THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260. No. 6, Issue of March 25, pp. 3812-3819, 1985 Printed in U.S.A.

Changes in the Nuclear and Cytoplasmic Levels of Type I and Type I1 Collagen RNAs during Growth of Chondrocytes in 5-Bromo-2’-deoxyuridine*

(Received for publication, October 9, 1984)

Stephen A. Saxe, Lewis N. Lukens, and Philip J. Pawlowski From the Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06457

Chick embryo chondrocytes were grown in cell cul- ture in the presence of 5-bromo-Z’-deoxyuridine. Un- der these conditions, the cells attached to the tissue culture plates and became polygonal in shape and con- currently shifted their synthesis of collagen from type I1 to type I subunits. The changes in the levels of the corresponding collagen RNAs in the nucleus and cyto- plasm were examined during this shift by dot blot and Northern blot analyses. It was found that the expres- sion of the cartilage collagen gene, al(II), was con- trolled at the transcriptional level. While the type I collagen genes may also be partially controlled at the transcriptional level, both genes appear to be tran- scribed in chondrocyte nuclei although chondrocytes do not synthesize type I collagen. In these chondro- cytes, the transcripts of the &(I) gene are polyadeny- lated and are apparently exported to the cytoplasm but are not translated.

The collagens represent a multigene family of extracellular matrix proteins, and the regulation of this family can affect not only tissue structure but also cell adhesion, cell migration, and development (for a review, see Bornstein and Sage, 1980). The coordinate regulation of these genes has been studied in normally developing embryos (Merlin0 et al., 1983) and some collagen diseases (de Wet et al., 1983). Several systems have been developed in which collagen synthesis can be selectively altered. These systems include growth of chondrocytes in the thymidine analog 5-bromo-2’-deoxyuridine (Mayne et al., 1975), infection of cells with Rous sarcoma virus (Howard et al., 1978; Adams et al., 1979; Sandmeyer and Bornstein, 1979), administration of glucocorticoids (Shull and Cutroneo, 1983; Sterling et al., 1983; Cutroneo et al., 1981), and hypertonic growth conditions (Koch et al., 1977).

We have been studying the effect of bromodeoxyuridine (BrdUrd’) on collagen synthesis in cultured chick embryo chondrocytes (Pawlowski et al., 1981; Pawlowski, 1976). These cells produce the collagen characteristic of cartilage, (al(II))3, and normally do not synthesize the type I collagen character- istic of fibroblasts. After treatment with BrdUrd, the chon- drocytes shift to the synthesis of type I collagen, (a l (1))~~2(1) ,

* This work was supported by Grant AM16162 from the National Institutes of Health (to L. N. L.) and by North Atlantic Treaty Organization Research Grant 1448 (to J. 0. Bishop and L. N. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: BrdUrd, 5-bromo-2’-deoxyuridine; SDS, sodium dodecyl sulfate; hp, base pair; Pipes, 1,4-piperazinedi- ethanesulfonic acid DBM, diazobenzyloxymethyl.

and a type I trimer, (c~l ( I ) )~ , while ceasing to make type 11 collagen. Although we have focused only on collagens, a number of other changes in the types of protein synthesized also occur, as well as changes in cell morphology (Bornstein and Sage, 1980). Previous work in our laboratory (Pawlowski et al., 1981) had compared the different collagen mRNA levels during this shift using a cell-free protein-synthesizing system and had shown that the translatable levels of these mRNAs closely followed the rates of synthesis of the different collagen protein subunits by these cultures. Findings in our own labo- ratory (Koch et al., 1977; Pawlowski, 1982) as well as other laboratories (Paglia et al., 1979; Paglia et al., 1981; Horlein, 1981) indicated that translational controls may be involved in some aspects of collagen synthesis, and we therefore sought to measure more directly the levels of collagen mRNAs.

In the work presented here, we have used specific cDNA clones complementary to the mRNAs coding for type I1 and type I collagen chains to measure these RNA levels in both the nucleus and the cytoplasm of chondrocytes undergoing a shift in collagen synthesis during growth in BrdUrd.

EXPERIMENTAL PROCEDURES

Cell Culture-Chondrocytes were isolated from 14-day-old embry- onic chick sterna using the floater selection procedure of Schiltz et al. (1973) as modified by Pawlowski et al. (1981). BrdUrd, when present, was at 6.5 X M. The doubling time of the cultured cells was slightly less than 2 days.

Cell Labeling and Gel Electrophoresis-Cells were labeled with [3H] proline as described in the legend to Fig. 2. The labeled protein was prepared and run on SDS-polyacrylamide gels as described by Paw- lowski et al. (1981). Radioactivity was displayed using the fluoro- graphic procedure of Laskey and Mills (1975).

RNA Extraction-Cells plus culture medium were scraped from the cell culture plates and pelleted by spinning 5 min at 4000 X g. The cell pellet was washed twice with cold saline. To prepare total RNA from these cells, the guanidine thiocyanate procedure of Chirg- win et al. (1979) was used. To prepare poly(A)+ RNA, total RNA at 5.0 mg/ml was run through a poly(U)-Sephadex G-10 column as described by Deeley et al. (1977). Total yeast RNA was added to the poly(A)+ fractions as carrier, and the RNAs were ethanol-precipi- tated. After ethanol precipitation, the poly(A)- and poly(A)+ fractions were resuspended in a volume of H20 equivalent to the volume of total RNA loaded onto the poly(U) Sephadex column.

