Regulation of Calbindin-DasK Gene Expression by 1,25-. Dihydroxyvitamin DB Is Correlated to Receptor Occupancy*. (Received for publication, June 19, 1986).
Vol. 261, No. 36, Issue of December 25, pp. 16943-16947.1986 Prinred in U.S A .
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc
Regulation of Calbindin-DasK GeneExpression by 1,25Dihydroxyvitamin DBIs Correlated to Receptor Occupancy* (Received for publication, June 19, 1986)
Georgia Theofan,Anh P. Nguyen, and AnthonyW. Norman$ From the Division of Biomedical Sciences and Department of Bwchemitry, University of California, Riverside,California 92521
1,25-Dihydroxyvitamin D3(1,25-(OH)2D3)induces proposed to mediate the primary biological action of 1,25the synthesis of calcium binding protein (calbindin(OH)2D3in chick intestine, namely calcium transport (6-8). D28~)and calbindin-DzeK-mRNA in the chick intestine. 1,25-(OH)2D3receptor occupancy has been correlated to calWe have examined the relationship between nuclear bindin-D=K induction (9), and calbindin-DZsK levelscorrelate uptake of 1,25-(OH)2D3,1,25-(OH)zD3receptor occu- with intestinal calcium absorption (8).1,25-(OH)zD3has been pancy levels, and transcription of the ~ a l b i n d i n - D ~shown ~ ~ to stimulate RNA synthesis in intestinal mucosa (10) gene and found all three parameters to be highly cor- and specifically induces calbindin-DpSK-mRNA as determined related. All three events occur rapidly (within 15-30 (11) and by hybridization analyses by cell-free translation min) following a single doseof 6.5 nmolof 1,25(12, 13). (OH)2D3 to vitaminD-deficient chicks, reaching peak In this study we wanted to directly measure calbindin-D=K values by 1-2 h; by 4 h, values of all three parameters gene transcription in response to 1,25-(OH)2D3by nuclear start to decline. Ca1bindin-DzeK-mRNA begins to accumulate by 3-5 h but does not peak until 12 h follow- transcription (run-off) assays. We have correlated calbindining hormone administration. The levels of calbindin- D2sK gene transcription with 1,25-(OH)2D3nuclear uptake DZsK start to increase by 5-8 h and do not peak until and 1,25-(OH)2D3receptor occupancy. We have also examined 4 8 h after the 1,25-(OH)& dose. These observations the temporal relationships between gene activity, mRNA acsuggest thatpost-transcriptionalregulatorymechacumulation, and synthesis of calbindin-DmK. nisms may be involved. Measurements of basal levels of ~ a 1 b i n d i n - D gene ~ ~ ~transcription show that there is MATERIALS ANDMETHODS virtually no transcription in the vitamin D-deficient Animal Treatments-White Leghorn cockerels (Lakeview Farms, chick intestine, and a12-fold induction in the intestine Lakeview, CA) were raised from hatch on either a standard vitamin of vitamin D-replete chicks. This basal level of tran- D-deficient rachitogenic diet (14) or a standard vitamin D-replete scription in vitamin D-replete chick intestine is not commercial diet (0. H. Kruse Grain and Milling, Ontario, CA). inhibited by cycloheximidepretreatment.These re- Vitamin D-deficient chicks treated with 1,25-(OH)&&received a sults confirm the thesis that a major component of the single intramuscular dose of 6.5 nmol of 1,25-(OH)zD3in 0.2 ml of mechanism of action of 1,25-(OH)2D3is functioning as ethano1:propanediol (1:l) at various times prior to killing. Vitamin D-replete chicks treated with cycloheximide (Sigma) received a dose a steroid hormone, effected through the direct action of the seco-steroid-receptor complex on the initiation of 600 pglchick 1 h prior to killing. At the time of killing, duodenal mucosa was collected from chicks and used either immediately for of transcriptionof specific genes.
