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Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells
194
Biochimica et Biophysica Acta, 1048 (1990) 194-201
Elsevier BBAEXP 92037
Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells Th6r+se Ouellet, Carmen Lampron,
Marc Lussier, Line Lapointe and Andr6 Royal
lnstitut du Cancer de Montrdal, Montreal (Canada)
(Received 18 August 1989)
Key words: Keratin; Intermediate filament; Retinoic acid; Dibutyryl cyclic AMP; Differentiation; Embryonal carcinoma
F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). c D N A s corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl c A M P (BtzcAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt2cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.
Introduction
Keratins are a large family of proteins forming intermediate filaments (IF) in all epithal cells [1] and in some other cell types [2-6]. They are subdivided into two classes: type I or acidic keratins, and type II or basic keratins [1]. The property that sets keratins apart from other IF proteins is the requirement for one subunit of each type for filament formation [7,8]. In vitro, any type I subunit can associate with any type II subunit [9], but in vivo, associations are more specific [10]. This implies that there are regulatory mechanisms to insure the coordinated expression of pair members. K8 and K18 are on such pair. In mouse, K8 is a type II keratin of 54193 Da [11] and K18 is a type I keratin of 47400 Da [12]. These two proteins are also called Endo A and Endo B [13], cytokeratins Y and X [14] and cytokeratins A and D [15]. Studies in a wide range of vertebrates have revealed that they are expressed in all simple epithelial cells [16-19], in some complex epithelia [19] and also in non-epithelial tissues [2-6,20-27]. In addition, they are almost always coexpressed [28]. During mouse development, K8 and K18 are the first IF proteins to be expressed. They are found in the outer cells of late morula and, subsequently, in the trophectoderm of blastocysts [14,29]. After implanta-
Correspondence: A. Royal, Institut du Cancer de Montr6al, 1560 rue Sherbrooke est, Montr6al, Quebec, Canada H2L 4M1.
tion, they appear in the visceral and parietal extraembryonic endoderms and in the embryonic ectoderm as these tissues differentiate from the inner cell mass of blastocyts [30]. K8 and K18 remain the major IF proteins expressed in the embryo until day 10 when vimentin expression begins [31]. Murine F9 EC cells can be used to study in vitro some of the events that occur during early development. RA treatment of F9 cells induces their differentiation into cells which resemble primitive endoderm [32,33]. In presence of RA and Bt2cAMP, F9 cells differentiate into parietal endoderm-like cells [34]. This differentiation is accompanied by many changes in gene expression including the induction of K8 and K18 [13,35]. Thus, F9 cells are a good model to study how the expression of a keratin pair is coordinately regulated during differentiation. Studies of keratin proteins and mRNAs in various tissues have led to the suggestion that regulation of their expression occurred chiefly at the transcriptional level [36,37]. However, other studies have stressed that post-transcriptional mechanisms could be involved as well [38 40]. In the work described here, we sought to determine to what extent K8 and K18 genes were coordinately regulated during epithelial differentiation. Using a very sensitive RNase protection assay [41] and specific c D N A probes, we have examined the changes in the abundance of K8 and K18 mRNAs that occur in differentiating F9 cells when treated with RA or with RA and BtzcAMP. We find that the two mRNAs are differently regulated.
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
195 Materials and Methods
Cell culture and differentiation The parietal endoderm-like P F H R 9 cell line [42] was cultivated in Dulbecco's modified Eagle's medium containing 1 mM sodium pyruvate and 10% heat-inactivated fetal bovine serum ( G I B C O / B R L , Burlington, Ontario), and was harvested with t r y p s i n / E D T A ( G I B C O / B R L ) . The EC F9-21 cell line and the parietal endoderm-like F9ACcl9 cell line [43] were cultivated as described previously [44]. The EC F9-21 cell line, a subclone of the F9 line [45], was isolated as a colony growing in soft agar. The capacity of the F9-21 clone to differentiate was ascertained by the disappearance of the EC cell-specific antigen SSEA-1 [46] and by the loss of their ability to form colonies in soft agar [47]. For the differentiation experiments, 2.5.106 F9-21 cells were plated in 150 mm petri dish and induced to differentiate by supplementing the medium with 1 0 - 6 M RA (all trans, Sigma Chemical Co, St. Louis, MO) and, where appropriate, with 10 3 M Bt2cAMP (Sigma). The cells were passaged on second and fourth day to prevent them from reaching confluence. RNA isolation Total RNA was isolated from subconfluent cultures as previously described [48]. For isolation of F9ACcl9 poly(A) ÷ RNA, polysomes were obtained by streptomycin sulfate precipitation [49] as described by Skup et al. [50]. After digestion with proteinase K, poly(A) ÷ RNA was purified by two passages through oligo(dT)cellulose [51]. Northern blots RNA was electrophoresed on 1% agarose gels in formaldehyde, according to Maniatis et al. [52], except that the running buffer was 10 mM phosphate buffer (pH 7.0). After electrophoresis, the RNA was transferred to nylon membranes without N a O H treatment. Hybridization conditions were described previously [53]. cDNA cloning Double-stranded c D N A was synthesized using plasmids pSV7186 and pSV1932 (Pharmacia LKB Biotechnology AB) as described by Okayama and Berg [54], with the following modifications. Starting with 10 lag of F9ACcl9 polysomal poly(A) ÷ RNA, the firststrand cDNA synthesis was performed at 4 2 ° C for 80 rnin. The reaction was monitored by incorporation of [a_ 32P]dCTP (3000 Ci/mmol, ICN Biomedicals Canada, Montrral, Qurbec) in a small scale synthesis conducted simultaneously. After tailing with dCTP and extraction, the cDNA was purified by adsorption and elution from a NENSORB-20 cartridge (NEN Research products, DuPont Co., Wilmington, DE). Transformations were carried out as described by Hanahan [55].
