0013-7227/91/1291-0396$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 1 Printed in U.S.A.
Effect of Glucocorticoids and 1,25-Dihydroxyvitamin D 3 on the Developmental Expression of the Rat Intestinal Vitamin D Receptor Gene* SOOJA LEE, MARGERY SZLACHETKA, AND SYLVIA CHRISTAKOS Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
be noted, however, that the up-regulation of VDR was accompanied by an increase in actin mRNA, suggesting that the effect is not specific for VDR. Similarly, when rats were bilaterally adrenalectomized on day 17 (killed on day 22), a 4-fold decrease in VDR mRNA was observed, accompanied by a decrease in actin mRNA. However, when rats were injected with 1,25(OH)2D3 (25 ng/day-100 g BW) from days 15-17, levels of intestinal VDR mRNA were significantly increased by 1.5-fold, and this change was specific for VDR mRNA. In summary, our results indicate that hydrocortisone and 1,25-(OH)2D3 can precociously induce intestinal VDR mRNA, suggesting the involvement of glucocorticoids and 1,25-(OH)2D3 in the regulation of VDR gene expression in the developing rat intestine. However, our results also indicate that the effect of glucocorticoids (unlike the effect of 1,25-(OH)2D3) is not specific for VDR mRNA, but may reflect general effects of glucocorticoids on intestinal maturation. (Endocrinology 129: 396-401,1991)
ABSTRACT. In this study the ontogenesis of rat intestinal vitamin D receptor (VDR) gene expression was examined. When Northern and slot blot analyses were used to examine the expression of intestinal VDR mRNA in 15-, 18-, 22-, and 28day-old rats, induction of VDR mRNA was not observed until 22 days postpartum. Since little is known, particularly in the neonate, concerning the in vivo regulation of VDR gene expression, we examined the possibility that glucocorticoids and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] could affect the developmental expression of the intestinal VDR gene. To examine the effect of glucocorticoids, rat pups received three sequential injections (one per day) of hydrocortisone (5, 2.5, and 2.5 mg/ 100 g BW). Hydrocortisone administration before day 14 or on days 19-21 was not effective in inducing VDR mRNA. However, a significant 3.8-fold increase in intestinal VDR mRNA was observed in rats injected with hydrocortisone from days 15-17. The hydrocortisone effective period coincides with the glucocorticoid-sensitive period of rat intestinal development. It should
1
and its regulation in the neonatal period. However, the factors involved in the induction of 1,25-(OH)2D3 receptor mRNA in the developing rat intestine are as yet unknown. Only recently has the molecular cloning of the cDNA encoding avian (8), rat (9), and human (10) VDR been reported, and little information is currently available concerning the in vivo regulation of VDR gene expression (7,11). Glucocorticoids have been reported to stimulate intestinal maturation (12, 13), and glucocorticoids and 1,25-(OH)2D3 have been reported to regulate the intestinal VDR (14-19). Based on these reports, we investigated the possibility that hydrocortisone or 1,25(OH)2D3 could affect the developmental expression of the VDR gene in neonatal rat intestine.
,25-DIHYDROXYVITAMIN D3 [1,25-(OH)2D3] plays a key role in intestinal calcium absorption, and this process is a receptor-mediated event (1). Before weaning, although the calcium requirement is high, active intestinal calcium transport, plasma 1,25-(OH)2D3 levels, and intestinal calcium-binding protein (calbindin) concentrations are relatively low (2-4). During the suckling period, rat intestine is relatively refractory to 1,25(OH)2D3, and during the first 2 postnatal weeks specific l,25-(OH)2D3-binding activity is relatively absent (5, 6). After weaning, there is an onset of active calcium transport responsiveness to 1,25-(OH)2D3 as well as a marked increase in intestinal 1,25-(OH)2D3 receptors (VDR) and serum 1,25-(OH)2D3 (2, 3). Induction of receptor mRNA at weaning (7) may be one of the essential factors involved in the development of active calcium transport
Materials and Methods Materials
Received December 26, 1990. Address all correspondence and requests for reprints to: Sylvia Christakos, Ph.D., Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103. * This work was supported by NIH Grant DK-38961 (to S.C.).
la,25-Dihydroxy-[23,24-N-3H]cholecalciferol was purchased from Amersham (Arlington Heights, IL). [a-32P]dCTP (3000 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Oligo(dT)-cellulose and oligonucleotide labeling kit 396
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 22:33 For personal use only. No other uses without permission. . All rights reserved.