Cytoplasmic RNA was prepared as described by Pawlowski (1976). To prepare nuclear RNA, the nuclear pellet remaining from the cytoplasmic RNA preparation was resuspended in 10.0 mM Tris-HC1, pH 9.0, 1.0% SDS and then phenol-CHCI3-extracted and ethanol- precipitated. This nuclear pellet was then run on a Cs trifluoroacetate (Pharmacia) gradient to separate RNA from DNA. The pellet was suspended in 0.1 M KC1, 0.1 M Tris-HC1, pH 8.0, 1.0 mM EDTA, and sufficient Cs trifluoroacetate to give a solution with a final density of approximately 1.78 g/ml. The gradients were centrifuged in an SW 41 rotor (Beckman Instruments) for 3 days at 28,000 rpm at 18 “C. DNA banded on the top of the gradient, and RNA banded at a density of 1.83 g/ml. Fractions with densities greater than 1.77 g/ml were

3812

Collagen RNA Levels in BrdUrd-treated Chondrocytes 3813

pooled as RNA. These were ethanol-precipitated and resuspended in

DNA Hybridization Probes-Two of the probes were portions of the cDNA-containing plasmids pCg54 and pCg45 which were a gift from Dr. Helga Boedtker of Harvard University (see Fuller and Boedtker, 1981). For the al(1) probe, a 1114-bp piece was cut out of pCg54 by digestion with HindIII and KpnI. For the a2(I) probe, a 466-bp BarnHI-Hind111 fragment was cut out of pCg45. The al(1I) probe was a 750-bp cDNA fragment cut out of pCgII-12 using PstI (Lukens et al., 1983). The type I11 probe was a gift from Dr. Benoit de Crombrugghe of National Institutes of Health. It is the segment C subclone of XC3-C1-24 (Yamada et al., 1983a). The probes were nick-translated with [cx-~'P]~GTP (New England Nuclear) to a spe- cific activity of 1 X 10' cpm/pg.

Hybridizations-Filters containing RNA dot blots were hybridized in individual bags. Normally, 20.0 ml of hybridization buffer (3.75 x SSC, 37.5 mM sodium phosphate, pH 6.5, 1 X Denhardt's solution (Denhardt, 1966), 0.1% SDS, 60% deionized formamide, 100.0 pg/ml denatured salmon sperm DNA (and 1% glycine for Northern blots)) were added to each bag, and the filter was prehybridized at 42 "C for 12 h. Then 5-10 X lo6 cpm of denatured [32P]DNA probe were added to each bag, and the filters were hybridized for 24 h at either 55 "C (for the 466-bp a2(I) probe) or 60 "C (for the 1114-bp al(1) and 750- bp al(I1) probes). For Northern blots, the filters were hybridized at 55 "C (a2(I) probe) or 60 "C with the collagen DNA probes, washed, and then rehybridized at 42 "C with nick-translated X and 4x174 DNA. The filters were washed for 2 h with four changes of 2 X SSC, 0.1% SDS at 55 "C followed by another 2 h of washing with four changes of 0.1 X SSC, 0.1% SDS at 55 "C. The filters were placed down with Kodak X-Omat AR film at -70 "C in the presence of a DuPont Cronex intensifying screen. The autoradiographs were scanned on an Ortec densitometer.

Hybrid Selected Dot Blots-Lyophilized DNA was resuspended in Hz0 at a concentration of 0.3 pg/pl (466-bp a2(I) fragment) or 0.44 pg/pl (750-bp al(I1) fragment). The samples were boiled for 5 min on a water bath and then quickly frozen in dry ice/methanol. 5.0 pl of these denatured DNAs were spotted onto each of three 8 X 8-mm DBM filters, prepared according to the supplier, Schleicher & Schuell. The filters were air-dried, cut into pieces approximately 1-mm square, and placed into Eppendorf tubes. The prehybridization and elution conditions were essentially as described by Maniatis et al. (1982), but the hybridizations were as described above for RNA dot blots. The RNA used was day 0 total RNA. 110 pg of this lyophilized RNA were resuspended in 198.0 pl of deionized formamide and heated 1 min at 60 "C, and then 132.0 pl of 2.5 X hybridization buffer were added (hybridization buffer is 0.6 M NaCl, 0.05 M Pipes, pH 6.4,0.2% SDS, 0.002 M EDTA). 150.0 p1 of this mixture were added to each Eppendorf tube. The a2(I) tube was incubated at 55 "C, and the al(1I) tube was incubated at 60 "C for 3 h. The hybridization mixture was then removed, and the filters were washed with nine 1-ml washes of 1 X SSC, 0.5% SDS at 60 "C. Each tube then received 200.0 pl of elution buffer and was incubated 2 min at 68 "C, and the elution buffer was transferred to clean Eppendorf tubes. To each of these tubes were added 200.0 pl of H20, 30.0 p1 of 3 M sodium acetate, pH 6.0, and 1100.0 pl of 95% ethanol. The tubes were placed at -75 "C for 1 day and were then centrifuged 15 min in an Eppendorf microfuge. The supernatants were discarded, the pellets were washed with 400.0 pl of 95% ethanol, and the tubes were respun for 15 min. The supernatants were discarded, and the pellets were lyophilized to dryness. Each pellet was resuspended in 7.5 pl of HzO, and this selected RNA was spotted in triplicate onto duplicate GeneScreen (New England Nu- clear) filters (1.0 pllspot), and the filters were hybridized as described with either the a2(I) or al(I1) 32P-labeled probes.

Dot Blot Analysis-For each experiment, 1.0 pl of each nuclear or cytoplasmic RNA sample, 2.5 and 5.0 pg/pl, respectively, was spotted in triplicate onto GeneScreen (New England Nuclear). Also, on filters to be hybridized with type I probes, 0.005, 0.01, 0.05, and 0.1 pg of poly(A)+ calvaria RNA were spotted in triplicate. These calvaria RNA dots were used both as a standard for comparing the ratio of al(1) to a2(I) and as a control to check the linearity of the densitom- eter readings. The Genescreen was pretreated by soaking for 1 h in 25.0 mM sodium phosphate, pH 6.5, and was then air-dried. RNA was spotted, and the filters were dried 15 min under a heat lamp and then baked for 2 h a t 80 "C in a vacuum oven.

