0013.7227/92/1316-2643$03.00/O Endocrinology Copyright 0 1992 by The Endocrine
Vol. 131, No. 6 Printed m US A
Society
Calbindin-D9K Gene Expression in the Lung Absence of Regulation by 1,25=Dihydroxyvitamin Estrogen* JEAN-MARIE DUPRET, FRANCOIS BERNAUDIN,
FABIENNE L’HORSET, CHRISTINE AND MONIQUE THOMASSET
PERRET,
of the Rat. D3 and
JEAN
INSERM U 120, H6pital Robert Debre, 75019 Paris; and Laboratoire d’Histologie-Biologie H6pital Tenon, 75020, Paris, France
Tumorale (J.-F.),
ABSTRACT Calbindin-D9K (CaBPSK) is classically considered to be the molecular expression of 1,25-dihydroxyvitamin Da. The hormone is known to regulate the rat CaBP9K gene in duodenal tissue at transcriptional and posttranscriptional levels. This study shows that the CaBP9K gene is expressed in the rat lung, and that this expression is probably not vitamin D- or estrogen-dependent. The CaBP9K gene is not expressed in alveolar macrophages, but CaBP9K messenger RNA (mRNA) was localized by in situ hybridization in alveolar epithelial cells. CaBP9K mRNA was detected as early as the 20th day of gestation. The quantity of CaBP9K mRNA gradually increased during growth, from 1-77 days
after birth, whereas the CaBP9K concentration dramatically increased from day 19 to day 20 of gestation. Vitamin D-deficient male rats (8 weeks old) were given a single injection of 1,25-dihydroxyvitamin D, (650 pmol/lOO g body wt) and killed 1 h and 24 h after injection. The hormonal treatment resulted in a rise in duodenal CaBP9K mRNA, but no significant change in lung extracted CaBP9K mRNA. Mature ovariectomized rats were injected with 17/3-estradiol (0.5 pg/lOO g body wt) and killed 24,48, and 72 h later. The CaBP9K mRNA concentration in the uterus was markedly dependent on estrogen; that of the lung was not. The factors regulating the CaBP9K gene expression in the lung remain to be determined. (Endocrinology 131: 2643-2648, 1992)
T
ization of CaBP9K messenger bridization.
vitamin D-dependent calcium-binding proteins (CaBP) or calbindin-D (see Ref. 1 for review) are a group of intracellular proteins which are considered to be one of the molecular expressions of the hormonal actions of the secosteroid 1,25-dihydroxyvitamin D3 [1,25(OH),D,] (for reviews see Refs. 2-4). These proteins bind Ca++ with a high affinity (association constant = lo6 M-‘), and share with other intracellular calcium-binding proteins a structural feature within their Ca++ binding regions called the EF hand domain (5). In mammals, vitamin D-dependent calbindin-D genes are expressed in the intestine (9-kilodalton CaBP, calbindinD9K or CaBP9K) (6, 7) and kidney (28-kilodalton CaBP, calbindin-D28K or CaBP28K) (8). The receptor-mediated action of 1,25(OHhD3 is believed to be exerted via direct interaction with DNA (9), as for osteocalcin (10) and osteopontin (11). The CaBP9K gene is also expressed in the uterus, where it is under the control of estrogen (12), and in other HE
tissues
(1) where
any
hormonal
control,
including
vitamin
Materials
by in situ hy-
and Methods
Materials Radioisotopes (32P- and %labeled) and hybridization membranes were purchased from Amersham Co. (Les Ullis, France). Enzyme for DNA labeling and RNA molecular mass markers were obtained from Boehringer Mannheim (Mannheim, Germany). 1,25(OHhD3 was kindly provided by Dr Uskokovic (Hoffmann LaRoche, Nutley, NJ). 17@Estradiol was purchased from Sigma (La Verpilliere, France). All other chemicals and reagents were analytical grade from standard suppliers.
Animals and treatments Male Sprague-Dawley rats were purchased from Charles River (St. Aubin Les Elbeuf, France). Three-week-old rats (-D group) were fed a vitamin D-free diet (Usine d’tllimentation Rationnelle, no. 6, Villemoisson sur Orge, France) with 0.50% calcium, 0.36% phosphorus, and were raised in the dark for 5 weeks. Hypocalcemia was used as index of vitamin D deficiency. The other rats were fed the same diet with vitamin D (2000 W/kg). 1,25(OH)2DS (650 pmol/lOO g body wt) was given ip into randomly sorted -D rats, and animals were killed 1 h and 24 h after injection. Mature ovariectomized rats were purchased from Charles River; 2 weeks later animals were given ip 17fi-estradiol (0.5 fig/100 g body wt) and were killed 24 h, 48 h, and 72 h later. Pregnant rats at known gestational stages (days 17-21) were subjected to cesarean section under pentobarbital sodium anesthesia, and the fetuses were rapid]! removed from the uterus. Each developmental study sample was obtained from the lungs of two animals. These studies were conducted in accordance with the NIH Guide for the Care and Use of Laborator! Animals.
D
dependency, remains to be determined. A recent immunological study suggested the expression of CaBP9K gene in the lung was independent of vitamin D (13). This study investigates the expression of the CaBP9K gene in the lung and the involvement of 1,25(OHhD3 and estrogen in its regulation. RNA from various rat tissues including the duodenum, uterus, kidney, and lung were extracted and hybridized with CaBP9K and CaBP28K cDNAs; lung CaBP9K levels were measured by RIA. We also examined the cellular localReceived April 2, 1992. Address all correspondence and requests for reprints Marie Dupret, INSERM U120, Hopital Robert Debre, Serurier, Bitiment Ecran +4, 75019 Paris, France. * In memoriam to Michel Rieutort.
RNA (mRNA)
to: Dr. Jean48 Boulevard
Tissuepreparation The rats were anesthetized with pentobarbital sodium and killed b\ decapitation. Blood from -D rats was collected for serum calcium 2643
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2644
CaBP9K
GENE
EXPRESSION
IN
THE
RAT
Endo. Voll31.
LUNG
measurement (complexometric titration). Tissues were rapidly and aseptically removed, washed, and snap-frozen in liquid nitrogen. Bronchoalveolar lavages were performed using 40 ml of a 0.9% NaCl solution. Alveolar macrophages were collected, sedimented by centrifugation, and frozen in liquid nitrogen. An aliquot of the macrophage suspension was stained (Field’s Staining Kit, Baxter, Maurepas, France) and counted. The yield was 230,000 cells/ml, free of blood and bronchial cell contamination. Total RNA was prepared by the Chomczynski and Sacchi method (14), poly(A)+ RNA was purified by chromatography on oligo (dT) cellulose (15), and lung CaBP9K quantified by RIA (16). For in situ hybridization lung tissue from mature rats was fixed by intratracheal instillation of 4% paraformaldehyde in PBS for 5 h. Ten-micrometer cryostat sections were subsequently performed.
Preparation
-pActin
qP2pglobuIin
of probes
Specific complementary DNA (cDNA) probes for rat CaBP9K and CaBP28K were prepared as previously described (17, 18). Mouse /3-actin and P-2 microglobulin cDNA probes were used as hybridization control. The estrogen-independent 1A cDNA (19) was also used as hybridization control. For in situ hybridization high specific activity 35S-labeled singlestranded antisense and sense CaBP9K-specific RNA probes were transcribed from the CaBP9K cDNA subcloned in the transcription vector pBluescript II SK (Stratagene, La Jolla, CA) (20). The specificity of the probes was checked in Northern blot analysis.