Screening and identification of cDNA clones A library containing ( 2 - 3 ) . 106 recombinants was obtained. The colonies were washed from the plates and stored at - 8 0 ° C. Then, (1-2) • 1 0 6 colonies were plated on two 22 cm 2 dishes, transferred to nylon membranes (1.2 /~m, Pall Canada, Mont Royal, Qurbec), and amplified. Hybridization was performed overnight at 5 0 ° C in 5 x SSC (1 x SSC is 0.15 M NaC1 and 0.015 M sodium citrate), 0.1 M sodium phosphate buffer (pH 7.0), 5 x Denhardt's solution, 0.1% SDS, 200 ~ g / m l sonicated herring sperm D N A and 100/~g/ml poly(A). Probes were gel-purified synthetic oligodeoxynucleotides designed from the conserved amino-acid sequences of mammalian keratins [37,56-67]. Four probes were used: A G ( G , A , C ) C G G G C A T T(A,G)TC(A,G)AT corresponding to the sequence IDNAR, G C G G T A G G T G G C(A,G)ATCTC and CTCCTC(A,T,G)CC(C,T) T C C A G ( A , C ) A G corresponding to the sequence E I A T Y R L L E G E , and C G C A C C T T ( A , G ) T C G A T G A A G G A corresponding to the sequence SFIDKV. Oligos were radiolabeled at their 5' end using [y-32p]ATP (5000 Ci/mmol, Amersham Canada, Oakville, Ontario) and T4 polynucleotide kinase. The filters were washed with 5 x SSC, 0.1 M sodium phosphate buffer (pH 7.0) and 0.1% SDS, four times at room temperature and twice at 50 ° C (30 min each). RNase protection assays In vitro transcriptions either with T7 or SP6 RNA polymerase were performed, with minor modifications, as described by Melton et al. [41]. Restriction fragments of cDNA inserts were subcloned into pGEM-1 (Promega Biotec, Madison, WI). Linear D N A templates (20 ~ g / m l ) were transcribed with 400 units/ml of polymerase for 1 h at 37°C, in the presence of 12/xM CTP and 1.6 m C i / m l [ct-aEp]CTP (800 C i / m m o l , Amersham Canada). Following RNA synthesis, the D N A template was removed by the addition of 10 mM Vanadyl Ribonucleoside Complex (BRL, Gaithersburg, MD) and 40 / l g / m l of RNase-free DNase (BRL). RNA was purified from unincorporated NTP by Sephadex G-50 chromatography. RNase protection assays were carried out as described [41], using 5-20 /~g of total cellular RNA, 1.105 cpm of in vitro transcribed RNA probe, 50 cpm of DNA marker and enough baker's yeast tRNA to give 25 /~g total RNA per assay. The D N A marker used was pAT153 digested with PstI and BamHI to give 1125 and 2532 bp fragments which were radiolabeled at their 5' end using [~,-32p]ATP and T4 polynucleotide kinase. The marker was used to evaluate losses in successive manipulations. Overnight ( > 15 h) hybridization was at 4 5 ° C and RNases digestion at 3 0 ° C for 1 h in the presence of 200 # g / m l of RNase A and 870 units/ml of RNase T1. After proteinase K digestion, extraction and precipitation, the protected RNA was dissolved in 90% foramide, 1 x TBE (0.089
196 M Tris, 0.089 M boric acid, 0.004 M EDTA (pH 8)) and dyes. Electrophoresis was carried out on a polyacrylamide/7 M urea denaturing gel (5% polyacrylamide for the K8 probe and 6% for the K18 probe). Densitometric analysis To quantitate the relative amounts of RNA and DNA, the bands on the autoradiograms were scanned with an Ultroscan XL laser densitometer (LKB, Bromma, Sweden) and the resulting data were analyzed with the LKB 2400 Gelscan XL software package. In a control experiment, known amounts of radioactive probe were fractionated by electrophoresis as described above, and autoradiographed. After scanning, the data obtained was used to determine the non-linear relation between radioactivity and image absorbance. Results
K8 and K18 m R N A are expressed at different levels Probes for K8 and K18 mRNAs were obtained from a cDNA library constructed using RNA from the F9derived cell line F9ACcl9 [43]. The library was screened with oligodeoxynucleotide probes specific for regions conserved in type-I and type-II keratins. Clones corresponding to K8 have been described before [11]. Clones homologous to K18 were obtained from the same library. The largest of these clones, pKB204, hybridized with a 1.6 kb RNA detectable in differentiating F9-21 cells and in differentiated cell lines (Fig. 1B). The size of the RNA it detected and the restriction map of the insert (Fig. 2C) when compared with published sequences [12,68], confirmed its identity. Differentiation of F9 cells by treatment with RA or with RA and Bt 2cAMP induced K8 (Fig. 1A, lanes a, b and c) and K18 (Fig. 1B, lanes a, b and c) mRNA expression in F9-21 cells. However, the signals obtained with the K8 probe were always more intense although the probes were approximately of the same size (Fig. 2) and of the same specific activity. This difference was observed not only in differentiating F9 cells but also in F9ACcl9 (Fig. 1, lanes d) and in PFHR9 cells (Fig. 1, lanes e). K8 m R N A is induced before K18 m R N A To further examine the change in abundance of K8 and K18 mRNAs in differentiating cells, RNAse protection assays were performed. For this purpose, two probes were designed from the cDNAs and subcloned into pGEM-I: a 540 bp PstI fragment from the K8 cDNA (Fig. 2B) and a 226 bp HindlII-SacI fragment spanning the 3' end of the K18 cDNA (Fig. 2D). By RNase protection assay on total RNA extracted from cells treated with 10 - 6 M RA for up to 8 days, expression of K8 m R N A was detected at all times including in undifferentiated cells (Fig. 3A). Examination of the
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18S Fig. 1. R N A blot analysis of the transcripts complementary to pKA56 and pKB204. Keratin c D N A clones pKA56 (A) and pKB204 (B) were used as probes in hybridization with 20 /tg of total RNA. (Lower panel) Quantification of the RNAs. To evaluate the relative quantity of total R N A in each sample, the probes were eluated and the filters were rehybridized with pMr974, a genomic clone containing a fragment of mouse r D N A homologous to the 18S r R N A [87]. (a) F9-21 cells; (b) F9 cells treated for 7 days with RA; (c) F9 cells treated for 6 days with RA and BtzcAMP; (d) F9ACC19 and; (e) P F H R 9 cell lines. The size of PstI and TaqI digests of pUC8 are indicated to the left, in kb. Exposure of (A) and (B) was 24 h.