REGULATION OF VDR mRNA (random primer labeling kit) were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Biotrans nylon membrane was purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). Guanidine isothiocyanate and agarose (gel electrohoresis grade) were purchased from Fisher Scientific (Springfield, NJ). All other chemicals were of analytical grade. 1,25(OH)aD:t was provided by Dr. M. Uskokovic of Hoffman-LaRoche (Nutley, NJ). Animals For developmental studies, timed pregnant and neonatal Sprague-Dawley rats (Taconic Farms, Germantown, PA) were fed a standard rat chow (Rodent Laboratory Chow 5001, Ralston-Purina Co., St. Louis, MO) ad libitum. To determine the influence of glucocorticoids on intestinal VDR gene expression, rat pups received three sequential sc injections (one per day) of hydrocortisone 21-hemisuccinate (Sigma Chemical Co., St. Louis, MO) in 0.9% sodium chloride (5, 2.5, and 2.5 mg/100 g BW). In separate experiments, in order to determine specificity, rat pups received three sequential injections (one per day) of 2 mg progesterone (Sigma Chemical Co.) in sesame oil. Bilateral adrenalectomy or sham operation was performed under ether anesthesia on littermates on day 17 postpartum, and animals were killed on day 22 postpartum. In additional studies, 1,25(OH)aDa supplementation was achieved by ip injection (25 ng/ 100 g BWday for 3 days or 200 ng/100 g BW-24 h before death). 1,25-(OH)2D3 injections were given in 0.1 ml 10% ethanol-90% propylene glycol. RNA isolation Total cellular RNA was extracted by the guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (20), and polyadenylated RNA was selected via oligo(dT)-cellulose affinity chromatography (21). For the slot blot hybridizaton assay, total RNA was blotted onto nylon filters (Biotrans, ICN) that were preequilibrated with 20 x SSC (1 X = 0.15 M NaCl and 0.015 M trisodium citrate). The filters were baked at 80 C for 2 h, prehybridized, and hybridized, as described previously (22), using 1-3 X 106 cpm/ml 32P-labeled cDNA. cDNA was labeled to a specific activity of 108-109 cpm/ mg DNA according to the random oligo-priming method of Feinberg and Vogelstein (23) using the random primed DNA labeling kit (Boehringer Mannheim). After hybridization at 45 C for 16 h, the filters were washed three times in 200 ml 2 X SSC-0.1% sodium dodecyl sulfate at room temperature for 5 min each and two times in 500 ml 0.1 X SSC-0.1% sodium dodecyl sulfate at 50 C for 30 min each. The blots were air dried and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C in the presence of intensifying screens. The slot intensities of autoradiographs of varying exposures were quantitated by densitometry using a Shimadzu CS-9000 scanning densitometer (Shimadzu Scientific Instruments, Columbia, MD). Northern blot analysis Poly(A)+ RNA was fractionated on a 1.2% formaldehydeagarose gel and transferred to a nylon filter in 20 x SSC (24). Filters were hybridized to the 32P-labeled probe and washed as
397
described above. All filters were stripped and rehybridized to )8-[:!2P]actin or p 2 P]l8S ribosomal RNA (rRNA) cDNA, which were used as control probes to detect RNA transfer problems and possible unequal loading of RNA on the gel. Probes
A 1.7-kilobase rat VDR DNA insert from the EcoRl site of pIBI76 (7, 25), a 1.4-kilobase chick a-tubulin cDNA insert from the Pstl site of pBR322 (26), and a 2.1-kilobase chick 0-actin cDNA insert from the Hindlll site of pBR322 (26) were obtained by restriction enzyme digestion of the respective plasmid preparation. The cDNA for 18S rRNA was obtained from Ramareddy Guntaka (University of Missouri, Columbia, MO). VDR assay The first 6 cm of the duodenum distal to the pylorus were excised and rinsed with PBS containing 50 IU aprotinin/ml (Trasylol, Mobay Chemical Co., New York, NY). Mucosa was scraped from serosa, blotted, weighed, and homogenized with a Potter-Elvehjem homogenizer equipped with a Teflon pestle in high salt buffer (0.3 M potassium chloride, 10 mM Tris-HCl, 1 mM EDTA, 0.5 mM dithiothreitol, and 10 mM Na molybdate, pH 7.4; KTEDM) containing 200 mg/ml soybean trypsin inhibitor. Cytosol was prepared by ultracentrifugation at 300,000 x g for 30 min at 4 C in a Beckman L5-75 ultracentrifuge (Palo Alto, CA). Kidneys were perfused with cold PBS containing aprotinin and handled similarly, except that kidney tissue was minced before homogenization. For the VDR binding assay, cytosol (0.2 ml) containing 1.3 nM 1,25-(OH)2-[3H]D3 alone or in the presence of a 200-fold excess of nonradioactive 1,25(OH)2D3 was incubated for 18 h at 4 C. Bound and free hormone were separated by hydroxylapatite, as previously described (27, 28). For receptor assays there were 5-10 replicates for each group. Results are reported as the mean ± SE of at least 2 separate experiments. Other methods Protein concentrations were determined by the method of Bradford (29). Significance was determined by Student's t test or Dunnet's multiple comparison t statistic (30).