Hybridization
1992 No 6
_‘]CaBP 28K - CaBP 9K
to filters
The relative amounts of CaBP9K and CaBP28K mRNA in total extracted RNA were estimated by Northern blot hybridization (21) and quantified by dot blot hybridization (22). @-Actin and p-2 microglobulin probes were used as controls. The amounts of mRNA were quantified by spectrodensitometric analysis of peak areas (Shimadzu Scientific Instruments, Columbia, MD); peak area has been shown to be directly proportional to the amount of radioactivity bound to the filters (6). Lung mRNA levels and uterine mRNA levels were normalized by hybridization with fl-actin and IA cDNA probes, respectively.
In situ hybridization Fixed rat lung cryostat sections were processed for in situ hybridization as previously described (23). The preparations were hybridized for 5 h at 50 C. After hybridization the slices were washed in 50% fornamide/2-fold standard saline citrate (SSC) at 52 C and incubated in a solution of RNase (Sigma, St. Louis, MO; 40 fig/ml) for 30 min at 37 C. For autoradiography slices were dipped in Amersham emulsion LMl and were developed after 2 weeks exposure. After development the autoradiographs were Giemsa stained.
FIG. 1. Calbindin-D
mRNA in tissues of vitamin D-replete rats. Eightweek-old rats were killed, and poly(A)+ RNA was extracted from the duodenal mucosa (D), liver (Li), spleen (S), lung (Lg), heart (H), and kidney (K). The RNAs were denatured with formaldehyde, fractionated on a 1.3% agarose gel (10 pg/sample), and blotted on a membrane filter. RNAs were then sequentially hybridized with the CaBP9K and CaBP28K [ol-a’P]cDNAs (bottom) and with the P-actin and p-2 microglobulin [Wa*P]cDNAs (top). A size scale was determined by coelectrophoresis of RNA markers.
1-n
Lg
Lg-M
M
I
D 1
- p Actin
Results Calbindin-D
mRNA detection
RNAs were extracted from the adult rat duodenal mucosa, liver, spleen, lung, heart, and kidney. Poly(A)+ RNA were then analyzed by Northern blot hybridization with 32Plabeled probes (Fig. 1). The CaBP9K cDNA detected a single 550-nucleotide mRNA speciesin the duodenum and lung RNA, whereasno signalwas detected in kidney, liver, spleen, and heart RNA. CaBP28K mRNA [a major 1.9-kilobase (kb) and two minor 2.8- and 3.2-kb species]was only found in the kidney; no signal was recorded elsewhere. @Actin (a 2.2kb species)and P-2 microglobulin (0.8- and 1-kb species) mRNA were detected in all the samples analyzed. No CaBP9K mRNA was detected in alveolar macrophage RNA, and no difference in CaBP9K mRNA concentration was found between intact lung and macrophage-cleared lung
-CaBP 9K
FIG. 2. CaBP9K
mRNA distribution in the lung of vitamin D-replete rats. Lungs from 8-week-old rats were given successive bronchoalveolar lavages and removed. Alveolar macrophages from three animals were collected as described in Materials and Methods. Total RNA (10 pg) from lung (Lg), macrophage-cleared lung (Lg-M), and macrophages (M) was prepared as described in the legend of Fig. 1. Total RNA from the duodenum (D) was used as control. RNAs were hybridized with CaBP9K [w”“P] and with /3-actin [w~‘P]cDNAs.
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CaBP9K
GENE
EXPRESSION
(Fig. 2). The presence of CaBP9K mRNA transcripts was localized by in situ hybridization in alveolar epithelial cells. No detection was observed in bronchiolar or pleural cells (Fig. 3). Lung CaBP9K
during
on lung CaBP9K
2645
were given a single injection of 17/3-estradiol there was a great increase in the uterine CaBP9K mRNA concentration (Fig. 7D), but extracts of CaBP mRNA from the lung (Fig. 7A) or duodenum (Fig. 7B) showed no change. Discussion
mRNA accumulation
A single 1,25(OHbD3 injection in vitamin D-deficient rats resulted in a dramatic increase in the duodenal CaBP9K mRNA. On the other hand, for CaBP9K extracted from the lung and after normalization with /3-actin levels, no statistical difference (by t test) could be recorded, either between vitamin D-replete (N) and vitamin D-deficient rats (OD) or between uninduced (OD) and vitamin D-induced rats (1 h and 24 h). (Fig. 6). Effects of 17/3-estradiol
RAT LUNG
the rat development
Lung CaBP9K mRNA was analyzed and quantified during the pre- and postnatal stages of rat development. CaBP9K mRNA was not detected until 20th day of pregnancy; the relative amounts then increased 20-fold until the rats were 77 days old, i.e. adult (Fig. 4). Northern blot analysis on a 1.3% denaturing agarose gel revealed no change in the electrophoresis properties of CaBP9K mRNA (Fig. 5). The lung CaBP9K concentration increasedrapidly (16 times) after the 19th day in utero and then more slowly (2-fold) until age 28 days (Table 1). Effects of 1,25(OH)zD3
IN THE
on lung CaBP9K
mRNA
accumulation
Total RNAs extracted from the duodenum, liver, lung, and uterus of mature ovariectomized rats were fractionated on a 2% agarosegel and analyzed by Northern blot hybridization with CaBP9K cDNA (Fig. 7, C-D). In all cases, a 550nucleotide mRNA was detected, and a second larger species was hybridized in uterus mRNA. When ovariectomized rats
FIG. 3. Autoradiograms of rat lung cryostat sections after in situ hybridization using 35S-labeled CaBPSK-specific antisense (A) and sense (B) RNA probes. In situ hybridization procedures are described in Materials and Methods. Alveolar epithelial cells are labeled with the antisense probe (arrows) (A), whereas they are not labeled with the sense probe (B) (X 400). The horizontal lines in the lower left corners of A and B represent 20 pm.
This study analyzes the molecular expression of the CaBP9K gene in the rat lung. The duodenum is the best known site of CaBP9K gene expression in the rat (1). Earlier developmental studies (16) reported that the rat duodenum contains small amounts of CaBP9K as early as gestational day 17, with a slight surge in CaBP9K concentration at birth, and a steady increase to the adult level reached by day 30 postpartum. CaBP9K mRNA was detected in the rat lung as early as gestational day 20. The CaBP9K concentration undergoes a dramatic increase between gestational days 19 and 20. The quantities of both CaBP9K and CaBP9K mRNA then increase throughout development. These data suggest that CaBP9K gene expression is not directly related to physiological events occurring during late fetal lung development, such as differentiation of alveolar epithelial cells to surfactant-secreting type II pneumocytes (24), or to such perinatal events as the increase in the alveolar fluid calcium concentration (25). Interestingly, CaBP9K mRNA has also been detected in human fetal lung, as early as 14 weeks of gestation with no significant change until the fetus is 32 weeks old (26). This in viva study also provides new evidence for tissuespecific regulation of the CaBP9K gene in the rat. We previously reported the transcriptional and posttranscriptional regulation of CaBP9K gene expression in the rat duodenum by 1,25(OH)2D3 (7). A single 1,25(OHhD3 injection induces a dramatic increase in duodenal CaBP9K mRNA in vitamin D-deficient rats, but the lung CaBP9K mRNA content does not change. These findings confirm earlier reports suggesting
a
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CaBP9K
2646
GENE EXPRESSION
IN THE RAT LUNG
rLung CaBP9K mRNA during development. Lung total RNAs were extracted from vitamin D-replete rats aged 19 days in utero to 77 days. Lungs from two animals were pooled for each sample; two samples were used in each analysis. Total RNAs were dotted on a membrane filter, fixed by heating, and sequentially hybridized to the CaBP9K and p-actin [LU-~‘P]CDNAS. All measurements of mRNA were carried out with total RNA in the range 1.25-10 ag/dot in the presence of excess DNA. CaBP9K mRNA was quantified by spectrodensitometric analysis of peak areas and was expressed as the ratio of CaBP9K to P-actin areas (arbitrary units). FIG.