Northern Blot Analysis-Total RNA was resuspended in HzO, and CH3HgOH was added to 11.0 mM. The RNA (5.0 pgllane) was electrophoresed on 3.0-mm thick vertical agarose gels containing 7.5

HzO. mM CH3HgOH (Bailey and Davidson, 1976). For size markers, 5.0 pg of a HindIII digest of X DNA and 5.0 pg of a HaeIII digest of 6x174 DNA were made 11.0 mM in CH3HgOH and run on the gel. The gels were made up in E buffer (0.05 M H3B03, 0.005 M NazB407. 10HzO, 0.01 M NazS04, 0.001 M Na3EDTA and adjusted to pH 8.2) and electrophoresed at 125 V with constant buffer recirculation. The gels were then soaked 40 min in E buffer + 10.0 mM P-mercaptoethanol followed by a 20-min soak in E buffer + 2.0 pg/ml ethidium bromide and then photographed. The gels were then destained 20 min in 0.2 M sodium acetate, pH 4.0, and then electroblotted in the same buffer onto DBM paper (Schleicher & Schuell) by a modification of Stellwag and Dahlberg (1980) for 2 h a t 20 V followed by 2 h at 50 V.

RESULTS

BrdUrd-induced Shift of Chondrocytes in Cell Culture- Chondrocytes which are initially plated out onto tissue culture plates are round and floating as seen in Fig. lA. After 8 days of growth in the presence of BrdUrd, the cells have attached to the tissue culture plates and have assumed a polygonal shape (Fig. 1B). These two types of cells synthesize different collagen subunits. To show this, the cells were labeled by incubation with ~-[2,3-~H]proline, and the cellular protein was extracted and run on a polyacrylamide gel. Fig. 2 shows that the cells at day 0 were synthesizing only type I1 collagen subunits, but by day 6 the cells had shifted and were primarily synthesizing al(1) and a2(I) subunits.

Stringency of Hybridization Required for Specific Measure- ment of Each Collagen RNA-Due to the homology of these

B

FIG. 1. Cultured sternal chondrocytes from 14-day-old chick embryos. A , day 0 chondrocytes. These grow as floating, round cells, and the majority remain floating even after 8 days of growth in Dulbecco's modified Eagle's medium in the absence of BrdUrd. B, chondrocytes grown for 8 days in the presence of BrdUrd. All of the cells have attached to the tissue culture plate and are polygonal in shape.

3814 Collagen RNA Levels in BrdUrd-treated Chondrocytes

0 2+ 2- 4+ 4- 6+ 6- 8+ 8-

DAYS IN CULTURE FIG. 2. Fluorograph showing the proteins synthesized by

chondrocytes grown with and without BrdUrd. Cells were labeled with ~-[2,3-~H]proline for 2 h, and the total cellular proteins were separated on a 5-12% polyacrylamide gel. + indicates cells were grown in the presence of BrdUrd; - indicates cells were grown in the absence of BrdUrd.

V - m - COOH

ter ina t I triple helical a, (I) .......

Q, (11) ““_ 1 “““““““““““““-a 0 1000 2000

Nucleotides

FIG. 3. Location of cDNA probes relative to full-length col- lagen mRNAs. The NHz-terminal region includes the 5“untrans- lated portion and the sequences coding for the signal peptide, the NHz-terminal propeptide, and the NHz-terminal telopeptide. The COOH-terminal region includes the sequences coding for the COOH- terminal telopeptide and COOH-terminal propeptide followed by the 3”untranslated sequences. Known lengths are indicated by a solid line, and estimated lengths are indicated by a dashed line. The black boxes indicate the regions corresponding to the cDNAs used as probes. The carbohydrate attachment site is indicated (V).

three collagen genes, there was a possibility that the DNA probes being used would cross-hybridize with the different collagen RNA species. Since we were attempting to quantify specifically the levels of the three separate RNAs, we made sure that our hybridization conditions prevented cross-hy- bridization. First, we chose cDNA sequences not likely to be homologous by virtue of their location near the 3‘ end of the RNAs (Fig. 3; data from Fuller and Boedtker, 1981; Highber- ger et al., 1982; Palmiter et al., 1979; Pesciotta et al., 1980; Boedtker et al., 1983; Sandell et al., 1984; Curran and Prockop, 1982; Lukens et al., 1983) in regions which are not highly conserved. Second, a series of hybridizations was performed at different stringencies to find conditions that gave negligible cross-hybridization. The different cDNA probes were hybrid- ized to RNA from day 0 cells (which make type I1 collagen) and day 8 + BrdUrd cells (which make type I collagen). At 60 “C and using a buffer containing 60% formamide and 0.56

M NaCl, the al(I1) probe hybridizes only to day 0 RNA and the al(1) probe hybridizes only to day 8 RNA. This level of stringency is therefore high enough to prevent cross-hybridi- zation of these two species. The a2(I) probe, however, is not sufficiently GC rich to hybridize under these conditions (the al(1) probe is 69.1% GC (Fuller and Boedtker, 1981), the al(I1) probe is 49.3% GC (Sandell et al., 1984; Lukens et al., 1983), and the a2(I) probe is 40.3% GC (Fuller and Boedtker, 1981)), but it will hybridize if the temperature is lowered to 55 “C. Although 55 “C and a buffer containing 60% formamide was the maximum stringency which allowed hybridization of a2(I) cDNA to day 8 RNA, we still found hybridization to day 0 RNA. To check whether this hybridization was due to cross-hybridization of the a2(I) probe to the type I1 RNA present at day 0 or whether it represented authentic hybridi- zation to a2(I) RNA unexpectedly present in day 0 cells, hybrid-select dot blot experiments were performed. In these experiments, al(I1) and a2(I) RNAs from day 0 chondrocytes were specifically selected by hybridization to their respective cDNAs immobilized on DBM paper. These RNAs were eluted and challenged with each cDNA by dot blot hybridizations. Fig. 4 shows that both probes hybridize to total unselected day 0 RNA, but the a2(I) probe hybridizes only to RNA selected with this cDNA and not to RNA selected with the al(I1) cDNA. Also, the al(I1) probe hybridizes strongly with RNA selected with the al(I1) DNA but only very weakly with RNA selected with the a2(I) DNA. These results indicate that the hybridization conditions are of sufficiently high stringency to reduce cross-hybridization to negligible levels.

