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POST-TRANSCRIPTIONAL REGULATION OF THE PROa l(I) COLLAGEN GENE IN PROal(DEFICIENT, CHEMICALLY TRANSFORMED SYRIAN HAMSTER EMBRYO FIBROBLASTS Elaine M. Schalk, Anna Gosiewska, Willie Prather and Beverly Peterkofsky’ Laboratory of Biochemistry, National Cancer Institute National Institutes of Health, Bethesda, MD 20892 Received

September

9,

1992

Summary: 4-Nitroquinoline-I -oxide-transformed Syrian hamster embryo frbroblasts (NQTSHE) synthesize the proa chain but not the proal subunit of type I procollagen, and they contain little proal(1) mRNA. This study shows that there was no accumulation of proal poly(A)+ mRNA in NQT-SHE libroblasts. BHK cells, a normal established line of hamster tibroblasts that synthesized collagen at approximately the same rate as NQT-SHE tibroblasts, nevertheless produced both subunits of type I collagen and contained proa 1(I) mRNA. Run-off transcription assays with isolated nuclei showed that both the proal(1) and proa2(1) genes were transcribed at about the same rate in NQT-SHE cells as well as in the normal BHK cells. These results suggest that a post-transcriptional defect, probably resulting from transformation, prevents the accumulation of proal mRNA in NQT-SHE cells. o 1992 Academic

Press,

Inc.

Type I procollagen is a major extracellular matrix protein of bone, tendon and skin and is a component of most tissues(1,2). It is secretedas a triple helical heterotrimer consisting of two proa l(I) chains and one proa2(1) chain. Generally, the synthesis of each subunit responds coordinately to perturbations that influence collagen synthesis.For example, glucocorticoids (3), vitamin D, (4) and cytokines such as interferon (5) and tumor necrosisfactor-a (6), decreasethe synthesis of the collagen subunits coordinately, while in some connective tissue diseasesthe synthesis of proa l(1) and proa2(1) chains is increased coordinately (7, 8). In viral transformation, the subunits are down-regulated coordinately (9, 10).

*To whom correspondence should be addressed at Building National Cancer Institute, Bethesda, MD 20892.

37,

Room 4C-18,

Abbreviations: SHE, Syrian hamster embryo; NQT-SHE, 4-nitroquinoline- 1-oxide-transformedSyrian hamster embryo; BHK, baby hamster kidney; MEM-S-PIE, Eagle’s minimal essential medium supplemented with 5% fetal calf senmr, 1 mM pyruvate, 2 Pg/ml insulin and 2 rig/ml epidermal growth factor; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphatebuffered saline; PCR, polymerase chain reaction. 0006-291X/92 Copyright All rights

$4.00

0 1992 by Academic Press, of reproduction in any form

Inc. reserved.

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SHE Iibroblasts synthesizenormal type I collagen, but a 4-nitroquinoline-l-oxide-transformed cell line (NQT-SHE) derived from these cellsexhibits a lower rate of total collagen synthesis and does not synthesize the proal(1) subunit of type I collagen (11). These cells secretemodified forms of the proa2(1) subunit (11,12) and analysisof total RNA showed that they contain mRNA for the proa2(1) but almost no mRNA for the proa l(1) subunit (13). In the present study, the mechanism for the lack of expression of the proa l(I) chain in NQTSHE cells was examined further. Poly(A)’ mRNA was used to analyze for functional procollagen transcripts and to quantitate steady state mRNA levels. In addition, the rate of transcription of the procollagen genes by isolated nuclei was determined. MATERIALS