4.
iz
i
Endo. Voll31.
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19d [
-3
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20d
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...+**r* ...* .** r....
14d
20d
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1992 No 6
.*r*...r
l
2
*.*
A
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28d
q# / *
1
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‘>
CaBP9K RNA BldTH TABLE
LS
-p Actin
e I
- CaBP9K +it
1
Days beforet
3
7 10 14 28 77I Days after
BIRTH FIG. 5. Northern blot hybridization of poly(A)+ RNA extracted from vitamin D-replete rats. Poly(A)+ RNA from the lung (Lg) was extracted from vitamin D-replete rats aged 19 days in utero to 77 days. RNA was denatured with formaldehyde, fractionated on a 1.3% agarose gel (1 pglsample), and blotted on a membrane filter. mRNA was then hybridized with the CaBP9K and P-actin [cx-~*P]cDNAs. Poly(A)+ RNAs from adult rat duodenum (D) and liver (Li) were used as positive and negative controls, respectively.
that the lung CaBP9K gene expression was not vitamin Ddependent. But these conclusions were only based upon measurements of CaBP9K by RIA or by immunoblotting. Thomassetand Tenenhouse (27), using 20-day and 30-dayold fifth-generation vitamin D-deficient rats, found no decreasein lung CaBP9K concentration. Verhaeghe et al. (28) also showed that the CaBP9K concentration of both the maternal and fetal lungs did not change when the fetal and maternal plasma 1,25(OHhD3 levels were altered. Recently, Walters et al. (13) used immunoblot analysis to demonstrate the vitamin D-independence of CaBP9K in the lung of 7 to 9-week-old second generation vitamin D-deficient rats. Nevertheless, these findings could not exclude a possible
1. Changes in Lung CaBP9K concentration Rat age (days) 17 19 20 21 1 3 7 10 14 20 28
' with rat age
CaBPSK (ng/mg protein) 2.8 3.5 57 94 Birth 60 92 104 105 82 98 124
Lung CaBP9K concentrations were determined by RIA from vitamin D-replete rats aged 17 days in utero to 28 days. Lungs from two animals were pooled for each sample. Two samples corresponding to those shown in mRNA analysis (Fig. 4) were used in each mean CaBP9K concentration for rats aged 17 days in utero is a single value obtained from four pooled fetuses.
translational effect of 1,25(OH)2D3upon vitamin D-dependent CaBP9K mRNA. Our data demonstrate that circulating 1,25(OH)2D3exerts no detectable effects upon the concentration of lung CaBP9K mRNA. Alveolar macrophages could also be involved in the synthesis of 1,25(OH)2D3 and consequently alter the vitamin D deficiency in a paracrine way. This is unlikely, sinceendogenous 1,25(OH)2D3synthesishas only been reported in pathological (29) or induced situations (30). It is noteworthy that 1,25(OH)zDs binding sites have only been detected in the rat lung during prenatal life (31), when the secosteroidplays a role in phospholipid synthesis and in surfactant secretion from type II pneumocytes (32). 17P-Estradiol stimulates uterine CaBP9K gene expression by induction of two mRNA species(12). Receptorsfor estrogen have also been detected in the rat lung (33). However, our data indicate that estrogen is unlikely to have any effect upon lung CaBP9K mRNA levels; current experiments seem
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CaBP9K
GENE
LUNG
r
EXPRESSION
-I-I
lh
00
II_II”
24h DUODENUM
1 -/3 Actin
a --
-CaBP 9K
FIG. 6. Effect of 1,25(OH)ZDs on CaBP9K mRNA concentration in the lung and duodenum of vitamin D-deficient rats. Vitamin D-deficient rats (OD) were killed 1 h or 24 h after a single injection of 1,25(OH)zD3 (650 pmol/lOO g). Ten micrograms of total RNA were denatured with formaldehyde, fractionated on a 1.3% agarose gel, transferred to a filter, and hybridized with the CaBP9K and /3-actin [cu-32P]cDNAs. Duodenum and lung were removed from the same individuals. Lung total RNAs from vitamin D-replete rats (N) were also blotted.
IUUU ovx
LUNG
DUODENUM
p
I
,
IL--JUU
ovx
24h 48h 72h
D L Lg Ut 03nnnn *=-
2647
24h
OD
@
RAT LUNG
to exclude glucocorticoid as a potential hormonal regulatory factor. The physiological significance of our results remains to be clarified. Pulmonary surfactant-producing cells, also named type II alveolar epithelial cells or type II pneumocytes (24), are potential sites for the expression of the CaBP9K gene. Indeed, the calcium concentration plays a role as an intracellular messengerin the secretory process(34) and in maintaining the extracellular structure and function of pulmonary surfactant (35, 36). Our data eliminate alveolar macrophages as putative candidates for CaBP9K gene expression. This finding is important since alveolar macrophages contain 1,25(OH)*D3 receptors (2) and are biological targets for 1,25(0H)2D3-induced cell fusion (37,38). Lung cells expressing CaBP9K mRNA have been identified as alveolar epitheha1cells; these cells could be a new model for the cell-specific regulation of CaBP9K gene expression.
I
-p Actin
N
IN THE
@ In
24h 48h 72h
UTERUS II 7 "*.-
-lA-
-CaBPSKUUUU
OVX 24 48 72 h FIG. 7. Effect of 17@-estradiol on CaBP9K mRNA concentration in the lung, duodenum, and uterus of ovariectomized rats. RNAs were extracted from the uterus (Ut), liver (L), duodenum (D), and lung (Lg) of mature ovariectomized rats. Ten micrograms of total RNA were fractionated on 1.3% (A and B) or 2% (C and D) denaturing agarose gels, transferred, and hybridized with CaBP9K [cr-32P]cDNA. Lung (A) and duodenum (B) mRNAs were also hybridized with @-actin [o~-~~P] cDNA, whereas uterine (D) and control mRNA [duodenum (D), liver (L), lung (Lg)] (C and D) were hybridized with 1A [cx-~‘P]cDNA. One microgram of duodenal RNA was used in C and D.