We also checked for the presence of type I11 RNA at days 0 and 8 using the type I11 cDNA probe provided by Dr. Benoit de Crombrugghe and found that it was not detectable in contrast to the results with day 10 decapitated embryos where comparable amounts of RNA gave a readily detectable signal (data not shown).

Measuremen? of Amounts of the Three Collagen RNAs dur- i n g the BrdUrd-induced Shift-To quantify the changes in levels of the collagen RNAs throughout the time course of 8 days, during which collagen synthesis shifts from type I1 to type I (Mayne et al., 1975; Benya et al., 1977; Pawlowski et al., 1981), RNA dot blots were hybridized with each of the three collagen cDNAs. The results for cytoplasmic RNAs are shown in Fig. 5, A-C. These graphs are based on assigning a value of 100% to the most intense spot obtained with each probe. The cytoplasmic data show a sharp decrease in the levels of al(I1) RNA which parallels the decrease in type I1 protein synthesis (Pawlowski et al., 1981). A sharp increase in the level of al(1) also parallels the observed increase in synthesis of this collagen subunit. The a2(I) RNA also shows an increase, but unlike the other two RNAs it is always present at appreciable levels even at day 0 when no a2(I) collagen subunits are synthesized by these cells. The similar although smaller changes in RNA levels which are seen with

32P-DNA probe 2(1) a1 ( 1 1 )

TOTAL RNA 0 0 0 .e e RNA

SELECTED ot2(1) DNA 0 0

wl(II) DNA e o 0

FIG. 4. Hybrid-selected RNA dot blots. Total RNA and RNA selected by hybridization with a2(I) or al(I1) cDNA was dotted onto Genescreen and then hybridized with 32P-labeled a2(I) or al(I1) cDNA.

Collagen RNA Levels in BrdUrd-treated Chondrocytes 3815 A CYTOPLASMIC PRO-al( l ) RNA B CYTOPLASMIC PRO-o2(1) RNA

100

c CYTOPLASMIC PRO-Ql( I I ) RNA . .

2 0 -

0 2 4 8 8

1 I 1 0 2 4 8 8

D~~~~~~~~ TYPE I RNA. RELATIVE TO D A V - 3 D~~~~~~~~ TYPE I RNA. RELATIVE TO D A V -

20-

0 2 4 8 8

DAYS IN CULTURE FIG. 5. Quantification of cytoplasmic collagen RNAs by dot blot hybridization. Cytoplasmic RNA was

extracted from cells at each of the time points indicated, spotted onto GeneScreen, and hybridized with [3'P] cDNA. Autoradiographs were obtained and scanned on a densitometer. A-C indicate relative amounts based on assigning a value of 100% to the most intense dot. 0, cells grown in the presence of BrdUrd; 0, control cells. D is based on normalizing the data for the type I probes such that day 0 has a value of 1. 0, a2(I) RNA from cells grown in the presence of BrdUrd 0, a2(I) RNA from control cells; X, al(1) RNA from cells grown in the presence of BrdUrd W, al(1) RNA from control cells.

the control cells are due to the fact that some chondrocytes attach to the cell culture plates and change their phenotype even in the absence of BrdUrd.

To show more clearly the relative changes of the two type I RNAs, the data are redrawn in Fig. 5 0 where all the day 0 points are assigned a value of 1 and the rest of the data are compared to that value. This figure emphasizes the dramatic increase in al(1) cytoplasmic RNA, about 30-fold greater at day 8 than at day 0 in BrdUrd-treated cells. The cytoplasmic a2(I) RNA increases only 3-fold from day 0 to day 8 in these cells.

From the dot blot data, the ratio of al(I):a2(1) RNA was calculated. To do this, poly(A)+ RNA from chick calvaria, a rich source of type I collagen RNA, was spotted onto the same filters as was the RNA from day 0 to day 8 cultured cells. On each filter, the day 0 to day 8 RNA hybridization levels were compared to the calvaria RNA hybridization levels. Ratios were determined by normalizing for a 2:l ratio of al(1) to a2(I) RNA present in calvaria (Vuust et al., 1983). Use of calvaria RNA as an internal standard eliminates artifactual changes in the extent of hybridization that could otherwise arise due to differences in lengths or specific activities of the probes. The cytoplasmic al(I):a2(1) RNA ratios are shown in Fig. 6. This ratio varies from a value of 0.3 at day 0 to 3.0 at day 8. The excess of al(1) RNA at day 8, as revealed by the ratio of 3.0 rather than 2.0, is in agreement with earlier work showing that al(1) chains are made in excess during treatment with BrdUrd, as is translatable al(1) mRNA (Pawlowski et al., 1981). The al(I):a2(1) RNA ratio of 0.3 at day 0 as compared to an expected ratio of 2.0 indicates that the a2(I) RNA is present in the cytoplasm of untreated day 0 chondro- cytes at approximately seven times the concentration ex- pected from the level of al(1) RNA.

