Molecular Cloning of mRNA from 3T3 Adipocytes REGULATION OF mRNA CONTENT FOR GLYCEROPHOSPHATE DEHYDROGENASE AND OTHER DIFFERENTIATION-DEPENDENT PROTEINS DURING ADIPOCYTE DEVELOPMENT* (Received for publication, March 11, 1983) Bruce M. SpiegelmanSB, Margaret Frankll, and Howard Green711 From the $Dana-FarberCancer Institute and the Department of Pharmacology, and the llDepartment of Physiology and Biophysics, Haruard Medical School, Boston, Massachusetts 021 I5 We have constructed a recombinant bacterial library containing cDNA prepared from mRNA of adipose 3T3 cells. We have screened this library by several methods and isolated colonies containing sequences complemen- tary to mRNAs for glycerophosphate dehydrogenase, two other major differentiation-dependent proteins of M, = 28,000 and 13,000, and actin. These recombi- nants were identified by hybrid selection of total adi- pocyte mRNA, translation in vitro and subsequent im- munoprecipitation, and two-dimensional electropho- resis of translated proteins. Three of the four cloned cDNAs hybridized to single adipocyte mRNA species of size close to that expected from the size of the poly- peptide it specifies, but the mRNA for glycerophos- phate dehydrogenase contained 3,550 bases, far larger than necessary to code for its polypeptide (Mr = 34,000). The increase in amount of mRNA during dif- ferentiation was 150-fold for the protein of M, = 13,000 and considerably greater for glycerophosphate dehydrogenase and the protein of M, = 28,000. The time course of mRNA accumulation was different for each of these mRNAs, indicating they do not respond synchronously during differentiation. In a process closely resembling the development of adipo- cytes in early life, certain clones of 3T3 cells can differentiate into adipocytes with high frequency incell culture (Green and Kehinde, 1974,1976). In this process, the cells develop enzyme activities related to fatty acid and triglyceride synthesis (Mackall et al., 1976; Kuri-Harcuch and Green, 1977; Coleman et al., 1978; Grimaldi et ai., 1978 Wise and Green, 1979), changes in cell morphology (Green and Kehinde, 1976; No- vikoff et al., 1980), and sensitivity to particular hormones (Rubin et al., 1977, 1978; Reed et al., 1977; Karlsson et al., 1979). These changes are part of aprogram in which the composition of cellular proteins is extensively revised (Sidhu, 1979; Spiegelman and Green, 1980) through changes in mRNAcontent(SpiegelmanandGreen, 1980; Spiegelman and Farmer, 1982). These changes are modulatedin response to lipogenic and lipolytic hormones (Spiegeiman and Green, 1980; Miller and Carrino, 1980; Weiss et al., 1980; Spiegelman and Green, 1981). ~~~ ” * 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. Supported by Grant AM 31405 from the National Institutes of Health. 11 Supported by Grant AM 31637 from the National Institutes of Health. We report here the construction and characterization of plasmids containing DNA complementary to several mRNAs whose concentration changes greatly during adipocyte differ- entiation. The cDNA clones are those coding for: 1) Glycer- ophosphate dehydrogenase, a key enzyme in triglyceride syn- thesis which provides the glycerophosphate backbone for ac- ylation. This enzyme accounts for about 2% of the soluble protein of adipose 3T3 cells. 2) A protein of M, = 13,000 that accounts for about 6% of soluble protein in the adipose cells (Spiegelman and Green, 1980). This is probably the same proteinastheone isolated from adipose 3T3-Ll cells by Ockner et al. (1982) and found to bind labeled oleic acid. It may be tentatively regarded as a fatty acid binding protein. 3) A protein of M, = 28,000 that accounts for about 5% of the soluble protein of adipose cells and whose function is un- known. Although scanning of gels had suggested that some of this protein was present in preadipose cells (Spiegelman and Green, 1980), we show here that no mRNA for this protein was detectable prior to adipose conversion. The study of changing mRNA content inadipose differen- tiation will be described for thesethree sequences whose protein products account for over 10% of the cytosolic protein of the adipocyte. EXPERIMENTAL PROCEDURES Cell Culture Condition.-3T3-F442A (Green and Kehinde, 1976) were grown in the Dulbecco-Vogt modification of Eagle’s medium supplemented with a mixture of 1% calf serum and 9% cat serum in order to minimize adipose conversion (Kuri-Harcuch and Green, 1978). For experiments, cells were trypsinized and 1 X lo5 cells were inoculated into 100-mm Petri dishes. To promote the formation of adipocytes, the same medium was supplemented with 10% fetal calf serum and 5 bg/ml of insulin; to maintain preadipocytes the supple- ment was 9% cat serum and 1% calf serum. Cultures were fed three times weekly. Confluence was attained approximately 4-5 days after inoculation. Protein Electrophoresis and Fluorography-Electrophoresis in the presence of SDS‘ was performed in polyacrylamide slab gels (Laemmli, 1970). Two-dimensional gel electrophoresis was performed according to the method of O’Farrell (1975). For fluorography, gels were impregnated with 2,5-diphenyloxazole according to the method of Bonner and Laskey (1974) and dried under vacuum. Kodak XR-2 film was prefogged (Laskey and Mills, 1975) and exposed to the gels at -70 “C. mRNA Isolation and Translation-Poly(A+) mRNA was isolated from adipocytes and preadipocytes by extraction with guanidine hydrochloride and chromatography on oligo(dT)-cellulose, as de- scribed previously (SpiegelmanandGreen, 1980; Spiegelman and Farmer, 1982). Translation of mRNA was carried out in nuclease- treated reticulocyte lysates (Pelham and Jackson, 1976) or wheat germ lysates (Roberts and Paterson, 1973). ~ The abbreviation used is: SDS, sodium dodecyl sulfate. 