AND METHODS

Cell m: The source of the NQT-SHE cell line was described previously (11). Primary SHE fibroblasts were kindly provided by Dr. Sarah Bruce, Johns Hopkins University. BHK-21 tibroblasts were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in MEM-S-PIE medium as described previously (11). m pf polv(A‘I’ R,&$ RNA was isolated by a modified guanidine hydrochloride extraction procedure (9) followed by purification on an oligo dT column (14). (collagen &a&&: The procedure for measuring the relative rate of collagen synthesis and for analysis of 14C-labeledproteins by SDS-PAGE were described previously (11). Briefly, cells were labeled with [‘4C]proline and procollagen was isolated from cell and medium fractions by precipitation at 33% saturated ammonium sulfate. Collagen was prepared by pepsin digestion of the procollagens. ,6&t a& &r&m J&t &&&&ns: RNA slot and Northern blots were prepared on Nytran membranes and hybridized with cDNA probes for proal( proa2(1), fibroncctin and B-actin as described previously (15). I&&i Isolation: Cells in mid- or late-logarithmic phase were released with 0.03% trypsin and were washed once in MEM-5-PIE at 4’C and twice in PBS. Cells were lysed in Buffer A (70 mM NaCl, 10 mM Tris-HCl, pH 8.4, 1.5 mM MgCl,) with 0.5% Nonidet P-40 for 5 min on ice. The suspension was diluted with an equal volume of 2 M sucrosein Buffer A, layered on 1.7 M sucrosein Buffer A and was centrifuged at 24,000 rpm for 40 min in an SW 27 rotor. The nuclear pellet was suspended in Buffer B (70 mM NaCl, 20 mM Tris-HCl, pH 8, 1 mM MnCl,, 10 mM MgCl,, 20% glycerol, 14 mM dithiotreitol),centrifuged at 12,000 x g for 5 min at 4°C and resuspended in Buffer C (50 mM Tris-HCl, pH 7.8, 10 mM MgCl,, 250 mM KCl, 20 mM creatinine phosphate, 40% glycerol) at 2 x lo7 nuclei per 100 ~1. Nuclei were stored over liquid N, until use. . . Run-offTranscnntlon: Nuclei were thawed and mixed with 1 mM dithiothreitol, 1 mM each of ATP, CTP and GTP, 10 ug/ml creatinine phosphokinase, 2.5 pl RNasin @omega, Madison, WI) and 250 I&i of 5’-[a-32P]-UTP (3000 Ci/mmol) in a final volume of 200 ul. Transcription was carried out for 45 min at 25°C. After digestion with RNase-free DNase I and proteinase K, the RNA was precipitated and redissolved in 10 mM Tris-HCl, pH 7.5/5 mM MgCl, and again treated with DNase I and proteinase K. RNA was extracted in hot phenol, precipitated in the presenceof 100 pg of yeast tRNA, and dissolved in hybridization buffer. Plasmids,cDNA inserts or PCR-amplified fragments were applied to Nytran membranes with a slot manifold. Membranes were incubated with 2-5 ml of prehybridization solution (50% formamide, 5X SSPE, 5X Denhardt’s solution, 0.5% SDS, 0.05% Na,P,O,, 0.1 mM UTP, 200 ug/ml denatured salmon sperm DNA, 200 @ml denatured yeast tRNA) at 42°C and then in fresh solution with radiolabeled RNA for 2 to 3 days at 42°C. The blots were washed 4 times in 1X SSC/O.l% SDS at 65°C and then were exposed to X-ray tihn with a Cronex intensifying screen at -70°C. 781

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Sinceprimary SHE libroblasts do not survive beyond passage4-5, we determined whether the established Syrian hamster libroblast line BHK would be a valid normal control. These cells are not transformed and have a normal tibroblast morphology and karyotype (American Type Culture Collection catalog). Collagen synthesisby SHE and BHK cellswas characterized by SDS-PAGE (Fig. 1). BHK cellssynthesized normal proal(1) and proa2(1) chains of type I collagen in an approximately 2 to 1 ratio, as in the primary SHE libroblasts. Both cell lines also synthesized small amounts of type V collagen (Fig. 1). The relative rate of collagen synthesis for SHE cells was 4.2%, while BHK cells exhibited a considerably lower rate at 0.84%, which was comparable to collagen synthesisby NQT-SHE cells at 0.70%. Secretion of type I procollagen also is regulated similarly in SHE and BHK cells, i.e. stimulated by ascorbate and inhibited by cis-hydroxyproline (12). Thus, BHK cellsappear to be a valid normal control cell line for studying regulation of the proa l(1) gene in NQT-SHE cells. Previously, analysisof total RNA from NQT-SHE cellsby dot-blot hybridization detected only a small amount of proa l(1) mRNA (13). To determine whether this RNA represented functional transcripts and to more accurately quantitate its concentration, poly(A)’ RNA was analyzed. A Northern blot of poly(A)‘mRNA(Fig.