the [3H]1,25-(OH)2D3nuclear uptake, receptor occupancy, and in vitro transcription studies, or snap frozen in liquid NPto be analyzed at a later date for calbindin-DBK-mRNAand calbindin-DBK levels. Preparation of Nuclei from Duodenal Mucosa-Duodenal mucosa The generally accepted mechanism of action of steroid (1g) was washed twice in 10 ml of phosphate-buffered saline, calcium hormones involves the high affinity binding of the steroid to and magnesium free and then once in 10 ml of lysis buffer (10 mM a specific, soluble intracellular binding protein, the interac- Tris-C1, pH 7.9,lO mM NaCI, 5 mM MgC12, 1mM dithiothreitol) with tion of this steroid-receptor complex with specific DNA se- 5-min spins(Beckman TJ6, topspeed) in between washes.The pellet quences, and the subsequent initiation of transcription of was resuspended in 10 ml of lysis buffer and 500 pl of 10% Nonidet specific genes (see Refs. 1 and 2 for review). There is accu- P-40 and was homogenized in a Potter-Elvehjem homogenizer. The mulating evidence that the biologically active metabolite of homogenate was centrifuged for 5 min at 4 “C at 2500 rpm (Sorvall), vitamin D3, 1,25-(OH)2D31functionsina similar manner. the pellet was resuspended in 1 ml of lysis buffer and centrifuged again. The final pellet was termed the “nuclear pellet.” 1,25-(OH)2D3 bindsto a specific, high affinity intracellular pH]l,25-(OH)93 Nuclear Uptake-Vitamin D-deficient chicks receptor, with properties similar to other steroid receptors (3, were dosed intramuscularly with 6.5 nmol of 1,25-(OH)2D3containing 4). The 1,25-(OH)2D3receptor is widely distributed in many 0.1 pCi of [3H]1,25-(OH)2D3(Amersham Corp., 100 Ci/mmol) a t tissues of the chick; many of these same tissues also produce various times prior to killing. Nuclei were prepared as described a 28,000-dalton vitamin D-dependent calcium binding protein above, except that an aliquot of the homogenate was removed prior (CaBP, now termed calbindin-DzsK) (5). Calbindin-DZsKis to centrifugation to determine DNA content by the method of Burton (15). The nuclear pellet was washed twice in lysis buffer followed by * This work was supported by United States Public Health Service 5-min centrifugation. The pellet was dissolved overnight in 1 mlof Grant AM-09012-022. The costs of publication of this article were TS-2 tissue solubilizer (Research Products International) and the defrayed in part by the payment of page charges. This article must tritium contentwas determined by liquid scintillation counting. The therefore be hereby marked “advertisement” in accordance with 18 data are expressed as cpm/pg DNA using nuclei from the intestines of three chicks per time point. U.S.C. Section 1734 solely to indicate this fact. Measurement of I,25-foH.&3 Receptor Occupancy-Vitamin D$ To whom correspondence should be addressed. The abbreviations used are: 1,25-(OH),D3,1,25-dihydroxyvitamin deficient chicks were dosed intramuscularly with 6.5 nmol of 1,25D3; CaBP, calcium binding protein, now termed calbindin-DBK; (OH)zD3at various times prior to killing. Quantitation of unoccupied and occupied 1,25-(OH)2D3receptors in the duodenal mucosa was ELISA, enzyme-linked immunosorbent assay.