Results The marked developmental induction of rat intestinal VDR mRNA at 22 days postpartum is illustrated in Fig. 1. A similar rapid induction of specific l,25-(OH)2D;r binding activity in rat intestine during the third postnatal week has previously been reported (31). The influence of glucocorticoids on intestinal VDR mRNA levels in the neonate was examined by sequential administration of hydrocortisone (as described in Materials and Methods). Hydrocortisone administration before day 14 or on days 19-21 was not effective in inducing VDR mRNA. However a 3.8-fold increase in intestinal VDR mRNA was observed in rats injected with hydrocortisone from days 15-17 and killed on day 18 postpartum (Fig. 2). Results of Northern blot analysis are shown
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 22:33 For personal use only. No other uses without permission. . All rights reserved.
REGULATION OF VDR mRNA
398 10
Endo • 1991 Voll29«Nol
Day 22
/o-octir mRNA
VDR mRNA
rHE *• £ 6
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Vol. 129, No. 1 Printed in U.S.A.
Effect of Glucocorticoids and 1,25-Dihydroxyvitamin D 3 on the Developmental Expression of the Rat Intestinal Vitamin D Receptor Gene* SOOJA LEE, MARGERY SZLACHETKA, AND SYLVIA CHRISTAKOS Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
be noted, however, that the up-regulation of VDR was accompanied by an increase in actin mRNA, suggesting that the effect is not specific for VDR. Similarly, when rats were bilaterally adrenalectomized on day 17 (killed on day 22), a 4-fold decrease in VDR mRNA was observed, accompanied by a decrease in actin mRNA. However, when rats were injected with 1,25(OH)2D3 (25 ng/day-100 g BW) from days 15-17, levels of intestinal VDR mRNA were significantly increased by 1.5-fold, and this change was specific for VDR mRNA. In summary, our results indicate that hydrocortisone and 1,25-(OH)2D3 can precociously induce intestinal VDR mRNA, suggesting the involvement of glucocorticoids and 1,25-(OH)2D3 in the regulation of VDR gene expression in the developing rat intestine. However, our results also indicate that the effect of glucocorticoids (unlike the effect of 1,25-(OH)2D3) is not specific for VDR mRNA, but may reflect general effects of glucocorticoids on intestinal maturation. (Endocrinology 129: 396-401,1991)
ABSTRACT. In this study the ontogenesis of rat intestinal vitamin D receptor (VDR) gene expression was examined. When Northern and slot blot analyses were used to examine the expression of intestinal VDR mRNA in 15-, 18-, 22-, and 28day-old rats, induction of VDR mRNA was not observed until 22 days postpartum. Since little is known, particularly in the neonate, concerning the in vivo regulation of VDR gene expression, we examined the possibility that glucocorticoids and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] could affect the developmental expression of the intestinal VDR gene. To examine the effect of glucocorticoids, rat pups received three sequential injections (one per day) of hydrocortisone (5, 2.5, and 2.5 mg/ 100 g BW). Hydrocortisone administration before day 14 or on days 19-21 was not effective in inducing VDR mRNA. However, a significant 3.8-fold increase in intestinal VDR mRNA was observed in rats injected with hydrocortisone from days 15-17. The hydrocortisone effective period coincides with the glucocorticoid-sensitive period of rat intestinal development. It should
1
and its regulation in the neonatal period. However, the factors involved in the induction of 1,25-(OH)2D3 receptor mRNA in the developing rat intestine are as yet unknown. Only recently has the molecular cloning of the cDNA encoding avian (8), rat (9), and human (10) VDR been reported, and little information is currently available concerning the in vivo regulation of VDR gene expression (7,11). Glucocorticoids have been reported to stimulate intestinal maturation (12, 13), and glucocorticoids and 1,25-(OH)2D3 have been reported to regulate the intestinal VDR (14-19). Based on these reports, we investigated the possibility that hydrocortisone or 1,25(OH)2D3 could affect the developmental expression of the VDR gene in neonatal rat intestine.