10083 by guest on April 21, 2018 http://www.jbc.org/ Downloaded from
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Molecular Cloning of mRNA from 3T3 Adipocytes REGULATION OF mRNA CONTENT FOR GLYCEROPHOSPHATE DEHYDROGENASE AND OTHER DIFFERENTIATION-DEPENDENT PROTEINS DURING ADIPOCYTE DEVELOPMENT*
(Received for publication, March 11, 1983)
Bruce M. SpiegelmanSB, Margaret Frankll, and Howard Green711 From the $Dana-Farber Cancer Institute and the Department of Pharmacology, and the llDepartment of Physiology and Biophysics, Haruard Medical School, Boston, Massachusetts 021 I5
We have constructed a recombinant bacterial library containing cDNA prepared from mRNA of adipose 3T3 cells. We have screened this library by several methods and isolated colonies containing sequences complemen- tary to mRNAs for glycerophosphate dehydrogenase, two other major differentiation-dependent proteins of M, = 28,000 and 13,000, and actin. These recombi- nants were identified by hybrid selection of total adi- pocyte mRNA, translation in vitro and subsequent im- munoprecipitation, and two-dimensional electropho- resis of translated proteins. Three of the four cloned cDNAs hybridized to single adipocyte mRNA species of size close to that expected from the size of the poly- peptide it specifies, but the mRNA for glycerophos- phate dehydrogenase contained 3,550 bases, far larger than necessary to code for its polypeptide (Mr = 34,000). The increase in amount of mRNA during dif- ferentiation was 150-fold for the protein of M, = 13,000 and considerably greater for glycerophosphate dehydrogenase and the protein of M, = 28,000. The time course of mRNA accumulation was different for each of these mRNAs, indicating they do not respond synchronously during differentiation.
In a process closely resembling the development of adipo- cytes in early life, certain clones of 3T3 cells can differentiate into adipocytes with high frequency in cell culture (Green and Kehinde, 1974,1976). In this process, the cells develop enzyme activities related to fatty acid and triglyceride synthesis (Mackall et al., 1976; Kuri-Harcuch and Green, 1977; Coleman et al., 1978; Grimaldi et ai., 1978 Wise and Green, 1979), changes in cell morphology (Green and Kehinde, 1976; No- vikoff et al., 1980), and sensitivity to particular hormones (Rubin et al., 1977, 1978; Reed et al., 1977; Karlsson et al., 1979). These changes are part of a program in which the composition of cellular proteins is extensively revised (Sidhu, 1979; Spiegelman and Green, 1980) through changes in mRNA content (Spiegelman and Green, 1980; Spiegelman and Farmer, 1982). These changes are modulated in response to lipogenic and lipolytic hormones (Spiegeiman and Green, 1980; Miller and Carrino, 1980; Weiss et al., 1980; Spiegelman and Green, 1981).
~~~ ”
* 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.
Supported by Grant AM 31405 from the National Institutes of Health.
11 Supported by Grant AM 31637 from the National Institutes of Health.
We report here the construction and characterization of plasmids containing DNA complementary to several mRNAs whose concentration changes greatly during adipocyte differ- entiation. The cDNA clones are those coding for: 1) Glycer- ophosphate dehydrogenase, a key enzyme in triglyceride syn- thesis which provides the glycerophosphate backbone for ac- ylation. This enzyme accounts for about 2% of the soluble protein of adipose 3T3 cells. 2) A protein of M , = 13,000 that accounts for about 6% of soluble protein in the adipose cells (Spiegelman and Green, 1980). This is probably the same protein as the one isolated from adipose 3T3-Ll cells by Ockner et al. (1982) and found to bind labeled oleic acid. It may be tentatively regarded as a fatty acid binding protein. 3) A protein of M , = 28,000 that accounts for about 5% of the soluble protein of adipose cells and whose function is un- known. Although scanning of gels had suggested that some of this protein was present in preadipose cells (Spiegelman and Green, 1980), we show here that no mRNA for this protein was detectable prior to adipose conversion.
The study of changing mRNA content in adipose differen- tiation will be described for these three sequences whose protein products account for over 10% of the cytosolic protein of the adipocyte.