Acknowledgments We are indebted to Martine Bovet for her expert technical assistance. We are grateful to Dr. Lyttle for 1A probe. We thank Institut National de la Santi et de la Recherche Midicale SC 6 personnel, Claudine Brunner, and Dr. Owen Parkes for their help in the preparation of the manuscript. References 1. Christakos S, Gabrielides C, Rhoten WB 1989 Vitamin D-dependent calcium-binding proteins: chemistry, distribution, functional consideration and molecular biology. Endocr Rev 10:3-25 2. Haussler MR 1986 Vitamin D receptors: nature and function. Annu Rev Nutr 6527-562 3. Minghetti PP, Norman AW 1988 1,25(OHh vitamin D3 receptors: gene regulation and genetic circuitry. FASEB J 2:3043-3053 4. Pike JW 1991 Vitamin D3 receptors: structure and function in transcription. Annu Rev Nutr 11:189-216 5. Kretsinger RH 1980 Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem 8:119-174 6. Perret C, Desplan C, Thomasset M 1985 Cholecalcin (a 9-kDa cholecalciferol-induced calcium-binding protein) messenger RNA. Distribution and induction by calcitriol in-the rathigestiveyract. Eur J Biochem 150:211-217 7. Dupret JM, Brun P, Perret C, Lomri N, Thomasset M, CuisinierGleizes P 1987 Transcriptional and post-transcriptional regulation of vitamin D-dependent calcium-binding protein gene expression in the rat duodenum by 1,25-dihydroxycholecalciferol. J Biol Chem 262:16553-16.557 8, Varghese S, Deaven LL, Huang YC, Gill RC, Iacopino AM, Christakos S 1989 Transcriptional regulation and chromosomal assignment of the mammalian calbindin-D28K gene. Mol Endocrino1 3:495-502 9. Beato M 1989 Gene regulation by steroid hormones. Cell 56:335344 10 Ozono K, Liao J, Kerner SA, Scott RA, Pike JW 1990 The vitamin D-responsive elements in the human osteocalcin gene. J Biol Chem 265:21881-21888 11. Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT 1990 Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Ssp-1 or osteopontin) gene expression. Proc Nat1 Acad Sci USA 87:99959999 12. L’Horset F, Perret C, Brkhier A, Thomasset M 1990 17/3-estradiol stimulates the calbindin-D9K (CaBP9K) gene expression at the transcriptional and post-transcriptional levels in the rat uterus. Endocrinology 127:2891-2897
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2648
CaBP9K
GENE
EXPRESSION
13. Walters MR, Bruns ME, Carter RM, Riggle PC 1991 Vitamin D independence of small calcium-binding proteins in nonclassical target tissues. Am J Physiol 260:E794-ES00 P, Sacchi N 1987 Single-step method of RNA isola14. Chomczynski tion by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 H, Leder P 1972 Purification of biologically active globin 15. Aviv messenger RNA by chromatography on oligothymidylic acid cellulose. Proc Nat1 Acad Sci USA 69:1408-1412 16. Thomasset M, Parkes 0, Cuisinier-Gleizes P 1982 Rat calciumbinding proteins: distribution, development and vitamin D dependence. Am J Physiol 243:E483-E488 17. Desplan C, Thomasset M, Moukhtar MS 1983 Synthesis, molecular cloning, and restriction analysis of DNA complementary to vitamin D-dependent calcium-binding protein mRNA from rat duodenum. J Biol Chem 258:2762-2765 N, Thomasset M 1989 Cloning and 18. Lomri N, Perret C, Gouhier analysis of calbindin-D28K cDNA and its expression in the central nervous system. Gene 80:87-98 19. Hsu CJ, Komm BS, Lyttle CR, Frankel F 1988 Cloning of estrogenregulated messenger ribonucleic acids from rat uterus. Endocrinology 122:631-639 20. Johnson MT, Johnson BA 1984 Efficient synthesis of high specific activity 35Slabeled human /3 globin pre mRNA. Biotechniques 2:156-162 21. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci, USA 77:5201-5205 FC 1982 Cytoplasmic dot hybridization: simple 22. White AB, Bancroft analysis of relative mRNA levels in multiple small cell or tissue sample. J Biol Chem 257:8569-8572 23. Lebargy F, Bulle F, Siegrist S, Guellain G, Bernaudin JF 1990 Localization by in situ hybridization of y glutamyl transpeptidase mRNA in the rat kidney using 35s labeled RNA probes. Lab Invest 62:731-735 24. Haagwood HP, Van Golde LM 1991 Synthesis and assembly of lung surfactant. Annu Rev Physiol 53:441-464 25. Eckenhoff RG 1989 Potential changes in lung surfactant calcium measured in situ. J Clin Invest 84:1295-1301 26. Brun P, Dupret JM, Perret C, Thomasset C, Mathieu H 1987 Vitamin D-dependent calcium-binding proteins (CaBI’s) in human fetuses: comparative distribution of 9K CaBP mRNA and 28K CaBP
IN
27
28
29.
30.
31.
32.
33. 34.
35.
36.
37.
38.
THE
RAT
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LUNG
1992 No 6
during development. Pediatr Res 21:362-367 Thomasset M, Tenenhouse A 1988 Vitamin D dependence of calbindinD9K and calbindin-D28K synthesis in various tissues. In: Norman AW, Schaefer K, Grigoleit HG, Herrath DV (eds) Vitamin D. Molecular, Cellular and Clinical Endocrinology. Walter de Gruyter, Berlin, New-York, pp 516-517 Verhaeghe J, Thomasset M, Brehier A, Van Assche FA, Bouillon R 1988 1,25(OH)rDa and Ca-binding protein in fetal rats: relationship to the maternal vitamin D status. Am J Physiol254:E505-E512 Adams IS, Sharma OP. Gacad MA. Sineer FR 1983 Metabolism of 25-hydroxyvitamin Da by cultured pulmonary alveolar macrophages in sarcoidosis. J Clin Invest 72:1856-1860 Koeffler HP, Reichel H, Bishop JE, Norman AW 1985 p-interferon stimulates production of 1,25-dihydroxyvitamin D3 by normal human macrophages. Biochem Biophys Res Commun 127:596-603 Marin L, Dufour ME, Tordet C, Nguyen TM 1990 1,25(OH)zD3 stimulates phospholipid biosynthesis and surfactant release in fetal rat lung explants. Biol Neonat 57:257-260 Nguyen TM, Guillozo H, Marin L, Dufour ME, Tordet C, Pike JW, Garabedian M 1990 1,25-dihydroxyvitamin D3 receptor in rat lung during the perinatal period: regulation and immunohistochemical localization. Endocrinology 127:1755-l 762 Morishige WK, Uetake CA 1978 Receptors for androgen and estrogen in the rat lung. Endocrinoloav 102:1827-1837 Voyno-YasenetskayaTA, Dobbs LG; Williams MC 1991 Regulation of ATP-dependent surfactant secretion and activation of second-messager systems in alveolar type II cells. Am J Physiol [4 Suppl] 261:105-109 Benson BJ, Williams MC, Sueishi K, Goerke J, Sargeant T 1984 Role of calcium ions in the structure and function of pulmonary surfactant. Biochim Biophys Acta 793:18-27 Haagwood HP, Hawgood S, Sargeant T, Bucklev D, White RT, Dridtamer K, Benson BJ 1987 The major lung surfactant protein; SF 28-36, is a calcium-dependent carbohvdrate-bindine I 1 “1 orotein. Biol Chem 262:13877-138’80 Abe E, Miyaura C, Tanaka H, Shiina Y, Kuribayashi T, Suda S, Nishii Y, DeLuca H, Suda T 1983 1,25-Dihydroxyvitamin D, promotes fusion of mouse alveolar macrophages both by a direct mechanism and by a spleen cell-mediated indirect mechanism. Proc Nat1 Acad Sci USA 80:5583-5587 Wientroub S, Winter C, Wahl SM, Wahl LM 1989 Effect of vitamin D deficiency on macrophage and lymphocyte function in the rat. Calcif Tissue Int 44:125-130 _
I
”
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Vol. 131, No. 6 Printed m US A
Society
Calbindin-D9K Gene Expression in the Lung Absence of Regulation by 1,25=Dihydroxyvitamin Estrogen* JEAN-MARIE DUPRET, FRANCOIS BERNAUDIN,
FABIENNE L’HORSET, CHRISTINE AND MONIQUE THOMASSET
PERRET,
of the Rat. D3 and
JEAN
INSERM U 120, H6pital Robert Debre, 75019 Paris; and Laboratoire d’Histologie-Biologie H6pital Tenon, 75020, Paris, France
Tumorale (J.-F.),
ABSTRACT Calbindin-D9K (CaBPSK) is classically considered to be the molecular expression of 1,25-dihydroxyvitamin Da. The hormone is known to regulate the rat CaBP9K gene in duodenal tissue at transcriptional and posttranscriptional levels. This study shows that the CaBP9K gene is expressed in the rat lung, and that this expression is probably not vitamin D- or estrogen-dependent. The CaBP9K gene is not expressed in alveolar macrophages, but CaBP9K messenger RNA (mRNA) was localized by in situ hybridization in alveolar epithelial cells. CaBP9K mRNA was detected as early as the 20th day of gestation. The quantity of CaBP9K mRNA gradually increased during growth, from 1-77 days
after birth, whereas the CaBP9K concentration dramatically increased from day 19 to day 20 of gestation. Vitamin D-deficient male rats (8 weeks old) were given a single injection of 1,25-dihydroxyvitamin D, (650 pmol/lOO g body wt) and killed 1 h and 24 h after injection. The hormonal treatment resulted in a rise in duodenal CaBP9K mRNA, but no significant change in lung extracted CaBP9K mRNA. Mature ovariectomized rats were injected with 17/3-estradiol (0.5 pg/lOO g body wt) and killed 24,48, and 72 h later. The CaBP9K mRNA concentration in the uterus was markedly dependent on estrogen; that of the lung was not. The factors regulating the CaBP9K gene expression in the lung remain to be determined. (Endocrinology 131: 2643-2648, 1992)
T
ization of CaBP9K messenger bridization.