2) showed that proa l(1) and proa2(1) transcripts of appropriate

sizes(16) were present in SHE and BHK cells.The proa2(1) transcript in NQT-SHE cellswas detected at the same position as transcripts from SHE and BHK cells. With the probe for proa l(I) mRNA, a faint band was observed with RNA from the transformed cell line, but it migrated slightly ahead of the

9-E CM

BIN CM

alvL *is a21 -

procollagensfrom SHEand BHK cells.C, cellular Eig. 1. SDS-PAGE analysisof [‘4C]proline-labeled fraction; M, medium fraction. Approximatelythe sameamount of radioactive,pepsin-derivedcollagen wasapplied to the gel in eachcase. 782

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I

CELL NPE

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&.Z. Northern blot hybridizationsofpoly(A)‘mRNA isolated from NQT-SHE, SHE and BHK cells. The cDNA probes used were: u lR2 for proa l(I); a2R2 for proa2(1); FN, tibronectin; and g-actin. The amounts (pg) of mRNA applied to the gel are indicated in the figure. Note that the amount used for SHE cells is only 3% of the amount used for the other cell types. The positions of the 28s and 18s ribosomal RNAs are indicated. The arrow on the right marks the positions of the transcripts from SHE and BHK tibroblasts that hybridized with the proal probe. For NQT-SHE, the transcript hybridizing with this prohe(arrow on the left), although faint, was visibleon the original autoradiogram and photograph. & 2. Slot-blot hybridizations of poly(A)’ mRNA isolated from NQT-SHE, DNA probes are described in the legend to Fig 2.

SHE and BHK cells.

transcripts from SHE and BHK cells.This result suggestedthat the probe may crosshybridize to a small extent with the proa2(1) transcript, which migrates slightly faster than the major proa l(1) transcript, and that therefore no proa l(1) mRNA was present in NQT-SHE

tibroblasts.

For comparison,

mRNA

for tibronectin, an extracelluar matrix protein that also has been shown to be reduced in established cell lines and in transformed cells (17), was analyzed. The normal 8 kb transcript for tibronectin (18) was present in all three cell types (Fig. 2). Slot-blot analysis of the poly(A)’ mRNAs was carried out to more accurately quantitate concentrations of the collagen transcripts (Fig. 3). The levelswere quantitated by computer imaging analysis of the autoradiograms. Proa l(1) mRNA was expressedin SHE cellsat 100 times the level in 783

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al R2 a2R2 p-actin pBR322 I& 4. Run-off transcription assays with nuclei isolated from NQT-SHE and BHK cells.Plasmids(2 pg) or insert cDNAs (500 ng) were applied to membranes as indicated and hybridization with radioactive in vitro transcribed RNA was carried out as described in Materials and Methods.

BHK cells.The low level of proa l(1) mRNA detected in NQT-SHE cells(0.1% of SHE) probably was due to cross-hybridization of the proal(1) DNA probe with proa2(1) RNA, as discussed above. In contrast, proa2(1) mRNA levelsin NQT-SHE cellswere twice ashigh as in BHK cells,although in both casesthe levelswere considerably lower than in SHE fibroblasts (10% and Y!!, respectively). Fibronectin mRNA levelsalso were significantly lower in BHK and NQT-SHE cellscompared to SHE cells(2% and 5%, respectively). A run-off transcription assaywas carried out to determine if the absence of proal(1) mRNA in NQT-SHE cellswas due to suppression of transcription (Fig. 4). The proa l(1) gene from NQT-SHE cellswas transcribed at a rate comparable to that of the proa2(1) gene, despite the fact that no proa l(1) mRNA could be detected in the cells. Relative to transcription of the p-actin gene, the rate of transcription of the proa l(1) gene in BHK nuclei was about live times greater than the rate in NQTSHE nuclei. The results with both the intact plasmid containing the DNA inserts (A.) or the isolated inserts (B.) were identical. Failure of NQT-SHE to accumulate proa l(1) mRNA thus appears to be due to a post-transcriptional defect. A similar situation is found in HeLa cells, which do not produce type I collagen nor accumulate mRNA for the proa l(1) and proa2(1) subunits, but nevertheless transcribe the genes for these subunits (19). In contrast, failure of a chemically transformed epithelial cell line to express the proa2(1) gene appears to be caused by a transcriptional defect (20). In normal BHK cells,the proa l(1) and proa2(1) genesalso were transcribed at comparable rates (Fig. 4). These results suggest that the mechanism for maintaining a 2:l ratio of the proal