EXPERIMENTAL PROCEDURES
Cell Culture Condition.-3T3-F442A (Green and Kehinde, 1976) were grown in the Dulbecco-Vogt modification of Eagle’s medium supplemented with a mixture of 1% calf serum and 9% cat serum in order to minimize adipose conversion (Kuri-Harcuch and Green, 1978). For experiments, cells were trypsinized and 1 X lo5 cells were inoculated into 100-mm Petri dishes. To promote the formation of adipocytes, the same medium was supplemented with 10% fetal calf serum and 5 bg/ml of insulin; to maintain preadipocytes the supple- ment was 9% cat serum and 1% calf serum. Cultures were fed three times weekly. Confluence was attained approximately 4-5 days after inoculation.
Protein Electrophoresis and Fluorography-Electrophoresis in the presence of SDS‘ was performed in polyacrylamide slab gels (Laemmli, 1970). Two-dimensional gel electrophoresis was performed according to the method of O’Farrell (1975). For fluorography, gels were impregnated with 2,5-diphenyloxazole according to the method of Bonner and Laskey (1974) and dried under vacuum. Kodak XR-2 film was prefogged (Laskey and Mills, 1975) and exposed to the gels a t -70 “C.
mRNA Isolation and Translation-Poly(A+) mRNA was isolated from adipocytes and preadipocytes by extraction with guanidine hydrochloride and chromatography on oligo(dT)-cellulose, as de- scribed previously (Spiegelman and Green, 1980; Spiegelman and Farmer, 1982). Translation of mRNA was carried out in nuclease- treated reticulocyte lysates (Pelham and Jackson, 1976) or wheat germ lysates (Roberts and Paterson, 1973).
~
The abbreviation used is: SDS, sodium dodecyl sulfate.
10084 Regulation of m R N A Content in Adipocyte Development
Construction of Adipocyte cDNA Clone 1,ihrap-cDNA was syn- thesized from mRNA with avian myeloblastosis virus reverse tran- scriptase (Payvar and Schimke, 1979), and the cDNA was made double-stranded with the Klenow fragment of Escherichia coli DNA polymerase I (Wickens et al., 1978). The conditions for both first and second strands were as described in Lemischka et a/. (1981). except the nucleic acids were initially denatured with 2 mM methyl mercury hydroxide at 25 "C for 10 min. This denaturation improved the size of the resulting product. Single-stranded regions were subsequently broken with nuclease S1 (Ullrich et al., 1977) and the double-stranded cDNA was tailed at 3' termini with approximately 15 deoxycytidylate residues using terminal transferase (Roychoudhyry et a/., 1976). Sim- ilarly, pHR 322 was linearized with restriction endonuclease I'stI and tailed with 15 deoxyguanidylate residues. The dC-tailed cDNA was annealed to the dG-tailed pRR 322 according to Villa-Kamaroff et a/. (1978). and the hyhrid plasmid was used to transform E. coli C600. Three thousand tetracycline-resistant colonies were obtained from 18 ng of input adipoc-yte-derived cDNA and 9 3 5 of these were sensitive to ampicillin, indicating the presence of new sequences inserted at the original Pstl site. Colonies were inoculated into broth containing 4 5 glycerol in 96-hole microtiter wells and were stored at -70 "C (Gergen et al., 1979).
Recombinant Bacterial Librap Screenint-Plasmids in bacterial colonies were amplified and transferred to Whatman 541 filters according to Gergen et al. (1979). Duplicate sets of these filters were hybridized for 24 h a t 37 "C to "2P-labeled cDNA (8 X lo5 dpm/l08- cm' filter) synthesized from total adipocyte or preadipocyte mRNA, or from a fraction enriched in glycerophosphate dehydrogenase mRNA obtained by electrophoresis in methyl mercury-agarose (see below). Hybridization buffer contained 50% formamide and 5 X SSC ( 1 X SSC = 0.15 M NaCI, 0.015 M sodium citrate, pH 7.5). Filters were washed in 2 X SSC at room temperature for 1 h and then 0.5 X SSC at 50 "C for 30 min. They were then exposed to film at -70 "C with intensifying screens.
Horterial Plasmid Isolation-Plasmids were isolated (with or with- out plasmid amplification with chloramphenicol) from bacteria by the SDS/NaOH method as described in Maniatis et nl. (1982a).
Ek~ctroph<JrC.si.s in Methyl Mercuw-Agarose Gels-Twenty micro- grams of poly(A+) mRNA from adipocytes were electrophoresed through 1.5% low melting point agarose gels containing 12.5 mM methyl mercury hydroxide (Bailey and Davidson, 1976). mRNA was extracted from 3-mm slices as described hy Maniatis et al. (1982b). and was translated or used as a substrate for cDNA synthesis in the presence of 0.1 mCi of ['"PIdCTP (1000 Ci/mmol) in a reaction volume of 25 p l .