,25-DIHYDROXYVITAMIN D3 [1,25-(OH)2D3] plays a key role in intestinal calcium absorption, and this process is a receptor-mediated event (1). Before weaning, although the calcium requirement is high, active intestinal calcium transport, plasma 1,25-(OH)2D3 levels, and intestinal calcium-binding protein (calbindin) concentrations are relatively low (2-4). During the suckling period, rat intestine is relatively refractory to 1,25(OH)2D3, and during the first 2 postnatal weeks specific l,25-(OH)2D3-binding activity is relatively absent (5, 6). After weaning, there is an onset of active calcium transport responsiveness to 1,25-(OH)2D3 as well as a marked increase in intestinal 1,25-(OH)2D3 receptors (VDR) and serum 1,25-(OH)2D3 (2, 3). Induction of receptor mRNA at weaning (7) may be one of the essential factors involved in the development of active calcium transport
Materials and Methods Materials
Received December 26, 1990. Address all correspondence and requests for reprints to: Sylvia Christakos, Ph.D., Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103. * This work was supported by NIH Grant DK-38961 (to S.C.).
la,25-Dihydroxy-[23,24-N-3H]cholecalciferol was purchased from Amersham (Arlington Heights, IL). [a-32P]dCTP (3000 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Oligo(dT)-cellulose and oligonucleotide labeling kit 396
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 22:33 For personal use only. No other uses without permission. . All rights reserved.
REGULATION OF VDR mRNA (random primer labeling kit) were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Biotrans nylon membrane was purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). Guanidine isothiocyanate and agarose (gel electrohoresis grade) were purchased from Fisher Scientific (Springfield, NJ). All other chemicals were of analytical grade. 1,25(OH)aD:t was provided by Dr. M. Uskokovic of Hoffman-LaRoche (Nutley, NJ). Animals For developmental studies, timed pregnant and neonatal Sprague-Dawley rats (Taconic Farms, Germantown, PA) were fed a standard rat chow (Rodent Laboratory Chow 5001, Ralston-Purina Co., St. Louis, MO) ad libitum. To determine the influence of glucocorticoids on intestinal VDR gene expression, rat pups received three sequential sc injections (one per day) of hydrocortisone 21-hemisuccinate (Sigma Chemical Co., St. Louis, MO) in 0.9% sodium chloride (5, 2.5, and 2.5 mg/100 g BW). In separate experiments, in order to determine specificity, rat pups received three sequential injections (one per day) of 2 mg progesterone (Sigma Chemical Co.) in sesame oil. Bilateral adrenalectomy or sham operation was performed under ether anesthesia on littermates on day 17 postpartum, and animals were killed on day 22 postpartum. In additional studies, 1,25(OH)aDa supplementation was achieved by ip injection (25 ng/ 100 g BWday for 3 days or 200 ng/100 g BW-24 h before death). 1,25-(OH)2D3 injections were given in 0.1 ml 10% ethanol-90% propylene glycol. RNA isolation Total cellular RNA was extracted by the guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (20), and polyadenylated RNA was selected via oligo(dT)-cellulose affinity chromatography (21). For the slot blot hybridizaton assay, total RNA was blotted onto nylon filters (Biotrans, ICN) that were preequilibrated with 20 x SSC (1 X = 0.15 M NaCl and 0.015 M trisodium citrate). The filters were baked at 80 C for 2 h, prehybridized, and hybridized, as described previously (22), using 1-3 X 106 cpm/ml 32P-labeled cDNA. cDNA was labeled to a specific activity of 108-109 cpm/ mg DNA according to the random oligo-priming method of Feinberg and Vogelstein (23) using the random primed DNA labeling kit (Boehringer Mannheim). After hybridization at 45 C for 