autoradiogram suggest that K8 m R N A started to accumulate during the third day. To confirm this observation, a quantitative evaluation was obtained by densitometric scanning. For each time point, the relative amount of K8 m R N A was calculated by taking into account the quantity of cellular RNA used in the assay, the size and the specific activity of the probe, the non-linear relationship of radioactivity to image absorbance of the film as determined in a control experiment (not shown) and the loss of material during
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Fig. 2. Restriction maps of K8 and K18 c D N A . (A) pKA56, a K8 c D N A clone. (B) the 540 bp PstI fragment of pKA56 inserted into pGEM-A36, pGEM-A36 was linearized at an EcoRl site located in the vector to synthesize the 560 bases R N A probe used in R N a s e protection assay. (C) pKB204, a K18 c D N A clone. (D) the 226 bp HindlII-SacI fragment of pKB203 inserted into pGEM-B1, pGEM-B1 was linearized at the F n u 4 H l site of the insert for synthesis of the 216 bases probe used in RNase protection assay. The PstI sites at the 5' end of the c D N A in (A) and (C) were introduced by the cloning procedure. The asterisk indicates a PvulI site present only in 50% of the K18 c D N A clones isolated; An, poly (A) tail; Ps, Pstl; B, BamHl; Pv, Pvull; H, HindllI; S, SacI; F, Fnu4HI.
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Fig. 3. Expression of K8 mRNA in differentiating F9 cells. RNase protection assay of K8 m R N A in cells treated (A) with RA or (B) with RA and Bt2cAMP. The size of the protected fragment is 540 bases. The 5' end-labeled 1125 bp pAT153 fragment used to monitor material losses (see Materials and Methods), is also shown. Exposure time: 72 h. (C) Northern blot analysis of the RNA used in the RNase protection assays. Upper row: RNA from RA treated cells; lower row: RNA from RA and Bt2cAMP treated cells. The blots were probed with pMr974 [87] to evaluate the relative quantity of total RNA in each sample. Exposure time for each row was 5 h. p, 560 bases K8 probe; c, control assay using the K8 probe and tRNA only; 0, F9-21 cells RNA, 20/tg; 1 to 8, RNA from F9 cells treated for 1 to 8 days, 20/~g (A) or 10/~g (B); f, F9ACcl9 cells RNA, 5 ~g (A) or 10/tg (B).
manipulations (see Materials and Methods). Examination of the results (Fig. 5A) indicates that K8 mRNA was significantly augmented only on day 4. Expression of K18 mRNA was also examined (Fig. 4A). As with K8 mRNA, the first days of treatment with RA did not result in any convincing increase in K18 mRNA abundance. Only on day 5 was K18 mRNA clearly more abundant than in undifferentiated cells, suggesting that it was induced later than K8 mRNA. This observation was confirmed by densitometric analysis (Fig. 5B). In cells treated with 10 -3 M Bt2cAMP keratin genes were not induced (results not shown). However, when cells were treated with RA and Bt2cAMP , K8 mRNA induction occurred during the first 24 h of treatment (Fig. 3B and Fig. 5A). K18 m R N A was also increased at that time. However, because of the low level of expression of K18 mRNA, this is difficult to judge from
the autoradiogram (Fig. 4). It is more clearly revealed by densitometric analysis (Fig. 5B). Bt2cAMP also affected the level of expression of the two mRNAs. After 8 days of treatment, K8 m R N A was 7.5-times more abundant in cells treated with RA and Bt2cAMP than in cells treated only with RA. K18 m R N A was 2-times more abundant in cells treated with the two inducers. Part of this difference was due to an increase in the number of differentiated cells. In a control experiment, the number of differentiated cells was evaluated by immunofluorescence, usmg the K8-specific monoclonal antibody Troma 1 [69]. After 6 days of treatment, it was found that there was 1.5-times more differentiated cells in the cultures treated with RA and Bt2cAMP (results not shown). K8 m R N A is more abundant than K 1 8 m R N A
Northern hybridization (Fig. 1) suggested that K8 m R N A was more abundant than K18 mRNA. The
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Fig. 4. Expression of K18 mRNA in differentiating F9 cells. RNase protection assay of K18 mRNA in cells treated (A) with RA or (B) with RA and Bt2cAMP. c, control assay using probe and tRNA only; 0, F9-21 cells RNA, 20 #g; 1 to 8, RNA from F9 cells treated for 1 to 8 days, 20 ttg; f, F9ACcl9 cells RNA, 5/tg. The size of the protected fragment is 200 bases. The band at 215 bases visible in all lanes is artefactual and results from interactions of the probe with itself as shown by its presence in the control lane (c). The 5' end-labeled 1125 and 2532 bases pAT153 fragments used to monitor material losses (see Materials and Methods), are also shown. Exposure time: 72 h.
results p r e s e n t e d in Fig. 5 c o n f i r m that i n t e r p r e t a t i o n . A t all times there was m o r e K8 m R N A . T a k i n g the average of the values o b t a i n e d f r o m cultures treated 7 a n d 8 d a y s with R A a n d from cultures t r e a t e d 6, 7 a n d 8 d a y s with R A plus B t 2 c A M P as the m a x i m u m o b t a i n e d in each treatment, the ratio of K8 to K18 was c a l c u l a t e d to be a b o u t 3.5 in R A - t r e a t e d cells a n d a b o u t 9 in cells treated with R A a n d B t 2 c A M P . These values are consistent with the results p r e s e n t e d in Fig. 1 for the d i f f e r e n t i a t e d cell line P F H R 9 a n d F 9 A C c l 9 , where K8 m R N A was a b o u t 4-times m o r e a b u n d a n t t h a n K18 m R N A . These two cell lines ressemble e x t r a e m b r y o n i c p a r i e t a l e n d o d e r m a n d are therefore similar to c o m pletely d i f f e r e n t i a t e d F9 cells. This is actually the case for F 9 A C c l 9 as this cell line was cloned from a R A t r e a t e d F9 cell c u l t u r e [43].