The results of the nuclear RNA dot blots are shown in Fig. 7. The type I1 collagen RNA shows a rapid decrease in nuclear

RATIO OF CYTOPLASMIC

L 1 1 0 2 4 6 8

D A Y S IN CULTURE FIG. 6. Ratio of cytoplasmic al(1) to a2(1) RNA. RNA dot

blots were performed as described in the legend to Fig. 5 but with the addition of extra dots of chick calvaria poly(A)+ RNA used as an internal standard. The poly(A)+ data were normalized to give a 2:l ratio of al(1) to a2(I); and the ratios of the BrdUrd (0) and control (0) RNA were determined by comparison to these values.

levels in BrdUrd-treated cells and becomes undetectable by day 6. This result suggests that transcription of this gene is shut off by exposure of cells to BrdUrd. The type I RNA data are not so clear cut. Whereas al(I1) RNA levels are high at day 0 and decrease to undetectable levels by day 6, type I collagen RNAs are present in the nucleus even at day 0. During continued growth in BrdUrd, while the cytoplasmic level of the type I RNAs increases (al(1) up to SO-fold), the nuclear level of al(1) RNA remains the same while that of a2(I) RNA decreases.

Fig. 8 shows the nuclear ratio of al(I):aB(I) RNA. Similar to the cytoplasmic ratio, the nuclear ratio also increases with growth in BrdUrd. The nuclear ratio, however, is lower than that in the cytoplasm at all days examined, reaching a maxi-

3816 Collagen RNA Levels in BrdUrd-treated Chondrocytes

FIG. 7. Quantification of nuclear collagen RNAs by dot blot data. See the legend to Fig. 5 for details.

A NUCLEAR PRO-a l(1) RNA B

NUCLEAR PRO-a2(1) RNA

2 0 - - 2 0 -

0 2 4 6 8 0 2 4 6 8

C NUCLEAR PRO -U l(11) RNA

I

RATIO OF NUCLEAR PRO--1 (I) RNA TO PRO-a2 (1) RNA I I

0 2 4 6 8

DAYS IN CULTURE FIG. 8. Ratio of nuclear al(1) to a2(I) RNA. See the legend to

Fig. 6 for details.

mum of 1.0 at day 6 at which time the cytoplasmic ratio is 3.0.

Characterization of Collagen RNAs by Northern Blot Anal- ysis-As an additional way of characterizing the RNAs that hybridize to the collagen probes, total RNA was extracted from cells grown in the presence of BrdUrd for periods ranging from 0 to 8 days. This RNA was electrophoresed on CH3HgOH gels, electroblotted onto DBM paper, and hybridized with each of the three probes (Fig. 9). The al(I1) cDNA detects a single band at 5000 bases which shows up strongly at day 0 and disappears by day 8. The al(1) cDNA gives a band at 5100 bases which is barely detectable at day 0 and becomes very strong at later days. Longer exposures show a second larger RNA band at 8100 bases. The Northern blot probed with a2(I) cDNA shows a doublet of 5600 and 5150 bases which is clearly apparent at days 0 and 2 and then increases throughout the time course of the experiment. On another Northern blot also probed with a2(I) cDNA in which the gel had been run longer to get a better separation of the two bands, the autoradiograph was scanned to compare the rela-

Q NUCLEAR TYPE I RNAs RELATIVE TO DAY 0

1 1 I , 0 2 4 6 8

DAYS IN CULTURE

tive amounts of the two bands (data not shown). The ratio of the 5150 to 5600 band remained constant at a value of about 1.6 throughout the course of the 8 days.

The sizes of the RNAs calculated from the Northern blots indicate that the hybridization given by the cDNA probes is due to the corresponding RNAs and not, for example, due to hybridization with contaminating DNA. The sizes for the RNAs are in reasonably good agreement with the results of other groups. Vuorio et al. (1982) reported a size of 5300 bases for the al(I1) RNA and also a second larger type I1 RNA species of 7000 bases. This larger species was not seen in our experiments. Adams et al. (1979) and Merlino et al. (1983) found sizes of 5000 and 7100 bases for the al(1) RNA. Merlino et al. (1983) reported sizes of 5100 and 5700 bases for the a2(I) RNA, and Adams et al. (1979) reported sizes of 5200 and 5700. Recently, from sequencing the a2(I) gene, Boedtker et al. (1983) calculates sizes, exclusive of the poly(A) tail, of 4522 and 4976 bases.

The Northern blot of a2(I) RNA confirms the fact that a2(I) RNA of the expected size is present in cells at day 0. The doublet seen is characteristic of a2(1) RNA as opposed to the single band seen for type I1 RNA. These Northern blots confirmed the surprising fact that a2(I) RNA is present in day 0 and day 2 cells although these cells are not synthesizing the ar2(I) collagen chain.

The 0-Day a2(1) RNA Is Polyadenylated-To determine if the unexpected a2(I) RNA at day 0 was poly(A)+ or if it were mostly poly(A)- (which could possibly explain the reason for it not being translated), day 0 and day 8 + BrdUrd total RNAs were run through a poly(U)-Sephadex G-10 column to separate them into poly(A)+ and poly@- fractions. These fractions were dot-blotted onto GeneScreen as were total day 0 and day 8 RNA. These dot blots were then hybridized to a2(1) cDNA probe. Fig. 10 shows that the poly(A)+ dots of day 0 and day 8 both showed up as intensely as did the total RNA for these days, whereas the poly(A)- dots were com- pletely blank. A control experiment showed that the carrier yeast RNA present in the poly(A)+ fractions does not hybrid- ize with the a2(1) cDNA probe (data not shown). These results

A RNA HYBRIDIZED WITH PRO-Wl(I I )

MARKER DAYS IN BrdUrd MARKER

23.1- b 4 9.4- b-4

0 2 4 6 8 t L

6.5- I 4.4- b 4 0 0 b

E t.?

RNA HYBRIDIZED WITH PRO &1(1)

MARKER DAYS IN Brd Urd MARKER

0 2

.1) * * u

4 6 8

I,

'RNA HYBRIDIZED WITH PRO-W2(1)

MARKER DAYS IN BrdUrd M A R K E R

Collagen RNA Levels in BrdUrd-treated Chondrocytes 3817

and locatedby autoradiography.