vitamin D-dependent calcium-binding proteins (CaBP) or calbindin-D (see Ref. 1 for review) are a group of intracellular proteins which are considered to be one of the molecular expressions of the hormonal actions of the secosteroid 1,25-dihydroxyvitamin D3 [1,25(OH),D,] (for reviews see Refs. 2-4). These proteins bind Ca++ with a high affinity (association constant = lo6 M-‘), and share with other intracellular calcium-binding proteins a structural feature within their Ca++ binding regions called the EF hand domain (5). In mammals, vitamin D-dependent calbindin-D genes are expressed in the intestine (9-kilodalton CaBP, calbindinD9K or CaBP9K) (6, 7) and kidney (28-kilodalton CaBP, calbindin-D28K or CaBP28K) (8). The receptor-mediated action of 1,25(OHhD3 is believed to be exerted via direct interaction with DNA (9), as for osteocalcin (10) and osteopontin (11). The CaBP9K gene is also expressed in the uterus, where it is under the control of estrogen (12), and in other HE
tissues
(1) where
any
hormonal
control,
including
vitamin
Materials
by in situ hy-
and Methods
Materials Radioisotopes (32P- and %labeled) and hybridization membranes were purchased from Amersham Co. (Les Ullis, France). Enzyme for DNA labeling and RNA molecular mass markers were obtained from Boehringer Mannheim (Mannheim, Germany). 1,25(OHhD3 was kindly provided by Dr Uskokovic (Hoffmann LaRoche, Nutley, NJ). 17@Estradiol was purchased from Sigma (La Verpilliere, France). All other chemicals and reagents were analytical grade from standard suppliers.
Animals and treatments Male Sprague-Dawley rats were purchased from Charles River (St. Aubin Les Elbeuf, France). Three-week-old rats (-D group) were fed a vitamin D-free diet (Usine d’tllimentation Rationnelle, no. 6, Villemoisson sur Orge, France) with 0.50% calcium, 0.36% phosphorus, and were raised in the dark for 5 weeks. Hypocalcemia was used as index of vitamin D deficiency. The other rats were fed the same diet with vitamin D (2000 W/kg). 1,25(OH)2DS (650 pmol/lOO g body wt) was given ip into randomly sorted -D rats, and animals were killed 1 h and 24 h after injection. Mature ovariectomized rats were purchased from Charles River; 2 weeks later animals were given ip 17fi-estradiol (0.5 fig/100 g body wt) and were killed 24 h, 48 h, and 72 h later. Pregnant rats at known gestational stages (days 17-21) were subjected to cesarean section under pentobarbital sodium anesthesia, and the fetuses were rapid]! removed from the uterus. Each developmental study sample was obtained from the lungs of two animals. These studies were conducted in accordance with the NIH Guide for the Care and Use of Laborator! Animals.
D
dependency, remains to be determined. A recent immunological study suggested the expression of CaBP9K gene in the lung was independent of vitamin D (13). This study investigates the expression of the CaBP9K gene in the lung and the involvement of 1,25(OHhD3 and estrogen in its regulation. RNA from various rat tissues including the duodenum, uterus, kidney, and lung were extracted and hybridized with CaBP9K and CaBP28K cDNAs; lung CaBP9K levels were measured by RIA. We also examined the cellular localReceived April 2, 1992. Address all correspondence and requests for reprints Marie Dupret, INSERM U120, Hopital Robert Debre, Serurier, Bitiment Ecran +4, 75019 Paris, France. * In memoriam to Michel Rieutort.
RNA (mRNA)
to: Dr. Jean48 Boulevard
Tissuepreparation The rats were anesthetized with pentobarbital sodium and killed b\ decapitation. Blood from -D rats was collected for serum calcium 2643
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2644
CaBP9K
GENE
EXPRESSION
IN
THE
RAT
Endo. Voll31.
LUNG
measurement (complexometric titration). Tissues were rapidly and aseptically removed, washed, and snap-frozen in liquid nitrogen. Bronchoalveolar lavages were performed using 40 ml of a 0.9% NaCl solution. Alveolar macrophages were collected, sedimented by centrifugation, and frozen in liquid nitrogen. An aliquot of the macrophage suspension was stained (Field’s Staining Kit, Baxter, Maurepas, France) and counted. The yield was 230,000 cells/ml, free of blood and bronchial cell contamination. Total RNA was prepared by the Chomczynski and Sacchi method (14), poly(A)+ RNA was purified by chromatography on oligo (dT) cellulose (15), and lung CaBP9K quantified by RIA (16). For in situ hybridization lung tissue from mature rats was fixed by intratracheal instillation of 4% paraformaldehyde in PBS for 5 h. Ten-micrometer cryostat sections were subsequently performed.
Preparation
-pActin
qP2pglobuIin
of probes
Specific complementary DNA (cDNA) probes for rat CaBP9K and CaBP28K were prepared as previously described (17, 18). Mouse /3-actin and P-2 microglobulin cDNA probes were used as hybridization control. The estrogen-independent 1A cDNA (19) was also used as hybridization control. For in situ hybridization high specific activity 35S-labeled singlestranded antisense and sense CaBP9K-specific RNA probes were transcribed from the CaBP9K cDNA subcloned in the transcription vector pBluescript II SK (Stratagene, La Jolla, CA) (20). The specificity of the probes was checked in Northern blot analysis.