Discussion T h e results p r e s e n t e d here i n d i c a t e that K8 a n d K18 m R N A s are i n d e p e n d e n t l y regulated. First, the two m R N A s were expressed at very different levels. In d i f f e r e n t i a t i n g cells, K8 m R N A was f o u n d to b e m o r e a b u n d a n t than K18 m R N A . This was also o b s e r v e d in
d i f f e r e n t i a t e d cell lines a n d in u n d i f f e r e n t i a t e d cells. In u n d i f f e r e n t i a t e d cells, the a m o u n t of b o t h m R N A s was very low. This is similar to o t h e r m R N A s , like H-2 for instance, which, b y n o r t h e r n analysis, are usually n o t d e t e c t a b l e in F9 EC cells, b u t c a n be d e m o n s t r a t e d using high specific activity p r o b e s a n d long e x p o s u r e s [70], or b y nuclease S1 resistance a s s a y [71]. In the w o r k d e s c r i b e d here, the b a s a l level of K8 m R N A e v a l u a t e d b y d e n s i t o m e t r y was 4 - 5 - t i m e s higher t h a n the b a s a l level of K18 m R N A . This is close to the ratio f o u n d in d i f f e r e n t i a t e d cells a n d suggest that it is due to the low level of s p o n t a n e o u s l y d i f f e r e n t i a t e d cells k n o w n to be present in F 9 cell cultures [72,73]. T h e higher level of e x p r e s s i o n of K 8 m R N A relative to K18 m R N A , 3.5 in R A - t r e a t e d cells a n d 9 in R A plus B t 2 c A M P - t r e a t e d cells, has to b e e x p l a i n e d since the ratio of t y p e I a n d t y p e II k e r a t i n s in I F is a p p r o x . 1 [74]. T h e K8 p r o t e i n m a y be m o r e a b u n d a n t t h a n K18 in d i f f e r e n t i a t i n g F 9 cells. A n excess of cellular K8 could reside in a n o n f i l a m e n t o u s pool. N o n f i l a m e n t o u s forms of K8 have b e e n h y p o t h e s i z e d to e x p l a i n the diffuse c y t o p l a s m i c staining seen b y i m m u n o f l u o r escence m i c r o s c o p y in m o u s e oocytes a n d cleavage-stage e m b r y o s [75,76]. T h e excess of K 8 c o u l d also form
199
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Fig. 5. Relative a m o u n t of K8 and K18 m R N A s in differentiating F9 cells. Autoradiograms, including those used in Fig. 3, and 4, were scanned with a laser densitometer. For all the time points, appropriately exposed autoradiograms were scanned. The 1125 pAT153 fragment was also evaluated by scanning and used to make corrections for material losses during manipulations. Relative R N A concentration of each sample was also evaluated by scanning the blot shown in Fig. 3C and used in the final calculations. The relative a m o u n t of m R N A was calculated with respect to the quantity of K8 m R N A in F9-21 cells. (A) K8 m R N A ; (B) K18 m R N A . Solid bars: RA treatment; open bars: RA plus Bt2cAMP treatment.
filaments with another type-I keratin, such as K19 which is also induced in differentiating F9 cells [77]. However, there is some evidence that strict regulation of keratin m R N A levels may not be essential. Transfection experiments have indicated that excess keratins are not stable in the absence of a partner subunit with which it can form filaments [39,40,78]. The second indication of independent regulation of K8 and K18 m R N A s expression during differentiation comes from the observation that the two m R N A s are not induced simultaneously in RA-treated cells. K8 m R N A accumulation started 1 day earlier. In cells treated with RA and Bt2cAMP, K8 and K18 m R N A s were already induced after 1 day of treatment. But this does not exclude that accumulation of one of the two m R N A s could have begun sooner than the other during
the first 24 h. Uncoupling of the expression of the members of a keratin pair by the earlier expression of the type-II subunit has been observed before in differentiating epithelial cells in culture. It was detected at the level of protein expression in maturing corneal epithelial cells [79] and in differentiating epidermal cells [80,81]. Also, in fibroblasts derived from an embryonal carcinoma, it was shown that after a treatment with 5-azacytidine some cells were expressing K8 alone, and that K18 was present only in K8 positive cells [82]. The third indication that K8 and K18 m R N A s are regulated independently comes from the effect of Bt 2cAMP. In the presence of Bt 2cAMP, the two m R N A s were not only induced earlier, they were also expressed at higher levels. However, this effect was more important for K8 m R N A . In cells treated 7 or 8 days with RA and Bt2cAMP, K8 m R N A was 4-8-times more abundant while K18 m R N A was only 2-times more abundant in cells treated With RA alone. Moreover, the number of differentiated cells was about 1.5-times higher in cultures treated with RA and BtEcAMP accounting for almost all the increase in K18 m R N A . Thus, the evidence presented here suggest that only K8 m R N A expression was affected by Bt2cAMP. The higher level of accumulation of K8 m R N A in presence of Bt2cAMP is in agreement with the results obtained with other markers of F9 differentiation, like plasminogen activator [83], laminins A and B [84,85] and collagen IV [85,86], which show a further increase in m R N A and protein levels when Bt2cAMP is added to RA-treated F9EC cells. Also, in the presence of BtzcAMP, collagen IV m R N A is induced sooner [86], like K8 and K18 mRNAs. The difference between the final levels of expression of K8 m R N A in the two treatments is consistent with the hypothesis that RA-treated cells are not in the same differentiation state as cells treated with RA and Bt2cAMP [34]. Since K8 and K18 form filaments together, it is paradoxical to find that expression of their m R N A s is independently regulated. However, because excess keratins are not stable in the absence of a partner subunit [39,40,78] filament assembly can in theory be regulated by controlling the expression of only one keratin m R N A . A question not addressed in this study is the level at which m R N A abundance is controlled: our results are consistent with a transcriptional as well as with a posttranscriptional mechanism including differential processing, transport or stability of the two mRNAs. Finally, in these cells as in m a n y other cells expressing K8 and K18, other keratins are also expressed. In differentiating F9 cells, the K19 gene is induced [77]. Possibly, different ratios of k18 to K19 result in filaments with slightly different properties. This would make sense, if, as noted above, cells treated with RA and cells treated with RA and Bt2cAMP were in a different differentiation state.