16943
16944
Calbindin-DP~K Gene Expression Is Regulated by 1,25-(OH),D,
performed by exchange assay using L-1-tosylamido-2-phenylethyl chloromethyl ketone to block unoccupied sites aspreviously described (9). Determination of Calbindin-DzsKGene Transcription-Nuclei were prepared as described above from duodenal mucosa of vitamin Ddeficient chicks treated for various times with 1,25-(OH)2D3.The nuclear pellets (100-200 pl) were incubated with transcription mixture containing 80-125 pCieach of [cY-~’P]CTP and [cY-~’P]GTP (3000 Ci/mmol, Amersham Corp.), treated with DNase I, protease K, and phenol-extracted as described by Spindler et al. (16). The RNA was further purified by the method of Evans et al. (17). Nitrocellulose filters (BA-85, Schleicher & Schuell) containing immobilized EcoRIwere cut pBR322 DNA or EcoRI-cut pBR322-calbindin-D~~-cDNA hybridized with the purified RNA and the filters were washed as is a described by Spindler et al. (16). pBR322-calbindin-D~~~cDNA 1700-base pair calbindin-DzsK-cDNA inserted into the PstI site of pBR322, which hybridizes in the 3’ untranslated region of the message. It was identified by screening a cDNA library with shorter cDNA probes already identified (18). Radioactivity bound to the filters was determined by counting in 4 ml of Filtron-X (National Diagnostics) for 20 min. Hybridizations typically contained input radioactivity of 3 to 25 X lo6 cpm. The amount of calbindin-DzsKmRNA synthesis (in ppm) was determined by subtracting the radioactivity of the filter containing pBR322 DNA (90-95 cpm) from the radioactivity bound to filterscontaining pBR322-calbindin-DZeKcDNA (100-200 cpm) and dividing by the total [32P]RNAinput. Determination of Total Calbindin-DzwmRNA Leuels-RNA was isolated from a five-chick duodenal mucosal pool by the method of Chirgwin et al. (19), and dot blot hybridization analyses were performed as previously described (20). Following autoradiography, the dots on the autoradiographs were quantitated by densitometric scanning. levels Determination of Calbindin-Dmkuek-ca1bindin-DzeK were quantitated in cytosol preparations from a five-chick duodenal mucosal pool by ELISA assay as described (21). Total protein was measured by Biuret assay. Statitics-Significance at various times after 1,25-(OH)ZD3treatment compared to untreated controls was determined by t test.
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FIG. 2. 1,25-(OH)& receptor occupancy levels. Chicks were dosed with 6.5 nmol of l,25-(OH)2D3and receptor occupancy was measured by exchange assay. Each value is the mean f S.E.of = 3 individual chicks. Significance triplicatedeterminationsinn levels are asdescribed in the legend to Fig. 1.
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RESULTS
[3H]1,25-(OH)2D3 administeredintramuscularly to intact chicks was rapidly taken up by intestinal nuclei, as shown in Fig. 1. Uptake of 3H was significant by 15 min ( p < 0.05), reached a plateau from 90 min to 3 h, and then started to decline by 4 h. The time course of 1,25-(OH)2D3receptor occupancy shows similar kinetics (Fig. 2). After in vivo administration of 1,25(OH)2D3,there was a significant amount of occupied receptor by 15 min ( p < 0.05), which reached a peak at 1 to 2 h corresponding to 75% receptor occupancy. Occupied receptor levels started to decline by 4 h. This increase in occupied
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TIME AFTER 1.25(OH)203 (hours)
FIG. 3. Calbindin-DzBK gene transcription. I n uitro transcription was measured in isolated nuclei following a dose of 1,25-(OH),D, as described under “Materials and Methods.” Each value is the mean f S.E. of three to six determinations. Significance levels are as described in the legend to Fig. 1.