FIG. 9. Northern blot hybridization of total RNA. Total RNA extracted from cells grown in the presence of BrdUrd was electropho- resed on CHSHgOH-0.8% agarose gels. The RNAs were electroblotted onto DBM paper, hybridized with the indicated [32P]cDNA probes,

TOTAL POLY A + POLY A-

o p ~" ~ " _ I 1 - I- ~ ~ < . ' " " ~ ~ " " ~ ~ I

Days

8+ il

1.0 1.0 1.0 2.0 3.0 1.0 2.0 3 .0

AJI RNA

FIG. 10. Dot blot analysis of the a2(I) RNA concentration in total, poly(A)+, and poly(A)- RNAs. RNA extracted from day 0 and day 8 + BrdUrd cells was separated into poly(A)+ and poly(A)- fractions. These RNA samples were resuspended in a volume of Hz0 equal to the volume of total RNA loaded onto the poly(U)-Sephadex column. 1.0 pl of the total RNA was spotted in duplicate, and 1.0-, 2.0-, and 3.0-p1 samples of the poly(A)+ and poly(A)- RNAs were spotted onto GeneScreen and hybridized with the a2(I) [32P]cDNA probe.

indicate that the vast majority of the a2(I) RNA which is present at day 0 (as well as at day 8) is polyadenylated.

DISCUSSION

Cytoplasmic Collagen RNA Leuels-Dot blot hybridizations were used to measure the relative levels of collagen type I1 and type I RNA over an %day period of cell growth in BrdUrd. The synthesis of these three collagen chains changes dramat- ically during this period of growth (see Fig. 2). The synthesis of al(I1) decreases to undetectable levels by day 6, while the synthesis of the al(1) subunit begins at about day 2 and increases in rate over the subsequent 6 days. The synthesis of the other type I collagen chain, a2(I), lags behind the al(1) chain and does not begin until day 4 or 5. The amount of increase in synthesis of this chain is less than the increase in synthesis of the al(1) chain, resulting in a deviation from the 2:l ratio expected for type I collagen chains. In our previous work, we used a cell-free protein-synthesizing system to assay the levels of translatable mRNAs coding for each of these three collagen chains and found that the levels of translatable RNA closely followed the in vivo subunit synthesis rates (Pawlowski et al., 1981).

The results obtained using cDNA probes to directly mea- sure cytoplasmic collagen RNA sequences are shown in Fig. 5. These results agree with previous work (Pawlowski et al., 1981) in that al(I1) RNA was found to decrease, while al(1) and a2(I) RNAs were found to increase during growth in BrdUrd. An unexpected finding, however, was the presence of a2(I) RNA at the earliest days tested (days 0 and 2). This would indicate that a2(I) RNA was present but untranslata- ble. Several lines of evidence indicate that, indeed, a2(I) message is present but not translatable. First, we were able to rule out the possibility of cross-hybridization of the a2(I) cDNA to the more abundant al(I1) RNA by using sufficiently stringent conditions of hybridization. As shown in Fig. 4, virtually no hybridization occurs between a2(I) cDNA and al(I1) RNA purified by hybrid selection. Second, Northern blot analyses (Fig. 9) showed that our a2(I) probe hybridized to distinctly sized RNA species characteristic of a2(I) mRNAs. The sizes of these a2(I) mRNAs (Fig. 9C) agree well with those found by other groups (Merlin0 et al., 1983; Adams et al., 1979) and differ from both the al(I1) and al(1) RNAs (Fig. 9, A and B) . Furthermore, the Northern blot analysis indicates the absence of appreciable amounts of contaminat- ing DNA in our RNA preparations although in any case the presence of genomic DNA would be expected to influence the hybridization of all three probes rather than the hybridization

3818 Collagen RNA Levels in BrdUrd-treated Chondrocytes

of only the a2(I) probe. Third, the possibility that the a2(I) RNA present in the untreated and day 2 chondrocytes is due to fibroblast contamination of these cultures is unlikely, since negligible amounts of type I collagen are synthesized and negligible amounts of al(1) RNA are present, as judged by dot blot hybridization assays.

The possible participation of post-transcriptional controls in the regulation of collagen synthesis has been demonstrated by other groups (Adams et al., 1982; Lozano et al., 1983; Yamada et al., 198313; Schmidt et al., 1984) as well as ourselves (Koch et al., 1977; Pawlowski, 1982). In chondrocytes derived from embryonic chick vertebrae, presumably similar to the sternum-derived chondrocytes used in our work, Adams et al. (1982) found that transformation with Rous sarcoma virus caused an increase in type I collagen RNAs which could be translated in a cell-free system but were not translated by the intact cells. A subsequent report suggests that at least some synthesis of al(1) collagen chains may occur in such cells although the synthesis of a2(I) chains is very low or absent (Gionti et al., 1983). Although untranslated a2(1) RNA may therefore be present in Rous sarcoma virus-transformed chon- drocytes as well as in the day 0 chondrocytes described here, the mechanisms of the post-transcriptional regulations pre- sumably differ, since the a2(1) RNA from Rous sarcoma virus- transformed chondrocytes can be translated in reticulocyte lysates, whereas the a2(I) RNA from day 0 chondrocytes cannot (Pawlowski et al., 1981).