Ei)rmnldeh\.de-Agarose Gels and Northern Blots-mRNA to be used for filter hybridization was quantitated by both absorhance at 260 nm and hybridization to [:'H]poly(lJ) (Bishop et nl., 1974). 0.5-2 pg of mRNA was denatured with formaldehyde, electrophoresed on 1.5rA agarose gels containing formaldehyde, and transferred to nitrocellu- lose according to the procedure of Seed and Goldberg as described in Maniatis et al. (1982~). Hybridization to plasmids labeled by nick translation (specific activity of 1 X 10"/pg of DNA; Maniatis et al., 1975) was carried out at 37 'C for 2-3 days in .50% formamide, 0.75 M sodium chloride, 0.15 M Tris-HCI, pH 7.5, 5 mM EDTA, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, 0.1 M sodium phosphate, pH 6.5,0.5% bovine serum albumin, 0.5% Ficoll400,0.5% polyvinylpyrrolidone, and 100 pg/ml of denatured salmon testis DNA. Blots were washed for 1-2 h in 0.1 X SSC, 0.1% SDS at room temperature, 0.5-1 h a t 50 "C in the same buffer, and were exposed to prefogged Kodak X-Omat film at -70 "C with a DuPont Lighting Plus intensifying screen. Bands on films were quantitated with an LKR soft laser densitometer equipped with a Hewlett-Packard peak integrator. Ry performing serial dilutions of mRNA, the response of film to radioactive bands on blots was determined to be linear from an optical density of 0.01 to 1.70 above a prefogged background of 0.30.
Material~-["~S]Methionine and [w: '~P]~CTP were obtained from New England Nuclear. Ampholines were from LKB Instruments, Inc., oligo(dT)-cellulose (type 3) from Collaborative Research Inc., and avian myeloblastosis virus reverse transcriptase from J. Beard, Life Sciences Inc. or Bethesda Research Laboratories. DNA polym- erase I (Klenow fragment), nuclease S1, and terminal transferase were from Rethesda Research Laboratories. Nick translation com- ponents and wheat germ mRNA translation systems were obtained from New England Nuclear and Rethesda Research Laboratories, respectively.
RESULTS
Isolation of Differentiation-dependent Cloned cDNAs T h e in vitro translation products obtained from the
poly(A+) mRNA used for the cDNA cloning are shown on a fluorograph of a polyacrylamide gel in Fig. 1. A comparison of the products translated from total adipocyte mRNA (slot a ) and total preadipocyte mRNA (slot b ) show large differen- tiation-dependent increases in several abundant proteins whose identities have been established earlier. These include malic enzyme (Malic), aldolase, glyceraldehyde 3-phosphate dehydrogenase ( G A P D ) , lactate dehydrogenase ( L I I H ) , and glycerophosphate dehydrogenase (GPD) (Spiegelman and Green, 1980). In addition, there are prominent increases in polypeptides of M , = 25,000, 28,000, 40,500, and 120,000 whose identity has not been established. The very abundant differentiation-dependent protein of M , = 13,000 (Spiegelman and Green, 1980) is not resolved from the front on this 115 polyacrylamide gel. There is also a decrease in the amount of actin. While these changes are the most prominent, many more changes in protein biosynthesis during differentiation are observed on two-dimensional gels (Sidhu, 1979).
Double-stranded cDNA was synthesized from the mRNA of adipocytes inserted into the PstI site of plasmid pRR 322 and used to transform E. coli C600. The average size of the inserted sequences was 750 base pairs, with a range of 200- 2900. Differentiation-dependent sequences were then identi- fied as described below.
I i
2 5 K - - c
L a L FIG. 1 (left). Translation of mRNA used for cloning. mRNA
was prepared from 90 100-mm cultures of preadipocytes or adipocytes (see "Experimental Procedures") by guanidine hydrochloride extrac- tion and oligo(dT)-cellulose chromatography. One microgram of mRNA was translated in 25 p l containing a rabbit reticulocyte lysate and 2-p l fractions were electrophoresed on an 11% SDS-polyacryl- amide gel. The gel was fluorographed, dried, and exposed to film at -70 "C for 16 h. Slot a. proteins synthesized from mRNA of adipo- cytes; slot b, proteins synthesized from mRNA of preadipocytes. 40..5K, 120K, B K , and 25K, denote position of unidentified proteins in kilodaltons.
FIG. 2 (right). Filter hybridization of DNA from bacterial colonies. Replicate sets of filters containing lysed bacterial colonies (Gergen et a/., 1979) were hybridized to equivalent amounts of :12P- laheled cDNA synthesized from total preadipocyte mRNA or total adipocyte mRNA. Filters were washed for 2 h with 2 X SSC at room temperature, 1 h at 0.5 X SSC at 50 "C, and were exposed to Kodak XAR film for 2 days at -70 "C with a DuPont Cronex intensifying screen. Arrou indicates the position of a colony yielding a much stronger signal from adipocyte cDNA than from preadipoc.yte cDNA.