16 h, the filters were washed three times in 200 ml 2 X SSC-0.1% sodium dodecyl sulfate at room temperature for 5 min each and two times in 500 ml 0.1 X SSC-0.1% sodium dodecyl sulfate at 50 C for 30 min each. The blots were air dried and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C in the presence of intensifying screens. The slot intensities of autoradiographs of varying exposures were quantitated by densitometry using a Shimadzu CS-9000 scanning densitometer (Shimadzu Scientific Instruments, Columbia, MD). Northern blot analysis Poly(A)+ RNA was fractionated on a 1.2% formaldehydeagarose gel and transferred to a nylon filter in 20 x SSC (24). Filters were hybridized to the 32P-labeled probe and washed as
397
described above. All filters were stripped and rehybridized to )8-[:!2P]actin or p 2 P]l8S ribosomal RNA (rRNA) cDNA, which were used as control probes to detect RNA transfer problems and possible unequal loading of RNA on the gel. Probes
A 1.7-kilobase rat VDR DNA insert from the EcoRl site of pIBI76 (7, 25), a 1.4-kilobase chick a-tubulin cDNA insert from the Pstl site of pBR322 (26), and a 2.1-kilobase chick 0-actin cDNA insert from the Hindlll site of pBR322 (26) were obtained by restriction enzyme digestion of the respective plasmid preparation. The cDNA for 18S rRNA was obtained from Ramareddy Guntaka (University of Missouri, Columbia, MO). VDR assay The first 6 cm of the duodenum distal to the pylorus were excised and rinsed with PBS containing 50 IU aprotinin/ml (Trasylol, Mobay Chemical Co., New York, NY). Mucosa was scraped from serosa, blotted, weighed, and homogenized with a Potter-Elvehjem homogenizer equipped with a Teflon pestle in high salt buffer (0.3 M potassium chloride, 10 mM Tris-HCl, 1 mM EDTA, 0.5 mM dithiothreitol, and 10 mM Na molybdate, pH 7.4; KTEDM) containing 200 mg/ml soybean trypsin inhibitor. Cytosol was prepared by ultracentrifugation at 300,000 x g for 30 min at 4 C in a Beckman L5-75 ultracentrifuge (Palo Alto, CA). Kidneys were perfused with cold PBS containing aprotinin and handled similarly, except that kidney tissue was minced before homogenization. For the VDR binding assay, cytosol (0.2 ml) containing 1.3 nM 1,25-(OH)2-[3H]D3 alone or in the presence of a 200-fold excess of nonradioactive 1,25(OH)2D3 was incubated for 18 h at 4 C. Bound and free hormone were separated by hydroxylapatite, as previously described (27, 28). For receptor assays there were 5-10 replicates for each group. Results are reported as the mean ± SE of at least 2 separate experiments. Other methods Protein concentrations were determined by the method of Bradford (29). Significance was determined by Student's t test or Dunnet's multiple comparison t statistic (30).
Results The marked developmental induction of rat intestinal VDR mRNA at 22 days postpartum is illustrated in Fig. 1. A similar rapid induction of specific l,25-(OH)2D;r binding activity in rat intestine during the third postnatal week has previously been reported (31). The influence of glucocorticoids on intestinal VDR mRNA levels in the neonate was examined by sequential administration of hydrocortisone (as described in Materials and Methods). Hydrocortisone administration before day 14 or on days 19-21 was not effective in inducing VDR mRNA. However a 3.8-fold increase in intestinal VDR mRNA was observed in rats injected with hydrocortisone from days 15-17 and killed on day 18 postpartum (Fig. 2). Results of Northern blot analysis are shown
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 22:33 For personal use only. No other uses without permission. . All rights reserved.
REGULATION OF VDR mRNA
398 10
Endo • 1991 Voll29«Nol
Day 22
/o-octir mRNA
VDR mRNA
rHE *• £ 6
-••••$**i
" •*•
18-
„««•*• .
•>•»,•
'!