The reasons for the inability of the a2(I) RNA in chondro- cytes to be translated are not yet understood. Several possible mechanisms appear unlikely, however, in view of our findings to date. This inability is apparently not due to lack of a 3‘- poly(A) tail since isolation of poly(A)+ RNA by column chro- matography on poly(U)-Sephadex G-10 followed by hybridi- zation of RNA dot blots to a2(I) cDNA showed that essen- tially all of the a2(I) RNA was in the poly(A)+ RNA fraction, in the case of both the 0 and 8-day RNA samples (Fig. 10). The finding of two discrete size classes of a2(I) RNA, due to different lengths in the 3”untranslated region of the mRNA (Ah0 et al., 1983), presented the possibility that they might be differently translated. This possibility was strengthened by the finding of Merlin0 et al. (1983) who demonstrated a shift in the ratio of the two a2(I) RNAs, but not in the ratio of the two al(1) RNAs, during chick embryonic development. The lack of translation of a2(I) RNA in chondrocytes cannot, however, be explained by such a mechanism, since we have measured the ratio of the 5100 nucleotide and 5700 nucleotide a2(I) RNAs during growth in BrdUrd and found that the ratio remains essentially unchanged from the value found in untreated chondrocytes. Additional possibilities that need investigation include the presence of a structural defect in this RNA (e.g. lack of a 5’-cap) or the presence or absence in chondrocytes of a factor that co-isolates with a2(I) RNA and inhibits or activates its translation in cell-free systems as well as in intact chondrocytes.

Nuclear Collagen RNA Levels-Before measuring nuclear collagen RNA levels by dot blot hybridizations, steps were taken to eliminate contamination by cytoplasmic RNA or by nuclear DNA. The nuclear isolation procedure used here has been found to give nuclear RNA preparations devoid of cy- toplasmic contamination, as judged by the absence of 18s rRNA.’ Moreover, in the case of the type I collagen RNAs, the nuclear and cytoplasmic levels change in opposite direc- tions, an outcome that suggests that the nuclear RNA samples cannot be heavily contaminated with cytoplasmic RNA. The

P. J . Pawlowski, unpublished data.

DNA was removed from nuclear samples on gradients of cesium trifluoroacetate under conditions where the DNA (at the top of the gradient) was widely separated from the RNA (at two thirds of the way down the gradient).

The most clear-cut alteration in nuclear levels of collagen RNAs was the decrease in al(I1) RNA to almost undetectable levels by day 6 in cells grown in BrdUrd (Fig. 7). This result indicates a shut down of al(I1) gene transcription. The data concerning the nuclear levels of al(1) and a2(1) RNA under- score a need for further study before the control of their synthesis can be understood. The nuclear levels of these RNAs did not start out low and subsequently increase during growth in BrdUrd as might be expected if only transcription were increased. It was found (Fig. 7) that both RNAs were present at early days and remained approximately the same (al(1)) or decreased (a2(I)) on subsequent days of growth in BrdUrd even though cytoplasmic levels were increasing. Several pos- sibilities exist which might account for these findings: a constant level of transcription with an increase in processing, transport, and cytoplasmic stability, or an increase in tran- scription again with an increase in processing, transport, and stability. That post-transcriptional controls may be operating is indicated by the discrepancy between the cytoplasmic and nuclear ratios of al(I):a2(1) RNAs. Experiments to distin- guish among these possibilities are now in progress.

Action of BrdUrd-How BrdUrd turns off or turns on different genes is unknown. Its action is thought to derive from its being incorporated into DNA and promoting tighter interaction between DNA and associated proteins (LaPeyre and Bekhor, 1974; Gordon et al., 1976; Lin et al., 1976; Schwartz, 1977; Bick and Devine, 1977). It has also been found that the rate of cytoplasmic appearance of mRNA is decreased in cells grown in BrdUrd (Pawlowski, 1976). Such findings, however, fail to explain the differential effects of BrdUrd in a system such as that used in this study. The action of BrdUrd in this system is apparently mimicked by a number of agents such as phorbol myristate acetate (Lowe et al., 1978), retinoic acid (Hassell et al., 1979), and chick embryo extract (Mayne et al., 1976) and by culture conditions (Benya and Shaffer, 1982). It appears therefore that these chondro- cytes can be readily switched by a variety of stimuli to the phenotype described here. The mechanism of action of any of these stimuli is not yet understood. The present paper shows, however, that the mechanisms that underlie the changes in collagen types are unexpectedly complex. While the cartilage collagen gene appears to stop being transcribed in BrdUrd, the two type I genes appear to be transcribed in chondrocytes even before exposure to BrdUrd. al(1) RNA, however, re- mains in the nucleus, and the a2(I) RNA that reaches the cytoplasm is not translated. After growth in BrdUrd, the al(1) RNA increases in the cytoplasm coordinately with increases in the synthesis of its protein. The a2(I) RNA is still exported and is now translated. It is also likely but not yet proven that the transcription rates of the type I genes increase.

Ackmwledgments-We gratefully acknowledge the gift of type I collagen clones from H. Boedtker and a type I11 clone from B. de Crombrugghe. We would like to thank John 0. Bishop for collabo- ration in the early stages of this work.

REFERENCES Adams, S. L., Alwine, J. C., de Crombrugghe, B., and Pastan, I. (1979)

Adams, S. L., Boettiger, D., Focht, R. J., Holtzer, H., and Pacifici,

Aho, S., Tate, V., and Boedtker, H. (1983) Nucleic Acids Res. 11,

Bailey, J. M., and Davidson, N. (1976) Anal. Biochem. 70, 75-85

J. Bwl. Chem. 264,4935-4938

M. (1982) Cell 30, 373-384

5443-5450

Collagen RNA Levels in BrdUrd-treated Chondrocytes 3819

Benya, P. D., and Shaffer, J . D. (1982) Cell 30, 215-224 Benya, P. D., Padilla, S. R., and Nimni, M. E. (1977) Biochemistry

Bick, M. D., and Devine, E. A. (1977) Nucleic Acids Res. 4 , 3687-

Boedtker, H., Fuller, F., and Tate, B. (1983) Znt. Reu. Connect. Tissue

Bornstein, P., and Sage, H. (1980) Annu. Rev. Biochem. 49, 957-

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W.