Hybridization
1992 No 6
_‘]CaBP 28K - CaBP 9K
to filters
The relative amounts of CaBP9K and CaBP28K mRNA in total extracted RNA were estimated by Northern blot hybridization (21) and quantified by dot blot hybridization (22). @-Actin and p-2 microglobulin probes were used as controls. The amounts of mRNA were quantified by spectrodensitometric analysis of peak areas (Shimadzu Scientific Instruments, Columbia, MD); peak area has been shown to be directly proportional to the amount of radioactivity bound to the filters (6). Lung mRNA levels and uterine mRNA levels were normalized by hybridization with fl-actin and IA cDNA probes, respectively.
In situ hybridization Fixed rat lung cryostat sections were processed for in situ hybridization as previously described (23). The preparations were hybridized for 5 h at 50 C. After hybridization the slices were washed in 50% fornamide/2-fold standard saline citrate (SSC) at 52 C and incubated in a solution of RNase (Sigma, St. Louis, MO; 40 fig/ml) for 30 min at 37 C. For autoradiography slices were dipped in Amersham emulsion LMl and were developed after 2 weeks exposure. After development the autoradiographs were Giemsa stained.
FIG. 1. Calbindin-D
mRNA in tissues of vitamin D-replete rats. Eightweek-old rats were killed, and poly(A)+ RNA was extracted from the duodenal mucosa (D), liver (Li), spleen (S), lung (Lg), heart (H), and kidney (K). The RNAs were denatured with formaldehyde, fractionated on a 1.3% agarose gel (10 pg/sample), and blotted on a membrane filter. RNAs were then sequentially hybridized with the CaBP9K and CaBP28K [ol-a’P]cDNAs (bottom) and with the P-actin and p-2 microglobulin [Wa*P]cDNAs (top). A size scale was determined by coelectrophoresis of RNA markers.
1-n
Lg
Lg-M
M
I
D 1
- p Actin
Results Calbindin-D
mRNA detection
RNAs were extracted from the adult rat duodenal mucosa, liver, spleen, lung, heart, and kidney. Poly(A)+ RNA were then analyzed by Northern blot hybridization with 32Plabeled probes (Fig. 1). The CaBP9K cDNA detected a single 550-nucleotide mRNA speciesin the duodenum and lung RNA, whereasno signalwas detected in kidney, liver, spleen, and heart RNA. CaBP28K mRNA [a major 1.9-kilobase (kb) and two minor 2.8- and 3.2-kb species]was only found in the kidney; no signal was recorded elsewhere. @Actin (a 2.2kb species)and P-2 microglobulin (0.8- and 1-kb species) mRNA were detected in all the samples analyzed. No CaBP9K mRNA was detected in alveolar macrophage RNA, and no difference in CaBP9K mRNA concentration was found between intact lung and macrophage-cleared lung
-CaBP 9K
FIG. 2. CaBP9K
mRNA distribution in the lung of vitamin D-replete rats. Lungs from 8-week-old rats were given successive bronchoalveolar lavages and removed. Alveolar macrophages from three animals were collected as described in Materials and Methods. Total RNA (10 pg) from lung (Lg), macrophage-cleared lung (Lg-M), and macrophages (M) was prepared as described in the legend of Fig. 1. Total RNA from the duodenum (D) was used as control. RNAs were hybridized with CaBP9K [w”“P] and with /3-actin [w~‘P]cDNAs.
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CaBP9K
GENE
EXPRESSION
(Fig. 2). The presence of CaBP9K mRNA transcripts was localized by in situ hybridization in alveolar epithelial cells. No detection was observed in bronchiolar or pleural cells (Fig. 3). Lung CaBP9K
during
on lung CaBP9K
2645
were given a single injection of 17/3-estradiol there was a great increase in the uterine CaBP9K mRNA concentration (Fig. 7D), but extracts of CaBP mRNA from the lung (Fig. 7A) or duodenum (Fig. 7B) showed no change. Discussion
mRNA accumulation
A single 1,25(OHbD3 injection in vitamin D-deficient rats resulted in a dramatic increase in the duodenal CaBP9K mRNA. On the other hand, for CaBP9K extracted from the lung and after normalization with /3-actin levels, no statistical difference (by t test) could be recorded, either between vitamin D-replete (N) and vitamin D-deficient rats (OD) or between uninduced (OD) and vitamin D-induced rats (1 h and 24 h). (Fig. 6). Effects of 17/3-estradiol
RAT LUNG
the rat development
Lung CaBP9K mRNA was analyzed and quantified during the pre- and postnatal stages of rat development. CaBP9K mRNA was not detected until 20th day of pregnancy; the relative amounts then increased 20-fold until the rats were 77 days old, i.e. adult (Fig. 4). Northern blot analysis on a 1.3% denaturing agarose gel revealed no change in the electrophoresis properties of CaBP9K mRNA (Fig. 5). The lung CaBP9K concentration increasedrapidly (16 times) after the 19th day in utero and then more slowly (2-fold) until age 28 days (Table 1). Effects of 1,25(OH)zD3
IN THE
on lung CaBP9K
mRNA
accumulation
Total RNAs extracted from the duodenum, liver, lung, and uterus of mature ovariectomized rats were fractionated on a 2% agarosegel and analyzed by Northern blot hybridization with CaBP9K cDNA (Fig. 7, C-D). In all cases, a 550nucleotide mRNA was detected, and a second larger species was hybridized in uterus mRNA. When ovariectomized rats
FIG. 3. Autoradiograms of rat lung cryostat sections after in situ hybridization using 35S-labeled CaBPSK-specific antisense (A) and sense (B) RNA probes. In situ hybridization procedures are described in Materials and Methods. Alveolar epithelial cells are labeled with the antisense probe (arrows) (A), whereas they are not labeled with the sense probe (B) (X 400). The horizontal lines in the lower left corners of A and B represent 20 pm.
This study analyzes the molecular expression of the CaBP9K gene in the rat lung. The duodenum is the best known site of CaBP9K gene expression in the rat (1). Earlier developmental studies (16) reported that the rat duodenum contains small amounts of CaBP9K as early as gestational day 17, with a slight surge in CaBP9K concentration at birth, and a steady increase to the adult level reached by day 30 postpartum. CaBP9K mRNA was detected in the rat lung as early as gestational day 20. The CaBP9K concentration undergoes a dramatic increase between gestational days 19 and 20. The quantities of both CaBP9K and CaBP9K mRNA then increase throughout development. These data suggest that CaBP9K gene expression is not directly related to physiological events occurring during late fetal lung development, such as differentiation of alveolar epithelial cells to surfactant-secreting type II pneumocytes (24), or to such perinatal events as the increase in the alveolar fluid calcium concentration (25). Interestingly, CaBP9K mRNA has also been detected in human fetal lung, as early as 14 weeks of gestation with no significant change until the fetus is 32 weeks old (26). This in viva study also provides new evidence for tissuespecific regulation of the CaBP9K gene in the rat. We previously reported the transcriptional and posttranscriptional regulation of CaBP9K gene expression in the rat duodenum by 1,25(OH)2D3 (7). A single 1,25(OHhD3 injection induces a dramatic increase in duodenal CaBP9K mRNA in vitamin D-deficient rats, but the lung CaBP9K mRNA content does not change. These findings confirm earlier reports suggesting
a
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CaBP9K
2646
GENE EXPRESSION
IN THE RAT LUNG
rLung CaBP9K mRNA during development. Lung total RNAs were extracted from vitamin D-replete rats aged 19 days in utero to 77 days. Lungs from two animals were pooled for each sample; two samples were used in each analysis. Total RNAs were dotted on a membrane filter, fixed by heating, and sequentially hybridized to the CaBP9K and p-actin [LU-~‘P]CDNAS. All measurements of mRNA were carried out with total RNA in the range 1.25-10 ag/dot in the presence of excess DNA. CaBP9K mRNA was quantified by spectrodensitometric analysis of peak areas and was expressed as the ratio of CaBP9K to P-actin areas (arbitrary units). FIG.