Regulation of mRNA Content in Adipocyte Development 10085
Isolation of cDNA Clones for the Proteins of M , = 28,000 and 13,000-PParallel sets of filters containing bacterial plas- mid DNA were hybridized to '"P-labeled cDNA synthesized from total preadipocyte or adipocyte poly(A+) RNA. As shown in Fig. 2, most colonies which gave a signal from preadipocyte cDNA also reacted with adipocyte cDNA, indicating that most mRNA species do not show a marked differentiation depend- ence. However, approximately 5% of the colonies reacted much more strongly with cDNA from the adipocytes (see arrows in Fig. 2). Plasmid DNA was isolated from several colonies showing the strongest differentiation dependence, immobilized on a nitrocellulose filter, and utilized to select hybridizable mRNA from a total adipocyte mRNA prepara- tion. As shown in a fluorograph of the reticulocyte-based translation products (Fig. 3A), plasmid pAd-5, with an insert size of 400 bases, selected an mRNA which directs translation of a protein of M , = 13,000 incompletely resolved from globin translated from endogenous mRNA (slot e). However, when mRNA hybrid-selected by pAd-5 was translated in a wheat germ lysate, the selected translation product clearly co-mi- grated with a major translation product of total adipocyte but not preadipocyte mRNA (Fig. 3R). Plasmid pAd-20, with an insert size of 940 bases, selected an mRNA which translates as a protein of M , = 28,000 (Fig. 3A, slot f). This protein co- migrated with a major protein in translations of total adipo- cyte mRNA (Fig. 3A, slot b). Neither of these proteins was synthesized when no mRNA was added or if control plasmid pRR 322 lacking an insert was used to hybrid-select the same
A B a b c d e f a b c "
.u. I - 2 8 K
FIG. 3 . Hyhrid selection of mRNAs corresponding to differ- entiation-dependent proteins of M , = 13,000 and 28,000. I'oly(A+) mllS.4 f r o m adipocytes was hyhridized to linearized im- mobilized recomhinant plasmids. After washing, the mRNA was eluted and translated (Miller et al., 1983). Translation products were separated on an I l r ; ( A ) or I:jrE ( H ) SDS-polyacrylamide gel. fluo- rographed. and exposed to film at -50 "C. A, translations using reticulocyte lysates are total preadipocyte mRNA (slot a) , total adi- pncyte mRNA Islot h) , no mRNA added (slot c), mRNA selected hy control PHR 322 plasmid (s lnt d ) , mRNA selected hv PAD-5 plasmid ( s l o t ( 3 ) . and mRNA selected hy PAD-20 plasmid ( s lo t /). H , transla- tions using wheat germ lysates are total preadipocyte mRNA ( s k ~ t a) , total adipocyte mRNA ( s l o t h ) , and mRNA selected hv pAd-5 plasmid (slot c). 1dK and W K denote position of' unidentified proteins in kilodaltons.
mRNA (Fig. 3A, slots c and d) . Peptide mapping (data not shown) indicates that the proteins translated after hybrid selection corresponded to the differentiation-dependent adi- pocyte proteins of equivalent size translated from total cell mRNA (Spiegelman and Green, 1980).
Isolation of a cIINA Clone for Glycerophosphate Dehydro- genase-No cDNA clone for the protein glycerophosphate dehydrogenase was isolated in the experiments described above. Since this enzyme increases greatly during adipose conversion and is commonly used as a differentiation marker, another attempt was made to obtain a cDNA clone encoding the enzyme. mRNA of adipose cells was fractionated by electrophoresis in agarose gels containing the denaturant methyl mercury hydroxide. After preliminary runs to deter- mine the approximate mobility of glycerophosphate dehydro- genase mRNA, the region corresponding to 2000-4500 bases was excised and the mRNA from each fraction was isolated and translated. A product with the electrophoretic mobility of glycerophosphate dehydrogenase was apparent from mRNA fractions with a size of approximately 3500 bases (Fig. 4, slots m and n). This protein was precipitated by antiserum to purified muscle glycerophosphate dehydrogenase (not shown). Shown on the same fluorogram is a protein with the mobility of actin, translated from mRNA with a size of approximately 2200 hases (slots d and e) consistent with the known size of this mRNA (Cleveland et al., 1980).
"'P-labeled cDNA was synthesized from the partially puri- fied glycerophosphate dehydrogenase mRNA and the bacte- rial library was screened. Two colonies not previously selected reacted with this probe. Plasmid from one of these colonies
2000 3000 4000 : bases a b l c d e f g h i ] I k I m n o x r s t u v
"_ "
- b .- ..
FIG. 4. Fractionation of adipocyte mRNA i n agarose gels containing methyl mercury. 20 pg of mRNA from adipocytes was denatured with 12.5 mM methyl mercury hydroxide and electropho- resed on 1.5% gels of low melting point agarose according to Bailey and Davidson (1976). The RNA was isolated from successive fractions (see "Experimental Procedures") and translated using a reticulocyte lysate. The products were electrophoresed on a 13% SDS-polyacryl- amide gel, fluorographed, and exposed to film for 2 days a t -70 "C. Rands with the electrophoretic mobility of authentic glycerophos- phate dehydrogenase were ohtained from slots m and n. The corre- sponding mRNA was estimated at 3400-3500 bases, using ribosomal RNA of 18 S (2000 hases) and 28 S (5000 bases) as standards. Slots a-t. mRNA fractions cut from methyl mercury gel; slot u, no mRNA added: slot L', unfractionated adipocyte mRNA. E , endogenous trans- lation hand: GPD, glycerophosphate dehydrogenase.