receptor was accompanied by a concomitant decrease in unoccupied 1,25-(OH)2D3receptors (data not shown). When ~ a 1 b i n d i n - Dgene ~ ~ ~transcription was measured in nuclei in vitro following administration of 1,25-(OH)2D3to chicks, a similar response profile was again observed (Fig. 3). Chicks raised in the absence of vitamin D3 showedno endogenous transcription of the calbindin-DzaKgene.Levels of ~ a 1 b i n d i n - Dgene ~ ~ ~transcription were elevated by 15 min, reaching significance by45 min ( p < 0.01). The peak of transcription occurred 2-3 h following administration of hormone and then started decline to at 4-6 h, approaching control levels by 12 h. Fig. 4 shows that there was good correlation among [3H] 1,25-(OH)& nuclear uptake, 1,25-(OH)2D3receptor occupancy, and ~ a 1 b i n d i n - Dgene ~ ~ ~transcription. Fig. 4A shows the correlation between [3H]1,25-(OH)2D3 nuclear uptake and receptor occupancy, with a correlation coefficient of 0.77. There was an excellent correlation between nuclear uptake and calbindin-DBKgene transcription (Fig. 4B) with a correlation coefficient of 0.96, and between receptor occupancy and calbindin-DZ8K gene transcription (Fig. 4c) with a correlation TIME AFTER ~ 2 5 ( O H ) 2 0 3(hours) coefficient of 0.87. FIG. 1. Nuclear uptake of [SH]1,25-(OH)aDs.Chicks were Fig. 5 shows the time course of accumulation of calbindindosed with 6.5 nmol of l,%-(OH)& containing 0.1 pCi of [3H]1,25- DZsK-mRNAas measured by dot blot hybridization of total (OH)2D3,and nuclear uptake in cpmlpg DNA was determined. Each value is the mean f S.E. of determinations using duodenal nuclei intestinal RNA following a dose of 1,25-(OH)zD3.Calbindinfrom six chicks. Significance from control (zero time): a, p < 0.05; b, DZaK-mRNAlevels start to increase by 3-5 h ( p < 0.01 at 3 h), reaching a peak at 12 h.Calbindin-DzaK-mRNAlevels then p < 0.01; c, p < 0.001.
Calbindin-DzsKGene Expression Is Regulated by 1,25-(0H)2D3 100
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TIME AFTER 1.25(0H)2D3 I h r d
FIG.5. Dot blot analysis of calbindin-DzsK-mRNAaccumulation. Chicks were dosed intramuscularly with 6.5 nmol of 1,25(OH),D3 and RNA was isolated from the duodenal mucosa for dot blot analysis of calbindin-DzaK-mRNA.Autoradiographs of dot blots were densitometrically scanned and the densities were expressed relative to the 12-h peak, which was arbitrarily set at 100%. Each value is the mean f S.E. of three determinations.
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TIME AFTER 1,25(0H)2D3 ( h r d
FIG. 6. Calbindin-DzaK accumulation. Calbindin-DwK levels in chick duodenal cytosol were measured by ELISA assay following an intramuscular dose of6.5 nmol of 1,25-(OH)zD3.Each value is the mean f S.E. of three determinations.
I2
c
T
CaBP GENE TRANSCRIPTION (ppm)
FIG.4. Correlation between the parameters measured in Figs. 1-3. A, correlation between [3H]1,25-(OH)pDgnuclear uptake and 1,25-(OH)& receptor occupancy. B, correlation between [3H] lr25-(OH)2D3nuclear uptake and cdbindin-DmK gene transcription. C, correlation between 1,25-(OH)& receptor occupancy and calbindin-DmK gene transcription.
decrease slowly to reach preinjection levels by 72 h. Fig. 6 shows the accumulation of calbindin-Dz8Kmeasured by ELISA assay following a dose of 1,25-(OH)2D3.CalbindinD28K levels start to increase by 5-8 h ( p < 0.05 at 5 h), reach a peak value at 48 h following hormone administration, and then decrease towards baseline levels by 7 2 h. The constitutive levels of calbindin-Dz8Kgene transcription in vitamin D-deficient and -replete chicks is shown in Fig. 7. Chicks maintained on a normal vitamin D-replete diet show an approximate 12-fold increase in basal levels of calbindinD28~gene transcription compared to chicks raised on a vitamin D-deficient diet. This basal level of calbindin-Dz8Kgene transcription in vitamin D-replete chick intestine cannot be inhibited by prior (1h) administration of 600 pg of cycloheximide. DISCUSSION
The data presented here confirm the hypothesis that the mechanism of action of the seco-steroid 1,25-(OH)zD3is like
a z a n
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+D
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+D +CYCLOHEXIMIDE
FIG.7. Basal levels of calbindin-DzeK gene transcription in vitamin D-deficient (43) and vitamin D-replete (+ D ) chick intestine. In vitro nuclear transcription of the calbindin-D2aK gene was measured in intestine of chicks maintained on either a -D or +D diet. Calbindin-DzaK gene transcription was also measured in +D chicks 1 h following a dose of 600 pg of cycloheximide. Each value is the mean f S.E. of three to six determinations.