4.
iz
i
Endo. Voll31.
.I
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19d [
-3
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20d
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...+**r* ...* .** r....
14d
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1992 No 6
.*r*...r
l
2
*.*
A
1
_
28d
q# / *
1
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‘>
CaBP9K RNA BldTH TABLE
LS
-p Actin
e I
- CaBP9K +it
1
Days beforet
3
7 10 14 28 77I Days after
BIRTH FIG. 5. Northern blot hybridization of poly(A)+ RNA extracted from vitamin D-replete rats. Poly(A)+ RNA from the lung (Lg) was extracted from vitamin D-replete rats aged 19 days in utero to 77 days. RNA was denatured with formaldehyde, fractionated on a 1.3% agarose gel (1 pglsample), and blotted on a membrane filter. mRNA was then hybridized with the CaBP9K and P-actin [cx-~*P]cDNAs. Poly(A)+ RNAs from adult rat duodenum (D) and liver (Li) were used as positive and negative controls, respectively.
that the lung CaBP9K gene expression was not vitamin Ddependent. But these conclusions were only based upon measurements of CaBP9K by RIA or by immunoblotting. Thomassetand Tenenhouse (27), using 20-day and 30-dayold fifth-generation vitamin D-deficient rats, found no decreasein lung CaBP9K concentration. Verhaeghe et al. (28) also showed that the CaBP9K concentration of both the maternal and fetal lungs did not change when the fetal and maternal plasma 1,25(OHhD3 levels were altered. Recently, Walters et al. (13) used immunoblot analysis to demonstrate the vitamin D-independence of CaBP9K in the lung of 7 to 9-week-old second generation vitamin D-deficient rats. Nevertheless, these findings could not exclude a possible
1. Changes in Lung CaBP9K concentration Rat age (days) 17 19 20 21 1 3 7 10 14 20 28
' with rat age
CaBPSK (ng/mg protein) 2.8 3.5 57 94 Birth 60 92 104 105 82 98 124
Lung CaBP9K concentrations were determined by RIA from vitamin D-replete rats aged 17 days in utero to 28 days. Lungs from two animals were pooled for each sample. Two samples corresponding to those shown in mRNA analysis (Fig. 4) were used in each mean CaBP9K concentration for rats aged 17 days in utero is a single value obtained from four pooled fetuses.
translational effect of 1,25(OH)2D3upon vitamin D-dependent CaBP9K mRNA. Our data demonstrate that circulating 1,25(OH)2D3exerts no detectable effects upon the concentration of lung CaBP9K mRNA. Alveolar macrophages could also be involved in the synthesis of 1,25(OH)2D3 and consequently alter the vitamin D deficiency in a paracrine way. This is unlikely, sinceendogenous 1,25(OH)2D3synthesishas only been reported in pathological (29) or induced situations (30). It is noteworthy that 1,25(OH)zDs binding sites have only been detected in the rat lung during prenatal life (31), when the secosteroidplays a role in phospholipid synthesis and in surfactant secretion from type II pneumocytes (32). 17P-Estradiol stimulates uterine CaBP9K gene expression by induction of two mRNA species(12). Receptorsfor estrogen have also been detected in the rat lung (33). However, our data indicate that estrogen is unlikely to have any effect upon lung CaBP9K mRNA levels; current experiments seem
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CaBP9K
GENE
LUNG
r
EXPRESSION
-I-I
lh
00
II_II”
24h DUODENUM
1 -/3 Actin
a --
-CaBP 9K
FIG. 6. Effect of 1,25(OH)ZDs on CaBP9K mRNA concentration in the lung and duodenum of vitamin D-deficient rats. Vitamin D-deficient rats (OD) were killed 1 h or 24 h after a single injection of 1,25(OH)zD3 (650 pmol/lOO g). Ten micrograms of total RNA were denatured with formaldehyde, fractionated on a 1.3% agarose gel, transferred to a filter, and hybridized with the CaBP9K and /3-actin [cu-32P]cDNAs. Duodenum and lung were removed from the same individuals. Lung total RNAs from vitamin D-replete rats (N) were also blotted.
IUUU ovx
LUNG
DUODENUM
p
I
,
IL--JUU
ovx
24h 48h 72h
D L Lg Ut 03nnnn *=-
2647
24h
OD
@
RAT LUNG
to exclude glucocorticoid as a potential hormonal regulatory factor. The physiological significance of our results remains to be clarified. Pulmonary surfactant-producing cells, also named type II alveolar epithelial cells or type II pneumocytes (24), are potential sites for the expression of the CaBP9K gene. Indeed, the calcium concentration plays a role as an intracellular messengerin the secretory process(34) and in maintaining the extracellular structure and function of pulmonary surfactant (35, 36). Our data eliminate alveolar macrophages as putative candidates for CaBP9K gene expression. This finding is important since alveolar macrophages contain 1,25(OH)*D3 receptors (2) and are biological targets for 1,25(0H)2D3-induced cell fusion (37,38). Lung cells expressing CaBP9K mRNA have been identified as alveolar epitheha1cells; these cells could be a new model for the cell-specific regulation of CaBP9K gene expression.
I
-p Actin
N
IN THE
@ In
24h 48h 72h
UTERUS II 7 "*.-
-lA-
-CaBPSKUUUU
OVX 24 48 72 h FIG. 7. Effect of 17@-estradiol on CaBP9K mRNA concentration in the lung, duodenum, and uterus of ovariectomized rats. RNAs were extracted from the uterus (Ut), liver (L), duodenum (D), and lung (Lg) of mature ovariectomized rats. Ten micrograms of total RNA were fractionated on 1.3% (A and B) or 2% (C and D) denaturing agarose gels, transferred, and hybridized with CaBP9K [cr-32P]cDNA. Lung (A) and duodenum (B) mRNAs were also hybridized with @-actin [o~-~~P] cDNA, whereas uterine (D) and control mRNA [duodenum (D), liver (L), lung (Lg)] (C and D) were hybridized with 1A [cx-~‘P]cDNA. One microgram of duodenal RNA was used in C and D.