10086 Regulation of mRNA Content in Adipocyte Development
(designated pGPD-1) had an insert size of 300 bases and selected an mRNA-directing translation of a polypeptide co- migrating with authentic glycerophosphate dehydrogenase (Fig. 5A, slots c and d). The identity of this polypeptide was confirmed by quantitative precipitation with antiserum spe- cific for glycerophosphate dehydrogenase (slots f and g; see also Spiegelman and Green, 1981), but not with nonimmune serum (slot e). This polypeptide also co-migrated with im- munoprecipitated mouse adipocyte glycerophosphate dehy- drogenase in two-dimensional electrophoresis by the O'Farrell procedure (data not shown).
Isolation of Clones Coding for Adipocyte Actin-The cDNA library prepared from adipose 3T3 cells was screened with "'P-labeled, PstI-excised insert from a chicken 6-actin clone (Cleveland et al., 1980). A single colony showed a strong reaction and the plasmid from this colony, pAct-1, selected an mRNA-directing translation of a protein with the electro-
A a b c d e f g a
4
GPD- .... &?-
phoretic mobility of actin (Fig. 5R). Two-dimensional electro- phoresis indicated that this plasmid, which has an insert of 1200 bases, selected mRNA for both 6- and y-actin (Fig. 5, C and I ) ) .
Regulation of Specific mRNAs during the Differentiation of 3T3 Adipocytes
The cloned probes described above were used to examine, by Northern blotting, the changes in specific mRNA content during adipocyte differentiation. Equivalent amounts of poly(A') mRNA quantitated by absorbance a t 260 nm (and in some experiments, hybridization to ["H]poly(U)) were elec- trophoresed through 1.5% agarose, transferred to nitrocellu- lose, and hybridized to the four probes. I t can be seen in Fig. 6 that there was a single mRNA for glycerophosphate dehy- drogenase with a size of 3,550 bases. This mRNA could first be detected 1 day after the cells became confluent, reached a
B b c d e f C -
. -
7 " . . . **
D
.
t
FIG. 5. Hybrid selection of mRNAs for glycerophosphate dehydrogenase and actin. Procedures were the same as for Fig. 3. Translation products from pGPD-1 were immunoprecipitated with chicken antiserum to glycerophosphate dehydrogenase or nonimmune serum as described by Spiegelman and Green (1981), electropho- resed through 11% ( A ) or 9% ( R ) polyacrylamide gels, and fluorographed. Translations of putative actin mRNAs were subjected to one-dimensional ( R ) and two-dimensional electrophoresis (C and I); O'Farrell, 1975). Isoelectric focusing gels contained ampholines of pH 3-10 and 5-7 in ratios of 40:60. Signal obtained from pBR-322 control selection of mRNA appears stronger in glycerophosphate dehydrogenase selections ( A ) compared to actin selections ( H ) because of the need to expose film longer (48 uersus 12 h) to obtain a clear signal from less abundant glycerophosphate dehydrogenase mRNA. A, translation using reticulocyte lysate and immunoprecipitation of glycerophosphate dehydrogenase. Slot a, total preadipocyte mRNA; slot b, total adipocyte mRNA; slot c, mRNA selected by control pRR 322 plasmid; slot d, mRNA selected by pGPD-1 plasmid; slot e, same mRNA as slot d , products immunoprecipitated with 5 pI of nonimmune sera; and slots f and g, same mRNA as slot d, products precipitated with 1 p1 (slot f ) or 5 p1 (slot g ) of chicken antiserum to glycerophosphate dehydrogenase. GPD, glycerophosphate dehydrogenase marker. R, translation of hybrid-selected actin mRNA using reticulocyte lysate. Slot a, preadipocyte mRNA; slot b, adipocyte mRNA; slot c, no mRNA; slot d, mRNA selected by pBR 322 plasmid and slots e and f , mRNA selected by pAct-1 plasmid (slot e ) or chicken actin plasmid (slot f ) . C, two-dimensional gel electrophoresis of translation products from total preadipocyte mRNA. 11, two-dimensional gel electrophoresis of products from mRNA selected by pAct-1 plasmid. 6 and y mark the mobility of authentic 6- and y-actins from 3T3 preadipocytes as previously described (Spiegelman and Farmer, 1982). E marks the endogenous translation product. Note that mRNA for hoth 6- and y-actin are hybrid-selected, as well as a small amount of material slightly more acidic than 6-actin.