200
Acknowledgements We would like to thank Jean-Francois Boulais for his technical assistance, Roger Duclos for photography and Dr. V. Bibor-Hardy for the scanning system. This work was supported by grants from the Medical Research Council of Canada (MRC), and by studentships from the MRC (T.O. and C.L.), the Cancer Research Society Inc. (M.L.) and the Universit6 de Montr6al (T.O.).
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Biochimica et Biophysica Acta, 1048 (1990) 194-201
Elsevier BBAEXP 92037
Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells Th6r+se Ouellet, Carmen Lampron,
Marc Lussier, Line Lapointe and Andr6 Royal
lnstitut du Cancer de Montrdal, Montreal (Canada)
(Received 18 August 1989)
Key words: Keratin; Intermediate filament; Retinoic acid; Dibutyryl cyclic AMP; Differentiation; Embryonal carcinoma
F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). c D N A s corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl c A M P (BtzcAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt2cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.
Introduction
Keratins are a large family of proteins forming intermediate filaments (IF) in all epithal cells [1] and in some other cell types [2-6]. They are subdivided into two classes: type I or acidic keratins, and type II or basic keratins [1]. The property that sets keratins apart from other IF proteins is the requirement for one subunit of each type for filament formation [7,8]. In vitro, any type I subunit can associate with any type II subunit [9], but in vivo, associations are more specific [10]. This implies that there are regulatory mechanisms to insure the coordinated expression of pair members. K8 and K18 are on such pair. In mouse, K8 is a type II keratin of 54193 Da [11] and K18 is a type I keratin of 47400 Da [12]. These two proteins are also called Endo A and Endo B [13], cytokeratins Y and X [14] and cytokeratins A and D [15]. Studies in a wide range of vertebrates have revealed that they are expressed in all simple epithelial cells [16-19], in some complex epithelia [19] and also in non-epithelial tissues [2-6,20-27]. In addition, they are almost always coexpressed [28]. During mouse development, K8 and K18 are the first IF proteins to be expressed. They are found in the outer cells of late morula and, subsequently, in the trophectoderm of blastocysts [14,29]. After implanta-
Correspondence: A. Royal, Institut du Cancer de Montr6al, 1560 rue Sherbrooke est, Montr6al, Quebec, Canada H2L 4M1.
tion, they appear in the visceral and parietal extraembryonic endoderms and in the embryonic ectoderm as these tissues differentiate from the inner cell mass of blastocyts [30]. K8 and K18 remain the major IF proteins expressed in the embryo until day 10 when vimentin expression begins [31]. Murine F9 EC cells can be used to study in vitro some of the events that occur during early development. RA treatment of F9 cells induces their differentiation into cells which resemble primitive endoderm [32,33]. In presence of RA and Bt2cAMP, F9 cells differentiate into parietal endoderm-like cells [34]. This differentiation is accompanied by many changes in gene expression including the induction of K8 and K18 [13,35]. Thus, F9 cells are a good model to study how the expression of a keratin pair is coordinately regulated during differentiation. Studies of keratin proteins and mRNAs in various tissues have led to the suggestion that regulation of their expression occurred chiefly at the transcriptional level [36,37]. However, other studies have stressed that post-transcriptional mechanisms could be involved as well [38 40]. In the work described here, we sought to determine to what extent K8 and K18 genes were coordinately regulated during epithelial differentiation. Using a very sensitive RNase protection assay [41] and specific c D N A probes, we have examined the changes in the abundance of K8 and K18 mRNAs that occur in differentiating F9 cells when treated with RA or with RA and BtzcAMP. We find that the two mRNAs are differently regulated.
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
195 Materials and Methods
Cell culture and differentiation The parietal endoderm-like P F H R 9 cell line [42] was cultivated in Dulbecco's modified Eagle's medium containing 1 mM sodium pyruvate and 10% heat-inactivated fetal bovine serum ( G I B C O / B R L , Burlington, Ontario), and was harvested with t r y p s i n / E D T A ( G I B C O / B R L ) . The EC F9-21 cell line and the parietal endoderm-like F9ACcl9 cell line [43] were cultivated as described previously [44]. The EC F9-21 cell line, a subclone of the F9 line [45], was isolated as a colony growing in soft agar. The capacity of the F9-21 clone to differentiate was ascertained by the disappearance of the EC cell-specific antigen SSEA-1 [46] and by the loss of their ability to form colonies in soft agar [47]. For the differentiation experiments, 2.5.106 F9-21 cells were plated in 150 mm petri dish and induced to differentiate by supplementing the medium with 1 0 - 6 M RA (all trans, Sigma Chemical Co, St. Louis, MO) and, where appropriate, with 10 3 M Bt2cAMP (Sigma). The cells were passaged on second and fourth day to prevent them from reaching confluence. RNA isolation Total RNA was isolated from subconfluent cultures as previously described [48]. For isolation of F9ACcl9 poly(A) ÷ RNA, polysomes were obtained by streptomycin sulfate precipitation [49] as described by Skup et al. [50]. After digestion with proteinase K, poly(A) ÷ RNA was purified by two passages through oligo(dT)cellulose [51]. Northern blots RNA was electrophoresed on 1% agarose gels in formaldehyde, according to Maniatis et al. [52], except that the running buffer was 10 mM phosphate buffer (pH 7.0). After electrophoresis, the RNA was transferred to nylon membranes without N a O H treatment. Hybridization conditions were described previously [53]. cDNA cloning Double-stranded c D N A was synthesized using plasmids pSV7186 and pSV1932 (Pharmacia LKB Biotechnology AB) as described by Okayama and Berg [54], with the following modifications. Starting with 10 lag of F9ACcl9 polysomal poly(A) ÷ RNA, the firststrand cDNA synthesis was performed at 4 2 ° C for 80 rnin. The reaction was monitored by incorporation of [a_ 32P]dCTP (3000 Ci/mmol, ICN Biomedicals Canada, Montrral, Qurbec) in a small scale synthesis conducted simultaneously. After tailing with dCTP and extraction, the cDNA was purified by adsorption and elution from a NENSORB-20 cartridge (NEN Research products, DuPont Co., Wilmington, DE). Transformations were carried out as described by Hanahan [55].