Acknowledgments We are indebted to Martine Bovet for her expert technical assistance. We are grateful to Dr. Lyttle for 1A probe. We thank Institut National de la Santi et de la Recherche Midicale SC 6 personnel, Claudine Brunner, and Dr. Owen Parkes for their help in the preparation of the manuscript. References 1. Christakos S, Gabrielides C, Rhoten WB 1989 Vitamin D-dependent calcium-binding proteins: chemistry, distribution, functional consideration and molecular biology. Endocr Rev 10:3-25 2. Haussler MR 1986 Vitamin D receptors: nature and function. Annu Rev Nutr 6527-562 3. Minghetti PP, Norman AW 1988 1,25(OHh vitamin D3 receptors: gene regulation and genetic circuitry. FASEB J 2:3043-3053 4. Pike JW 1991 Vitamin D3 receptors: structure and function in transcription. Annu Rev Nutr 11:189-216 5. Kretsinger RH 1980 Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem 8:119-174 6. Perret C, Desplan C, Thomasset M 1985 Cholecalcin (a 9-kDa cholecalciferol-induced calcium-binding protein) messenger RNA. Distribution and induction by calcitriol in-the rathigestiveyract. Eur J Biochem 150:211-217 7. Dupret JM, Brun P, Perret C, Lomri N, Thomasset M, CuisinierGleizes P 1987 Transcriptional and post-transcriptional regulation of vitamin D-dependent calcium-binding protein gene expression in the rat duodenum by 1,25-dihydroxycholecalciferol. J Biol Chem 262:16553-16.557 8, Varghese S, Deaven LL, Huang YC, Gill RC, Iacopino AM, Christakos S 1989 Transcriptional regulation and chromosomal assignment of the mammalian calbindin-D28K gene. Mol Endocrino1 3:495-502 9. Beato M 1989 Gene regulation by steroid hormones. Cell 56:335344 10 Ozono K, Liao J, Kerner SA, Scott RA, Pike JW 1990 The vitamin D-responsive elements in the human osteocalcin gene. J Biol Chem 265:21881-21888 11. Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT 1990 Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Ssp-1 or osteopontin) gene expression. Proc Nat1 Acad Sci USA 87:99959999 12. L’Horset F, Perret C, Brkhier A, Thomasset M 1990 17/3-estradiol stimulates the calbindin-D9K (CaBP9K) gene expression at the transcriptional and post-transcriptional levels in the rat uterus. Endocrinology 127:2891-2897
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2648
CaBP9K
GENE
EXPRESSION
13. Walters MR, Bruns ME, Carter RM, Riggle PC 1991 Vitamin D independence of small calcium-binding proteins in nonclassical target tissues. Am J Physiol 260:E794-ES00 P, Sacchi N 1987 Single-step method of RNA isola14. Chomczynski tion by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 H, Leder P 1972 Purification of biologically active globin 15. Aviv messenger RNA by chromatography on oligothymidylic acid cellulose. Proc Nat1 Acad Sci USA 69:1408-1412 16. Thomasset M, Parkes 0, Cuisinier-Gleizes P 1982 Rat calciumbinding proteins: distribution, development and vitamin D dependence. Am J Physiol 243:E483-E488 17. Desplan C, Thomasset M, Moukhtar MS 1983 Synthesis, molecular cloning, and restriction analysis of DNA complementary to vitamin D-dependent calcium-binding protein mRNA from rat duodenum. J Biol Chem 258:2762-2765 N, Thomasset M 1989 Cloning and 18. Lomri N, Perret C, Gouhier analysis of calbindin-D28K cDNA and its expression in the central nervous system. Gene 80:87-98 19. Hsu CJ, Komm BS, Lyttle CR, Frankel F 1988 Cloning of estrogenregulated messenger ribonucleic acids from rat uterus. Endocrinology 122:631-639 20. Johnson MT, Johnson BA 1984 Efficient synthesis of high specific activity 35Slabeled human /3 globin pre mRNA. Biotechniques 2:156-162 21. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci, USA 77:5201-5205 FC 1982 Cytoplasmic dot hybridization: simple 22. White AB, Bancroft analysis of relative mRNA levels in multiple small cell or tissue sample. J Biol Chem 257:8569-8572 23. Lebargy F, Bulle F, Siegrist S, Guellain G, Bernaudin JF 1990 Localization by in situ hybridization of y glutamyl transpeptidase mRNA in the rat kidney using 35s labeled RNA probes. Lab Invest 62:731-735 24. Haagwood HP, Van Golde LM 1991 Synthesis and assembly of lung surfactant. Annu Rev Physiol 53:441-464 25. Eckenhoff RG 1989 Potential changes in lung surfactant calcium measured in situ. J Clin Invest 84:1295-1301 26. Brun P, Dupret JM, Perret C, Thomasset C, Mathieu H 1987 Vitamin D-dependent calcium-binding proteins (CaBI’s) in human fetuses: comparative distribution of 9K CaBP mRNA and 28K CaBP
IN
27
28
29.
30.
31.
32.
33. 34.
35.
36.
37.
38.
THE
RAT
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LUNG
1992 No 6
during development. Pediatr Res 21:362-367 Thomasset M, Tenenhouse A 1988 Vitamin D dependence of calbindinD9K and calbindin-D28K synthesis in various tissues. In: Norman AW, Schaefer K, Grigoleit HG, Herrath DV (eds) Vitamin D. Molecular, Cellular and Clinical Endocrinology. Walter de Gruyter, Berlin, New-York, pp 516-517 Verhaeghe J, Thomasset M, Brehier A, Van Assche FA, Bouillon R 1988 1,25(OH)rDa and Ca-binding protein in fetal rats: relationship to the maternal vitamin D status. Am J Physiol254:E505-E512 Adams IS, Sharma OP. Gacad MA. Sineer FR 1983 Metabolism of 25-hydroxyvitamin Da by cultured pulmonary alveolar macrophages in sarcoidosis. J Clin Invest 72:1856-1860 Koeffler HP, Reichel H, Bishop JE, Norman AW 1985 p-interferon stimulates production of 1,25-dihydroxyvitamin D3 by normal human macrophages. Biochem Biophys Res Commun 127:596-603 Marin L, Dufour ME, Tordet C, Nguyen TM 1990 1,25(OH)zD3 stimulates phospholipid biosynthesis and surfactant release in fetal rat lung explants. Biol Neonat 57:257-260 Nguyen TM, Guillozo H, Marin L, Dufour ME, Tordet C, Pike JW, Garabedian M 1990 1,25-dihydroxyvitamin D3 receptor in rat lung during the perinatal period: regulation and immunohistochemical localization. Endocrinology 127:1755-l 762 Morishige WK, Uetake CA 1978 Receptors for androgen and estrogen in the rat lung. Endocrinoloav 102:1827-1837 Voyno-YasenetskayaTA, Dobbs LG; Williams MC 1991 Regulation of ATP-dependent surfactant secretion and activation of second-messager systems in alveolar type II cells. Am J Physiol [4 Suppl] 261:105-109 Benson BJ, Williams MC, Sueishi K, Goerke J, Sargeant T 1984 Role of calcium ions in the structure and function of pulmonary surfactant. Biochim Biophys Acta 793:18-27 Haagwood HP, Hawgood S, Sargeant T, Bucklev D, White RT, Dridtamer K, Benson BJ 1987 The major lung surfactant protein; SF 28-36, is a calcium-dependent carbohvdrate-bindine I 1 “1 orotein. Biol Chem 262:13877-138’80 Abe E, Miyaura C, Tanaka H, Shiina Y, Kuribayashi T, Suda S, Nishii Y, DeLuca H, Suda T 1983 1,25-Dihydroxyvitamin D, promotes fusion of mouse alveolar macrophages both by a direct mechanism and by a spleen cell-mediated indirect mechanism. Proc Nat1 Acad Sci USA 80:5583-5587 Wientroub S, Winter C, Wahl SM, Wahl LM 1989 Effect of vitamin D deficiency on macrophage and lymphocyte function in the rat. Calcif Tissue Int 44:125-130 _
I
”
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