Regulation of m R N A Content in Adipocyte Development 10087
GPD Act in 28K 13K DAYS A F T E R CONFLUENCE:-I 0 I 2 3 4 -I 0 I 2 3 4 -I 0 I 2 3 4 -101 2 3 4
- l0,OOO
- 3,550
-2,150 a - 1,050 rl) -650
FIG. 6. Blots showing specific mRNA levels during adipocyte differentiation. Cells were maintained in the preadipose state in medium containing 9% cat serum, 1% calf serum. Two days before confluence ( 3 days after plating), medium was changed to 10% fetal calf serum and 5 g / m l of insulin. Poly(A+) mRNA was isolated from 10-30 100-mm cultures of :3T3-F442A cells daily from 1 day before confluence to 4 days after confluence. Two micrograms of poly(A+) were denatured with formaldehyde and electrophoresed on a 1.5% agarose gel containing formaldehyde. RNA was transferred to nitrocellulose and hybridized to plasmids containing characterized inserts radiolabeled with :v2P by nick translation (Maniatis et ol., 1975) to a specific activity of 10“ dpm/pg of DNA. Rlots were washed and autoradiographed as described under “Experimental Procedures.” Day 0 = day on which cultures hecame confluent. The size of the major mRNA bands is shown at right, as determined from standards of ribosomal RNA (5000 and 2000 bases), X phage DNA digested with HindIII, and @X174 DNA digested with HaeIII. mRNA for (;PI) and 28K were blotted onto parallel strips of nitrocellulose from the same gel while Actin and 13K were blotted from separate gels. GPD. glvcerophosphate dehydrogenase; 2RK and 13K denote position of unidentified . . . .
proteins in kilodaltons.
maximum at 2-3 days postconfluence and decreased by 22% at day 4. It was not detectable in growing preadipocytes, even when the films were greatly overexposed. An earlier study demonstrated that this mRNA could not be detected by in citro translation of message from cells maintained for several days in the preadipose state (Spiegelman and Green, 1980). The mRNA for the protein of M, = 28,000 contained 1,050 bases. I t was also undetectable before differentiation began but became detectable at 2 days after confluence and contin- ued to increase to 4 days postconfluence. From the sensitivity of the detection method, we estimate that the amount of these mRNAs in preadipocytes must be less than 0.5% of the maximum attained in adipocytes and is likely to be consider- ably less.
The mRNA for the protein of M, = 13,000 is 650 bases in length. I t was seen as a very faint band in growing preadipo- cvtes, hut t,he amount increased very markedly at 1 day after confluence and rose slowly between days 2-4 postconfluence. Densitometry of prefogged films indicated that the quantita- tive increase of this mRNA during differentiation was 150- fold. The linear relationship between amount of mRNA and film response was established by densitometric scanning of autoradiograms containing a series of dilutions of the adipo- cyte mRNA (see “Experimental Procedures”). These results were confirmed by direct scintillation counting of radioactive bands after excision from blots.
Actin mRNA (2250 bases) decreased 2-3-fold during differ- entiation. Most of this decrease occurred by 1 day after the cells become confluent, as had been observed earlier in studies of cellular biosynthesis (Spiegelman and Farmer, 1982). There was also a very faint band a t approximately 10 kilobases which could represent a polyadenylated nuclear precursor for the actin mRNA. Quantitatively, the decrease in actin mRNA was somewhat smaller than the 4-5-fold change previously reported (Spiegelman and Farmer, 1982); this is due to the presence of insulin in the culture medium (data not shown).
The differences observed between preadipocytes and differ- entiated cells are not simply due to the resting state of the latter. In addition to growing preadipose cells, mRNA from
Days After Confluence FIG. 7. Time course of changes in specific mRNA levels
duringdifferentiation. The blots shown in Fig. 6. were quantitated hy scanning prefogged autoradiograms with an LKR soft laser den- sitometer equipped with an automatic peak integrator. All measure- ments were performed within the film’s range of linear response with respect to radioactivity (see “Experimental Procedures”). The results
U, glycerophosphate dehydrogenase; O ” - o , 1:bkDa protein; are expressed as a percentage of the maximal signal. A-A, actin;
A-A, 28-kDa protein.
resting preadipose cells was extracted and blotted with all four probes and yielded results identical with those from growing cells.
The amounts of these mRNAs as determined from densi- tometric tracings of the blots shown in Fig. 6 are plotted in Fig. 7. It is apparent from these curves that each mRNA has its own characteristic time course of accumulation. While mRNA for glycerophosphate dehydrogenase and the protein of M, = 13,000 begin to appear simultaneously, the differences between them on days 3 and 4 are significant and could also be demonstrated by hybridizing a mixture of the two clones
10088 Regulation of mRNA Content in Adipocyte Development
TABLE I Sizes and quantitative changes in specific mRNAs during 3T3
adipocyte differentiation The size of coding region of mRNAs was estimated from the size
of the polypeptide, assuming 3 bases codes for 110 Da of polypeptide. The size of the noncoding region was estimated as the total size of the mRNA (see footnotes b and c below) minus the estimated coding region. The fold change during differentiation was estimated by soft laser densitometer scanning of prefogged films exposed to Northern blots.