that of other classical steroid hormones, exerting its effects by regulation of transcription of specific genes through a receptor-mediated mechanism. The results of this study indicate that there is a close temporal relationship between nuclear localization of the hormone, receptor binding, and initiation of transcription. The time courses of the increases
16946
Calbindin-DzsKGene Expression Is Regulated by
1,25-(0H)zDa
in all three parameters are highly correlated with one another. major factor in the induction of several mRNAs in mouse All three events begin to occur immediately after administra- kidney by androgens (33). Estrogens can stabilize vitellogenin tion of 1,25-(OH)2D3. Nuclear uptake of 1,25-(OH)zD3reaches mRNA in Xenopus liver (34), while inhibiting albumin mRNA a maximum by 90 min, receptor occupancy by 1 h, and in the same tissue by both transcriptional and post-transcrip~ a 1 b i n d i n - Dgene ~~~ transcription by 2 h. All three events are tional mechanisms (35). Steroid hormones have also been attenuated by 4 h. These resultssuggest that rapid binding of shown to have extranuclear effects on mRNA translation. 1,25-(OH)2D3 to thereceptor occurs following nuclear uptake. Estradiol induces peptide chain elongation on rat uterine Previous experiments have demonstrated that unoccupied ribosomes, suggesting the existence of estradiol receptor in1,25-(OH)2D3receptors are apparently localized in thenuclei teraction with ribosomes (36). More relevant to the present of chick intestine (22). The rapidity of the gene response study, 1,25-(OH)& has a stimulatory effect on the peptide suggests a directeffect of the 1,25-(OH)2D3.receptorcomplex chain initiation process in chick intestinal mucosa (37). It is on initiation of calbindin-DzsKgene transcription, and also possible, therefore, that only the first few hours of the 1,25that the receptor resides on ornear the site at which it (OH),D3-inducedincrease in calbindin-DzsK-mRNAobserved activates expression of the ~ a 1 b i n d i n - D gene. ~ ~ ~To our knowl- in the present study may be due to stimulation of transcripedge, this is the firstdemonstration of such a rapid and direct tion, and increases observed at later times after transcription effect of 1,25-(0H),D3 on the expression of a specific gene. has ceased may bedue to post-transcriptional effects mediated Similar results have been observed in other steroid hormone- by the hormone. The evidence suggests that both effects on regulated systems. In the chick oviduct, there is excellent mRNA stability and translationmay be involved. correlation between nuclear estrogen receptor concentrations, The datapresented in Fig. 7 show that intestinal calbindintemplate-engaged RNA polymerase 11, and ovalbumin gene DaK gene transcription is also vitamin D-dependent in the transcription (23). The rates of estrogen and progesterone- intact chick. The basal level of ~ a 1 b i n d i n - D gene ~ ~ transcrip~ induced synthesis of ovalbumin and conalbumin mRNAs are tion in vitamin D-replete chicks is approximately 12-fold consistent with the levels of receptor present (24). In the higher than in vitamin D-deficient chicks, where it is almost mouse mammary tumor virus system, glucocorticoids rapidly undetectable. The fact that cycloheximide is unable to inhibit induce synthesis of viral mRNA via a mechanism mediated calbindin-DaK gene transcription in the vitamin D-replete by the glucocorticoid receptor and independent of protein chick intestinefurther indicates that the action of 1,25synthesis (1).Recent studies have demonstrated a direct and (OH)?DS is occurring directly at thelevel of transcription and selective interaction between steroid-receptor complexes and is not mediated by some other factor. The lack of inhibition sites within or near hormone-responsive genes. These studies of calbindin-DBK gene transcription by cycloheximide also have demonstrated selective binding of the estrogen receptor confirms our previous hypothesis (20) that the inhibition