Screening and identification of cDNA clones A library containing ( 2 - 3 ) . 106 recombinants was obtained. The colonies were washed from the plates and stored at - 8 0 ° C. Then, (1-2) • 1 0 6 colonies were plated on two 22 cm 2 dishes, transferred to nylon membranes (1.2 /~m, Pall Canada, Mont Royal, Qurbec), and amplified. Hybridization was performed overnight at 5 0 ° C in 5 x SSC (1 x SSC is 0.15 M NaC1 and 0.015 M sodium citrate), 0.1 M sodium phosphate buffer (pH 7.0), 5 x Denhardt's solution, 0.1% SDS, 200 ~ g / m l sonicated herring sperm D N A and 100/~g/ml poly(A). Probes were gel-purified synthetic oligodeoxynucleotides designed from the conserved amino-acid sequences of mammalian keratins [37,56-67]. Four probes were used: A G ( G , A , C ) C G G G C A T T(A,G)TC(A,G)AT corresponding to the sequence IDNAR, G C G G T A G G T G G C(A,G)ATCTC and CTCCTC(A,T,G)CC(C,T) T C C A G ( A , C ) A G corresponding to the sequence E I A T Y R L L E G E , and C G C A C C T T ( A , G ) T C G A T G A A G G A corresponding to the sequence SFIDKV. Oligos were radiolabeled at their 5' end using [y-32p]ATP (5000 Ci/mmol, Amersham Canada, Oakville, Ontario) and T4 polynucleotide kinase. The filters were washed with 5 x SSC, 0.1 M sodium phosphate buffer (pH 7.0) and 0.1% SDS, four times at room temperature and twice at 50 ° C (30 min each). RNase protection assays In vitro transcriptions either with T7 or SP6 RNA polymerase were performed, with minor modifications, as described by Melton et al. [41]. Restriction fragments of cDNA inserts were subcloned into pGEM-1 (Promega Biotec, Madison, WI). Linear D N A templates (20 ~ g / m l ) were transcribed with 400 units/ml of polymerase for 1 h at 37°C, in the presence of 12/xM CTP and 1.6 m C i / m l [ct-aEp]CTP (800 C i / m m o l , Amersham Canada). Following RNA synthesis, the D N A template was removed by the addition of 10 mM Vanadyl Ribonucleoside Complex (BRL, Gaithersburg, MD) and 40 / l g / m l of RNase-free DNase (BRL). RNA was purified from unincorporated NTP by Sephadex G-50 chromatography. RNase protection assays were carried out as described [41], using 5-20 /~g of total cellular RNA, 1.105 cpm of in vitro transcribed RNA probe, 50 cpm of DNA marker and enough baker's yeast tRNA to give 25 /~g total RNA per assay. The D N A marker used was pAT153 digested with PstI and BamHI to give 1125 and 2532 bp fragments which were radiolabeled at their 5' end using [~,-32p]ATP and T4 polynucleotide kinase. The marker was used to evaluate losses in successive manipulations. Overnight ( > 15 h) hybridization was at 4 5 ° C and RNases digestion at 3 0 ° C for 1 h in the presence of 200 # g / m l of RNase A and 870 units/ml of RNase T1. After proteinase K digestion, extraction and precipitation, the protected RNA was dissolved in 90% foramide, 1 x TBE (0.089
196 M Tris, 0.089 M boric acid, 0.004 M EDTA (pH 8)) and dyes. Electrophoresis was carried out on a polyacrylamide/7 M urea denaturing gel (5% polyacrylamide for the K8 probe and 6% for the K18 probe). Densitometric analysis To quantitate the relative amounts of RNA and DNA, the bands on the autoradiograms were scanned with an Ultroscan XL laser densitometer (LKB, Bromma, Sweden) and the resulting data were analyzed with the LKB 2400 Gelscan XL software package. In a control experiment, known amounts of radioactive probe were fractionated by electrophoresis as described above, and autoradiographed. After scanning, the data obtained was used to determine the non-linear relation between radioactivity and image absorbance. Results
K8 and K18 m R N A are expressed at different levels Probes for K8 and K18 mRNAs were obtained from a cDNA library constructed using RNA from the F9derived cell line F9ACcl9 [43]. The library was screened with oligodeoxynucleotide probes specific for regions conserved in type-I and type-II keratins. Clones corresponding to K8 have been described before [11]. Clones homologous to K18 were obtained from the same library. The largest of these clones, pKB204, hybridized with a 1.6 kb RNA detectable in differentiating F9-21 cells and in differentiated cell lines (Fig. 1B). The size of the RNA it detected and the restriction map of the insert (Fig. 2C) when compared with published sequences [12,68], confirmed its identity. Differentiation of F9 cells by treatment with RA or with RA and Bt 2cAMP induced K8 (Fig. 1A, lanes a, b and c) and K18 (Fig. 1B, lanes a, b and c) mRNA expression in F9-21 cells. However, the signals obtained with the K8 probe were always more intense although the probes were approximately of the same size (Fig. 2) and of the same specific activity. This difference was observed not only in differentiating F9 cells but also in F9ACcl9 (Fig. 1, lanes d) and in PFHR9 cells (Fig. 1, lanes e). K8 m R N A is induced before K18 m R N A To further examine the change in abundance of K8 and K18 mRNAs in differentiating cells, RNAse protection assays were performed. For this purpose, two probes were designed from the cDNAs and subcloned into pGEM-I: a 540 bp PstI fragment from the K8 cDNA (Fig. 2B) and a 226 bp HindlII-SacI fragment spanning the 3' end of the K18 cDNA (Fig. 2D). By RNase protection assay on total RNA extracted from cells treated with 10 - 6 M RA for up to 8 days, expression of K8 m R N A was detected at all times including in undifferentiated cells (Fig. 3A). Examination of the
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autoradiogram suggest that K8 m R N A started to accumulate during the third day. To confirm this observation, a quantitative evaluation was obtained by densitometric scanning. For each time point, the relative amount of K8 m R N A was calculated by taking into account the quantity of cellular RNA used in the assay, the size and the specific activity of the probe, the non-linear relationship of radioactivity to image absorbance of the film as determined in a control experiment (not shown) and the loss of material during
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Fig. 2. Restriction maps of K8 and K18 c D N A . (A) pKA56, a K8 c D N A clone. (B) the 540 bp PstI fragment of pKA56 inserted into pGEM-A36, pGEM-A36 was linearized at an EcoRl site located in the vector to synthesize the 560 bases R N A probe used in R N a s e protection assay. (C) pKB204, a K18 c D N A clone. (D) the 226 bp HindlII-SacI fragment of pKB203 inserted into pGEM-B1, pGEM-B1 was linearized at the F n u 4 H l site of the insert for synthesis of the 216 bases probe used in RNase protection assay. The PstI sites at the 5' end of the c D N A in (A) and (C) were introduced by the cloning procedure. The asterisk indicates a PvulI site present only in 50% of the K18 c D N A clones isolated; An, poly (A) tail; Ps, Pstl; B, BamHl; Pv, Pvull; H, HindllI; S, SacI; F, Fnu4HI.