and

proa2(1) polypeptides operates after transcription. This conclusion is in agreement with that of Olsen and Prockop (21), who observed wide variations in the proa l(I):proaZ(I) ratios of transcription rates in nuclei from human Bbroblasts at different stagesofgrowth, although the proa l(I):proa2(I) ratio for mRNA isolated from cells was maintained at 2: 1. 784

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The post-transcriptional defect in proa l(1) mRNA accumulation in NQT-SHE cellsprobably can be attributed to the fact that these cells are transformed, rather than to their being an established cell line. BHK cells, an established but normal line of hamster fibroblasts, synthesized both type I collagen subunits, albeit at a lower rate than primary SHE fibroblasts, and contain mRNA for both subunits. The defect in NQT-SHE fibroblasts must be relatively specific, since proa2(1) mRNA continues to be expressed,and thus it might result from a mutation in the proa l(1) gene induced by the carcinogen used for transformation. A mutation in the 3’-untranslated region, for example, might alter the polyadenylation signal or lead to formation of an AUUUA type of sequence,both of which could lead to degradation of any transcribed proa l(I) mRNA (22,23).

REFERENCES 1. Bornstein, P., and Sage, H. (1980) Ann. Rev. Biochem. 49,957-1003. 2. Bomstein, P., and Sage,H. (1989) Progressin Nucleic Acid Researchand Molecular Biology 37,67106. 3. Cockayne, D., and Cutroneo, K.R. (1988) Biochemistry 27, 2736-2745. 4. Lichtler, A., Stover, M.L., Angilly, J., Kream, B., and Rowe, D.W. (1989) J.Biol. Chem. 264,30723077. 5. Stephenson, M.L., Krane, S.M., Amento, E.P., McCroskery, P.A., and Byrne, M. (1985) FEBS Letters 180,43-50. 6. Solis-Herruzo, J.A., Brenner, D.A., and Chojkier, M. (1988) J. Biol. Chem. 263, 5841-5845. 7. Ohta, A., and Uitto, J. ( 1987) Arthritis and Rheumatism 30,404-411. 8. Raghow, R., Gossage, D., Seyer,J.M., and Kang, A.H. (1984) J. Biol. Chem. 259, 12718-12723. 9. Bateman, J.F., and Peterkofsky, B. (1981) Proc. Natl. Acad. Sci. USA 78, 60286032. 10. Sandmeyer, S., Gallis, B., and Bomstein, P. (1982) J. Biol. Chem. 256, 5022-5028. 11. Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826. 12. Peterkofsky, B., and Prather, W. (1992) J. Biol. Chem. 267,5388-5395. 13. Majmudar, G., Schalk, E., Bateman, J., and Peterkofsky, B. (1988) J. Biol.Chem. 263, 5555-5559. 14. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412. 15. Takeda, K., Gosiewska, A., and Peterkofsky, B. (1992) J. Cell. Physiol. in press. 16. Chu, M-L., Myers, J.C., Bernard, M.P., Ding, J-F., and Ramirez, F. (1982) Nucleic Acid Res. 10, 5925-5934. 17. Yamada, K.M., and Olden, K. (1978) Nature 275,179-184. 18. Komblihtt, A.R., Vibe-Pedersen, K., and Baralle, F.E. (1984) Nucleic Acid Res. 12, 5853-5868. 19. Furth, J.J., Wroth, T.H., and Ackerman, S. (1991) Exptl. Cell Res. 192, 118-121. 20. Smith, B.D., and Marsilio, E. (1988) B&hem. J. 253,269-273. 21. Olsen, A.S., and Prockop, D.J. (1989) Matrix 9,73-81. 22. Nevins, J.R. (1983) Ann. Rev. Biochem. 52,441~466. 23. Shaw, G., and Kamen, R. (1986) Cell 46,659-667.

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