Curran, S., and Prockop, D. J. (1982) Biochemistry 21 , 1482-1487 Cutroneo, K. R., Rokowski, R., and Counts, D. F. (1981) Collagen

Relat. Res. 1,557-568 Deeley, R. G., Gordon, J. I., Burns, A. T. H., Mullinix, K. P., Bina-

Stein, M., and Goldberger, R. F. (1977) J . Biol. Chem. 252 , 8310- 83 19

Denhardt, D. T. (1966) Biochem. Biophys. Res. Commun. 2 3 , 641- 646

de Wet, W. J., Chu, M., and Prockop, D. J. (1983) J . Biol. Chem.

Fuller, F., and Boedtker, H. (1981) Biochemistry 20,996-1006 Gionti, E., Capasso, O., and Cancedda, R. (1983) J . Biol. Chem. 258 ,

Gordon, J. S., Bell, G. I., Martinson, H. C., and Rutter, W. J. (1976)

Hassell, J . R., Pennypacker, J. P., Kleinman, H. K., Pratt, R. M.,

Highberger, J. H., Corbett, C., Dixit, S. N., Yu, W., Seyer, J. M.,

Horlein, D., McPherson, J., Goh, S. H., and Bornstein, P. (1981)

Howard, B. H., Adams, S. L., Sobel, M. E., Pastan, I., and de

Koch, F., Pawlowski, P. J., and Lukens, L. N. (1977) Arch. Biochem.

LaPeyre, J.-N., and Bekhor, I. (1974) J . Mol. Biol. 8 9 , 137-162 Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56,335-341 Lin, S., Lin, D., and Riggs, A. D. (1976) Nucleic Acids Res. 3, 2183-

16,865-872

3700

Res. 10 , l -63

1003

J . (1979) Biochemistry 18,5294-5299

2 5 8 , 14385-14389

7190-7194

Biochemistry 15,4778-4786

and Yamada, K. M. (1979) Cell 17,821-826

Kang, A. H., and Gross, J . (1982) Biochemistry 21,2048-2055

Proc. Natl. Acad. Sci. U. S. A. 78, 6163-6167

Crombrugghe, B. (1978) J . Biol. Chem. 2 5 3 , 5869-5874

Biophys. 178,373-380

2191 LOW;,". E., Pacifici, M., and Holtzer, H. (1978) Cancer Res. 38 ,

2350-2356 Lozano, G., Helle, O., and Muller, P. K. (1983) EMBO J. 2 , 1223-

Lukens, L. N., Frischauf, A. M., Pawlowski, P. J., Brierley, G. T., 1227

and Lehrach, H. (1983) Nucleic Acids Res. 11,6021-6039 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

Cloning: A Laboratory Manual, pp. 332-333, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Mayne, R., Vail, M. S., and Miller, E. J. (1975) Proc. Natl. Acad. Sci.

Mayne, R., Vail, M. S., and Miller, E. J. (1976) Deu. Biol. 5 4 , 230- 240

Merlino, G. T., McKeon, C., de Crombrugghe, B., and Pastan, I. (1983) J . Biol. Chem. 258 , 10041-10048

Paglia, L., Wilczek, J., Diaz de Leon, L., Martin, G. R., Horlein, D., and Muller, P. (1979) Biochemistry 18 , 5030-5034

Paglia, L. M., Wiestner, M., Duchene, M., Ouellette, L. A., Horlein, D., Martin, G. R., and Muller, P. K. (1981) Biochemistry 20,3523- 3527

Palmiter, R. D., Davidson, J . M., Gagnon, J., Rowe, D. W., and Bornstein, P. (1979) J . Biol. Chem. 254 , 1433-1436

Pawlowski, P. J. (1976) J . Cell. Physiol. 8 9 , 19-27 Pawlowski, P. J. (1982) Biochemistry 2 1,34-38 Pawlowski, P. J., Brierley, G. T., and Lukens, L. N. (1981) J . Biol.

Pesciotta, D. M., Silkowitz, M. H., Fietzek, P. P., Graves, P. N., Berg,

Sandell, L. J., Prentice, H. L., Kravis, D., and Upholt, W. B. (1984)

Sandmeyer, S., and Bornstein, P. (1979) J . Biol. Chem. 254 , 4950-

Schiltz, J. R., Mayne, R., and Holtzer, H. (1973) Dijjerentiation 1 ,

Schmidt, A., Yamada, Y., and de Crombrugghe, B. (1984) J . Biol.

Schwartz, S. A. (1977) Biochemistry 16,4101-4108 Shull, S., and Cutroneo, K. R. (1983) J . Biol. Chem. 258,3364-3369 Stellwag, E. J., and Dahlberg, A. E. (1980) Nucleic Acids Res. 8,299-

Sterling, K. M., Jr., Harris, M. J., Mitchell, J. J., DiPetrillo. T. A..

U. S. A. 72,4511-4515

Chem. 256,7695-7698

R. A., and Olsen, B. R. (1980) Biochemistry 19 , 2447-2454

J . Biol. Chem. 259 , 7826-7834

4953

97-108

Chem. 259,7411-7415

317

Delaney, G. L., and Cutroneo, K. R. (1983) J..Biol. Chem. 2 5 8 , 7644-7647

Vuorio, E., Sandell, L., Kravis, D., Sheffield, V., Vuorio, T., Dorfman,

Vuust, J., Sobel, M. E., and Martin, G. R. (1983) Collagen Relat. Res.

Yamada, Y., Kuhn, K., andde Crombrugghe, B. (1983a) Nucleic Acids

Yamada, Y., Mudryj, M., and de Crombrugghe, B. (1983b) J . Biol.

A., and Upholt, W. (1982) Nucleic Acids Res. 10,1175-1192

3 , 70

Res. 11, 2733-2744

Chem. 258,14914-14919