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Fig. 3. Expression of K8 mRNA in differentiating F9 cells. RNase protection assay of K8 m R N A in cells treated (A) with RA or (B) with RA and Bt2cAMP. The size of the protected fragment is 540 bases. The 5' end-labeled 1125 bp pAT153 fragment used to monitor material losses (see Materials and Methods), is also shown. Exposure time: 72 h. (C) Northern blot analysis of the RNA used in the RNase protection assays. Upper row: RNA from RA treated cells; lower row: RNA from RA and Bt2cAMP treated cells. The blots were probed with pMr974 [87] to evaluate the relative quantity of total RNA in each sample. Exposure time for each row was 5 h. p, 560 bases K8 probe; c, control assay using the K8 probe and tRNA only; 0, F9-21 cells RNA, 20/tg; 1 to 8, RNA from F9 cells treated for 1 to 8 days, 20/~g (A) or 10/~g (B); f, F9ACcl9 cells RNA, 5 ~g (A) or 10/tg (B).
manipulations (see Materials and Methods). Examination of the results (Fig. 5A) indicates that K8 mRNA was significantly augmented only on day 4. Expression of K18 mRNA was also examined (Fig. 4A). As with K8 mRNA, the first days of treatment with RA did not result in any convincing increase in K18 mRNA abundance. Only on day 5 was K18 mRNA clearly more abundant than in undifferentiated cells, suggesting that it was induced later than K8 mRNA. This observation was confirmed by densitometric analysis (Fig. 5B). In cells treated with 10 -3 M Bt2cAMP keratin genes were not induced (results not shown). However, when cells were treated with RA and Bt2cAMP , K8 mRNA induction occurred during the first 24 h of treatment (Fig. 3B and Fig. 5A). K18 m R N A was also increased at that time. However, because of the low level of expression of K18 mRNA, this is difficult to judge from
the autoradiogram (Fig. 4). It is more clearly revealed by densitometric analysis (Fig. 5B). Bt2cAMP also affected the level of expression of the two mRNAs. After 8 days of treatment, K8 m R N A was 7.5-times more abundant in cells treated with RA and Bt2cAMP than in cells treated only with RA. K18 m R N A was 2-times more abundant in cells treated with the two inducers. Part of this difference was due to an increase in the number of differentiated cells. In a control experiment, the number of differentiated cells was evaluated by immunofluorescence, usmg the K8-specific monoclonal antibody Troma 1 [69]. After 6 days of treatment, it was found that there was 1.5-times more differentiated cells in the cultures treated with RA and Bt2cAMP (results not shown). K8 m R N A is more abundant than K 1 8 m R N A
Northern hybridization (Fig. 1) suggested that K8 m R N A was more abundant than K18 mRNA. The
198
c
0
1
1.5 2
25
3
4
5
6
7
8
I
A
-2532 ~,~
- 1125
-220 -200
El -2532 -1125
-220 -200
Fig. 4. Expression of K18 mRNA in differentiating F9 cells. RNase protection assay of K18 mRNA in cells treated (A) with RA or (B) with RA and Bt2cAMP. c, control assay using probe and tRNA only; 0, F9-21 cells RNA, 20 #g; 1 to 8, RNA from F9 cells treated for 1 to 8 days, 20 ttg; f, F9ACcl9 cells RNA, 5/tg. The size of the protected fragment is 200 bases. The band at 215 bases visible in all lanes is artefactual and results from interactions of the probe with itself as shown by its presence in the control lane (c). The 5' end-labeled 1125 and 2532 bases pAT153 fragments used to monitor material losses (see Materials and Methods), are also shown. Exposure time: 72 h.
results p r e s e n t e d in Fig. 5 c o n f i r m that i n t e r p r e t a t i o n . A t all times there was m o r e K8 m R N A . T a k i n g the average of the values o b t a i n e d f r o m cultures treated 7 a n d 8 d a y s with R A a n d from cultures t r e a t e d 6, 7 a n d 8 d a y s with R A plus B t 2 c A M P as the m a x i m u m o b t a i n e d in each treatment, the ratio of K8 to K18 was c a l c u l a t e d to be a b o u t 3.5 in R A - t r e a t e d cells a n d a b o u t 9 in cells treated with R A a n d B t 2 c A M P . These values are consistent with the results p r e s e n t e d in Fig. 1 for the d i f f e r e n t i a t e d cell line P F H R 9 a n d F 9 A C c l 9 , where K8 m R N A was a b o u t 4-times m o r e a b u n d a n t t h a n K18 m R N A . These two cell lines ressemble e x t r a e m b r y o n i c p a r i e t a l e n d o d e r m a n d are therefore similar to c o m pletely d i f f e r e n t i a t e d F9 cells. This is actually the case for F 9 A C c l 9 as this cell line was cloned from a R A t r e a t e d F9 cell c u l t u r e [43].