Size of Size of mRNA Protein Polypep- mRNA mRNA noncod- Change during
differentiation tide size" size codlng region ing re-
Polypeptide size estimated from SDS-polyacrylamide gels. 'mRNA size estimated from Northern blots of formaldehyde-
mRNA size estimated from mRNA mobility in methyl mercury- agarose gels.
agarose gels assayed by in vitro translation.
simultaneously to the same nitrocellulose blot. These results, therefore, indicated a substantial but not complete synchrony in the response of these 2 mRNAs.
The differences between the responses of mRNA for gly- cerophosphate dehydrogenase and the M , = 13,000 protein and the responses of the other two mRNAs are very obvious. The increase in mRNA for the protein of M , = 28,000 was delayed by 1-2 days, in comparison to the mRNA for glycer- ophosphate dehydrogenase, while actin underwent an early and abrupt decrease (Fig. 7).
The sizes of these mRNAs and their relative amounts of coding and noncoding sequence are summarized in Table I. The most striking feature is the highly unusual size of the mRNA for glycerophosphate dehydrogenase mRNA. This protein of M, = 34,000 polypeptide has an mRNA of 3,500 bases, as determined by mobility in both formaldehyde gels and methyl mercury-agarose gels. It can be estimated that only 930 bases are required to code a polypeptide of this size, leaving some 2,500-2,600 bases for noncoding sequence. As has been noted earlier (Cleveland et al., 1980), actin has a somewhat larger than usual noncoding segment (950-1,000 bases) while the proteins of M, = 13,000 and 28,000 have mRNAs which contain a typical noncoding segment of ap- proximately 200-300 bases.
DISCUSSION
During adipose differentiation, changes in amounts of mRNAs complementary to the cloned sequences were very large. In the case of glycerophosphate dehydrogenase, this had been anticipated from previous studies of enzyme activity (Wise and Green, 1979; Pairault and Green, 1979) and protein content (Spiegelman and Green, 1980). The increase in mRNA for the proteins of M, = 13,000 and 28,000 was also very great, 150-fold for the former and even greater for the latter. On SDS-polyacrylamide gels, the changes at the protein level seemed smaller (Spiegelman and Green, 1980), suggest- ing the presence of co-migrating, nonidentical proteins of similar molecular weight. While we do not know the function of the M , = 28,000 protein, the protein of M , = 13,000 may
be the differentiation-dependent fatty acid binding protein recently described in rat liver and 3T3 adipocytes (Ockner et al., 1982) on the basis of size and its accumulation during differentiation.
The size of the mRNA for glycerophosphate dehydrogenase is unusually large (3,500 bases) for a polypeptide having a molecular weight of 34,000. This large size does not seem to be an artifact of one particular type of measurement, since the results are virtually identical when the mRNA is dena- tured by methyl mercury and assayed by in vitro translation, or denatured with formaldehyde and assayed by hybridization to a cloned probe. I t can be estimated that the noncoding region must be approximately 2,600 bases, almost three times the size of the coding segment (Table I). A more typical size for the noncoding region of eukaryotic mRNA is 100-400 bases (Lewin, 1980). While unusual, the very large noncoding region of glycerophosphate dehydrogenase mRNA is not unique. Fatty acid synthetase in the goose liver and uropygial gland has a noncoding region of 9,000 bases (Morris et al., 1982). Although the function of most of the noncoding region of mRNA is unknown, the presence of these large noncoding segments in mRNA for two key regulatory enzymes of tri- glyceride synthesis (fatty acid synthetase and glycerophos- phate dehydrogenase) suggests that they may exert an impor- tant though presently undetermined regulatory function.
The time course of change of the different mRNA species indicates that although each is linked to the differentiation of the adipocyte, they are not under tight coordinate control. This may not apply to all lipogenic proteins participating in adipocyte differentiation since certain enzyme activities seem to increase coordinately (Mackall et al., 1976; Coleman et al., 1978). However, the varying kinetics of appearance of the mRNAs described here is consistent with our earlier findings that the response of several differentiation-dependent pro- teins could be quantitatively uncoupled from each other by deprivation of biotin or insulin (Spiegelman and Green, 1980) or by the presence of exogenous agents affecting cellular content of cyclic AMP agents (Spiegelman and Green, 1981).
The large increases in amounts of mRNA for differentia- tion-specific proteins suggests that the corresponding genes undergo large changes in transcription rate. The marked delay in accumulation of mRNA for the protein of M , = 28,000, relative to the other two mRNAs, raises the question whether the corresponding gene might become active only some time later than the other two. Such a sequence of gene activation could either be the result of the intrinsic nature of the pro- gram, or a late gene could require, for its activation, the products of genes activated earlier in the process of differen- tiation.
Acknowledgments-The authors thank Dr. Bryan Roberts for ad- vice and the facilities he provided for construction of the adipocyte cDNA library, and Drs. Philip Sharp and Ihor Lemischke for advice and materials provided at the initial stages of this project. The technical assistance of Carol Ginty is gratefully acknowledged for the isolation of the mouse adipocyte actin close.
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proteinsfor glycerophosphate dehydrogenase and other differentiation-dependent Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content
1983, 258:10083-10089.J. Biol. Chem.
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