of to the prolactin gene (25) and the chicken vitellogenin gene calbindin-DaK-mRNA observed after cycloheximide treat(26), binding of the progesterone receptor to chick oviduct ment occurs via inhibition of protein synthesis and not by a genes (27, 28) andthe rabbit uteroglobin gene (29), and direct effect on transcription. binding of the glucocorticoid receptor to mammary tumor In conclusion, we have demonstrated for the first time the virus sequences (30, 31). It will be interesting to determine direct effect of 1,25-(OH)zD3on the transcription of a specific whether 1,25-(OH)zD3.receptor complexes regulate gene gene. Transcription of the ~ a 1 b i n d i n - Dgene ~ ~ ~ is directly expression in the same manner, by binding to specific steroid correlated to nuclear uptake and binding of 1,25-(OH)& to regulatory elements on the calbindin-Dzs~ gene. its receptor. These events are followed by the accumulation In the present study we also measured the accumulation of of the mature message in the cytosol andits subsequent calbindin-DzSK-mRNAby dot blot hybridization analysis of translation to calbindin-DBK. The long lag times before these total isolated RNA. The peak of ca1bindin-DzsK-mRNAac- latter two events occur may reflect the involvement of postcumulation occurred 12 h following administration of 1,25- transcriptional mechanisms in the regulation of calbindin(OH),D, to chicks. This reflects a l a g of 9 h between the peak D B K gene expression. Presumably, transcription of the calbinof calbindin-D28Kgene transcription (3 h) and the peak of din-DfSKgene results from a direct interactionof the occupied ~ ~ ~1,25-(OH)2D3receptor with specific regulatory regions of the mRNA accumulation (12 h). The peak of ~ a 1 b i n d i n - Daccumulation did not occur until 48 h after hormone adminis- gene as has been demonstrated for other steroid hormones; tration, reflecting a lag of 36 h between the peak of mRNA however, this remains to be determined experimentally. accumulation and the protein peak. These long lag times suggest the possibility of post-transcriptional regulation of Acknowledgments-We wish to thank June E. Bishop for carrying calbindin-D28Kgene expression, possibly directly mediated by out the receptor exchange assays, and Dr. Michael W. King, in 1,25-(OH)&. These post-transcriptional effects may include collaboration with whom the results in Figs. 5 and 6 were obtained. stabilization of the mRNA, effects on RNA processing, and/ REFERENCES or mRNA translation efficiency. We have previously de1. Ringold, G. M. (1985) Annu. Rev. Pharmucol. ToxicoL 2 5 , 529scribed the rapid degradation of calbindin-DzsK-mRNAin 566 vitamin D-replete chicks following a dose of cycloheximide 2. Yamamoto, K. R. (1985) Annu. Reo. Genet. 19,209-252 (20). This indicates the involvement of protein synthesis in 3. Pike, J. W., and Haussler, M. R. (1979) Proc. Natl. Acad. Sci. U. maintaining the stability of this message, an effect which S.A. 76,5485-5489 could be mediated by 1,25-(OH)2D3.It has been demonstrated 4. Walters, M. R. (1985)Endocr. Rev. 6,512-543 in several instances that steroid hormones can increase the 5. Norman, A. W., Fbth, J., and Orci, L. (1982) Endocr. Rev. 3, 331-366 stability of mRNAs as well as inducing their transcription. 6. Kretsinger, R. H., Mann, J. E., and Simmonds, J. G. (1982) in For example, in the chick oviduct, estrogen and progesterone Vitamin D: Chemical, Biochemical, and Clinical Endocrinology have significant effects on ovalbumin and conalbumin mRNA of CalciumMetabolism (Norman, A. W., Schaefer, K., von stability, as well as on mRNA synthesis (24). Regulation of Herrath, D., and Grigoleit, H.-G., eds) pp. 233-248, Walter de al-acid glycoprotein by dexamethasone in hepatoma cells Gruyter, Berlin appears to be exclusively post-transcriptional and involves 7. Morrissey, R. L., and Wasserman, R. H. (1971) Am. J. Physiol. 220,1509-1515 stabilization of nuclear RNA (32). mRNA stabilization is a
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