Discussion T h e results p r e s e n t e d here i n d i c a t e that K8 a n d K18 m R N A s are i n d e p e n d e n t l y regulated. First, the two m R N A s were expressed at very different levels. In d i f f e r e n t i a t i n g cells, K8 m R N A was f o u n d to b e m o r e a b u n d a n t than K18 m R N A . This was also o b s e r v e d in
d i f f e r e n t i a t e d cell lines a n d in u n d i f f e r e n t i a t e d cells. In u n d i f f e r e n t i a t e d cells, the a m o u n t of b o t h m R N A s was very low. This is similar to o t h e r m R N A s , like H-2 for instance, which, b y n o r t h e r n analysis, are usually n o t d e t e c t a b l e in F9 EC cells, b u t c a n be d e m o n s t r a t e d using high specific activity p r o b e s a n d long e x p o s u r e s [70], or b y nuclease S1 resistance a s s a y [71]. In the w o r k d e s c r i b e d here, the b a s a l level of K8 m R N A e v a l u a t e d b y d e n s i t o m e t r y was 4 - 5 - t i m e s higher t h a n the b a s a l level of K18 m R N A . This is close to the ratio f o u n d in d i f f e r e n t i a t e d cells a n d suggest that it is due to the low level of s p o n t a n e o u s l y d i f f e r e n t i a t e d cells k n o w n to be present in F 9 cell cultures [72,73]. T h e higher level of e x p r e s s i o n of K 8 m R N A relative to K18 m R N A , 3.5 in R A - t r e a t e d cells a n d 9 in R A plus B t 2 c A M P - t r e a t e d cells, has to b e e x p l a i n e d since the ratio of t y p e I a n d t y p e II k e r a t i n s in I F is a p p r o x . 1 [74]. T h e K8 p r o t e i n m a y be m o r e a b u n d a n t t h a n K18 in d i f f e r e n t i a t i n g F 9 cells. A n excess of cellular K8 could reside in a n o n f i l a m e n t o u s pool. N o n f i l a m e n t o u s forms of K8 have b e e n h y p o t h e s i z e d to e x p l a i n the diffuse c y t o p l a s m i c staining seen b y i m m u n o f l u o r escence m i c r o s c o p y in m o u s e oocytes a n d cleavage-stage e m b r y o s [75,76]. T h e excess of K 8 c o u l d also form
199
80-
1
60"
40-
o
B i
10-
o
n - , ,=1"-I~ r - I J - l ~ l - ] 0 1 1.5 2 2.5 3 4 5 DAYS OF TBEATMENT
6
7
8
Fig. 5. Relative a m o u n t of K8 and K18 m R N A s in differentiating F9 cells. Autoradiograms, including those used in Fig. 3, and 4, were scanned with a laser densitometer. For all the time points, appropriately exposed autoradiograms were scanned. The 1125 pAT153 fragment was also evaluated by scanning and used to make corrections for material losses during manipulations. Relative R N A concentration of each sample was also evaluated by scanning the blot shown in Fig. 3C and used in the final calculations. The relative a m o u n t of m R N A was calculated with respect to the quantity of K8 m R N A in F9-21 cells. (A) K8 m R N A ; (B) K18 m R N A . Solid bars: RA treatment; open bars: RA plus Bt2cAMP treatment.
filaments with another type-I keratin, such as K19 which is also induced in differentiating F9 cells [77]. However, there is some evidence that strict regulation of keratin m R N A levels may not be essential. Transfection experiments have indicated that excess keratins are not stable in the absence of a partner subunit with which it can form filaments [39,40,78]. The second indication of independent regulation of K8 and K18 m R N A s expression during differentiation comes from the observation that the two m R N A s are not induced simultaneously in RA-treated cells. K8 m R N A accumulation started 1 day earlier. In cells treated with RA and Bt2cAMP, K8 and K18 m R N A s were already induced after 1 day of treatment. But this does not exclude that accumulation of one of the two m R N A s could have begun sooner than the other during
the first 24 h. Uncoupling of the expression of the members of a keratin pair by the earlier expression of the type-II subunit has been observed before in differentiating epithelial cells in culture. It was detected at the level of protein expression in maturing corneal epithelial cells [79] and in differentiating epidermal cells [80,81]. Also, in fibroblasts derived from an embryonal carcinoma, it was shown that after a treatment with 5-azacytidine some cells were expressing K8 alone, and that K18 was present only in K8 positive cells [82]. The third indication that K8 and K18 m R N A s are regulated independently comes from the effect of Bt 2cAMP. In the presence of Bt 2cAMP, the two m R N A s were not only induced earlier, they were also expressed at higher levels. However, this effect was more important for K8 m R N A . In cells treated 7 or 8 days with RA and Bt2cAMP, K8 m R N A was 4-8-times more abundant while K18 m R N A was only 2-times more abundant in cells treated With RA alone. Moreover, the number of differentiated cells was about 1.5-times higher in cultures treated with RA and BtEcAMP accounting for almost all the increase in K18 m R N A . Thus, the evidence presented here suggest that only K8 m R N A expression was affected by Bt2cAMP. The higher level of accumulation of K8 m R N A in presence of Bt2cAMP is in agreement with the results obtained with other markers of F9 differentiation, like plasminogen activator [83], laminins A and B [84,85] and collagen IV [85,86], which show a further increase in m R N A and protein levels when Bt2cAMP is added to RA-treated F9EC cells. Also, in the presence of BtzcAMP, collagen IV m R N A is induced sooner [86], like K8 and K18 mRNAs. The difference between the final levels of expression of K8 m R N A in the two treatments is consistent with the hypothesis that RA-treated cells are not in the same differentiation state as cells treated with RA and Bt2cAMP [34]. Since K8 and K18 form filaments together, it is paradoxical to find that expression of their m R N A s is independently regulated. However, because excess keratins are not stable in the absence of a partner subunit [39,40,78] filament assembly can in theory be regulated by controlling the expression of only one keratin m R N A . A question not addressed in this study is the level at which m R N A abundance is controlled: our results are consistent with a transcriptional as well as with a posttranscriptional mechanism including differential processing, transport or stability of the two mRNAs. Finally, in these cells as in m a n y other cells expressing K8 and K18, other keratins are also expressed. In differentiating F9 cells, the K19 gene is induced [77]. Possibly, different ratios of k18 to K19 result in filaments with slightly different properties. This would make sense, if, as noted above, cells treated with RA and cells treated with RA and Bt2cAMP were in a different differentiation state.
200
Acknowledgements We would like to thank Jean-Francois Boulais for his technical assistance, Roger Duclos for photography and Dr. V. Bibor-Hardy for the scanning system. This work was supported by grants from the Medical Research Council of Canada (MRC), and by studentships from the MRC (T.O. and C.L.), the Cancer Research Society Inc. (M.L.) and the Universit6 de Montr6al (T.O.).
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