound region rapidly secrete an extensive fibronectin matrix possibly required for the organization of collagen [32--341. In addition, other matrix components are codeposited with ronectin suggesting that its presence is essential in the organization of the gra ation tissue matrix. Fibronectin is a high-molecular-weight protein that is able to interact with several ma~romolecuies using different structural domains of its subunits (reviewed Hn [35]). One of these is a binding domain that is able to interact wit receptor, a glycoprotein involved in the transmembra~e linkage between fibronectin and actin (for review see [28]). ~ibro~e~ti~ receptor, in its ca as a transmembrane linker between the extracellular matrix nd the cytoskefeton, is presumed to function in cell migration in embryos an probably in wound at the /?-subunit hng [36]. Moreover, it has been recently WA is expressed in gastrula and neurula sta embryos WI. A~~~~~~~~~Q~ of fibronectin receptor mRNA at the gastrula stage coincides with the onset of cell ion. Fibronectin levels also increase during this period [37] an en shown to occur in contact with an extracellular matrix ric tin [37-391. The increase in gene expression of actin can be c changes in cytoskeleton and cellular motility occurring rapi Several studies have shown that these initial. cytoskeletal changes are parried by the structural organization of actin filaments into stress fibers 1. Interestingly, we have observed t myosin, a ~orn~o~~~t of the stress fibers [43] and an important modu~at is also indua [44]. The multiplicity of tropomyosin isoforms in ~bro~lasts [45, 461 raises t possibility that specific associations of giv isoforms with actin le to different anges in relative Ieve of different actin structures. It is possible then that forms of tropomyosin in the cell may alter the organization of the actin microhlaments [44] ~ Altogether, these results would suggest that the immediate changes in actin, tropomyosin, tibronectin, and fibronectin ceptor expression after st~~~~~at~o~ of quiescent fibroblasts are correlated with e normal response of cebis in several complex biological processes. I. 2. 3. 4. 5.
Cochran, B. H., Reffel, A. C., and Stiles, C. D. (1983) Ceil 33, 939-947. Lau, L. F., and Nathans, D. (1985) EMBO J. 4, 3145-3151. Lau, L. F., and Nathans, D. (1987) Proc. Mutl. Acad. Sci. USA 84, 1182-11%. Lim, R. W., Varnum, B. C., and Herschman, H. R. (1987) Oncogeze I, 263-270. Aimendral, J. M., Sommer, D., Macdonald-Bravo, H., Burckhardt, J., Perera, J., and Bravo, R. (1988) Mol. Cell. Bid. 8, 214%2148.
Expression
of cytoskeletal
genes during GOIGI
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