Communication THE JOURNAL OF BKXOGICAL CHEMISTRY Vol. 260, No. 9, Issue of May 10, pp. 5209%5212,1985

0 1985 by The American Society of Biological Chemists, Inc. Printed in U.S.A.

l&,25-Dihydroxyvitamin D- induced Modification of a Cytosolic Protein in Embryonic Chick Intestine*

(Received for publication, October 22,1984) Charles W. Bishop+, Nancy C. Kendrick, Margaret C. Dame, and Hector F. DeLuca

From the Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences, Madison, Wisconsin 53706

Two-dimensional electrophoresis together with ra- diolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin DI. 1,25-Dihydroxyvitamin DB caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI,5.1) after a IO-min pulse with radioisotope followed by a 4-h chase. Decreased [‘4C]leucine content of the same pro- tein was also observed at various times following 1,25- dihydroxyvitamin Da addition to culture media; a sig- nificant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The re- sults argue that 1,25-dihydroxyvitamin Ds causes either a decreased synthesis rate or a post-transla- tional modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin Ds.

The hormonally active metabolite of vitamin DS, namely 1,25-dihydroxyvitamin Ds (1,25-(OH)2D,‘), acts on the small intestine to stimulate calcium and phosphate absorption. The mechanisms by which this hormone achieves these physiolog- ical responses are presently unknown, but recent studies with chicks have identified rapid changes within the intestinal cell that occur after its administration. De novo synthesis of calcium-binding protein has been demonstrated by 2 h in the d-week-old chick (l-4) and by 1 h in the chick embryo (5). Increased synthesis of brush-border and mitochondrial mem- brane proteins has also been reported by 2-4 h (6, 7). Other rapid changes include alterations in phospholipid metabolism (a), increases in ornithine decarboxylase and spermidine- binding activities (9, lo), and variations in the intracellular levels of CAMP and cGMP (11, 12). Further study of these changes is expected to lead to a better understanding of vitamin D action.

* This work was supported by Program Project Grant AM-14881 and Postdoctoral Fellowship AM 06692 from the National Institutes of Health and by the Harry Steenbock Research Fund of the Wiscon- sin Alumni Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Proctor and Gamble, 11511 Reed Hartman High, Cincinatti, OH 45206.

1 The abbreviation used is: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3.

In our continuing investigation of calcium-binding protein, we have become interested in other cellular proteins that are influenced by 1,25-(OH)zD3. Using the highly sensitive tech- niques of autoradiography and fluorography in combination with two-dimensional polyacrylamide gel electrophoresis, we have discovered two new proteins in the embryonic chick duodenum that are rapidly changed by the hormone.

MATERIALS AND METHODS

Chemicals and Media-X-Omat AR-5 x-ray film, GBX developer, and GBX fixer were obtained from Eastman Kodak Co. L-[U-‘~C] Leucine (336 mCi/mmol) and L-[4,5-3H]leucine (157 Ci/mmol) were purchased from Amersham, and EN3HANCE was purchased from New England Nuclear. Crystalline 1,25-(OH)2D3 was donated by the Hoffmann-La Roche, and streptomycin, penicillin, and Waymouth’s 752/l medium were purchased from Gibco. Medium containing 150 nM 1,25-(OH)zDa was prepared by adding the hormone in ethanol, and control medium was prepared by adding an equivalent volume of ethanol alone (<O.l%). Media with radiolabel was prepared according to the published formulation for Waymouth’s 752/l medium (13) with reductions in the leucine concentration and containing 300 PM [‘?]leucine at a final specific activity of 336 mCi/mmol or 38 pM 13H]leucine at a final specific activity of 13.2 Ci/mmol. Penicillin (50 units/ml) and streptomycin (50 fig/d) were added to all media before use.

Tissue Cultures and Sample Preparation-Duodena from 19-day- old chick embryos (Sunnyside Hatcheries, Oregon, WI) were cultured by the method of Corradino (14) as modified by Franceschi and DeLuca (15). After culturing, the duodena were rinsed in 4 “C buffer (50 InM POI, 150 mM NaCl, pH 7.4), placed in separate vials, and frozen on dry ice. At a later time, the contents of each vial were defrosted and transferred to a Potter-Elvehjem homogenizer, contain- ing an additional 0.5 ml of buffer, and homogenized on ice with a motor-driven pestle. The homogenates were centrifuged (100,000 x g; 45 min; 4 “C) and the supernatants containing the cytosolic proteins were recovered. Cytosolic extracts were analyzed for radiolabel incor- poration (16) and protein content (17) and were lyophilized and resuspended in sodium dodecyl sulfate-free lysis buffer (18) to a final concentration of 6 mg/ml.

Polyacrylamide Gel Electrophoresis-A portion of each cytosolic extract was resolved by two-dimensional polyacrylamide gel electio- phoresis (18) by the Kendrick Laboratory (Madison, WI). Isoelectric focusing was carried out at 400 V for 12 h and 800 V for the following 30 min, and molecular weight separation was completed in 9 or 12% acrylamide gels (120 X 150 x 0.75 mm). The pH gradient achieved during isoelectric focusing was monitored by acetylated cytochromes (Calbiochem-Behring) applied to separate tube gels. Molecular weight separation was monitored by the migration of internal standards (myosin, 220,000; phosphorylase a, 94,000; catalase, 60,000; and actin, 43,000) added to the top of each slab gel before electrophoresis in the second dimension. The finished gels were placed in an aqueous solution containing 10% (w/v) trichloroacetic acid, 10% (v/v) glacial acetic acid, and 30% (v/v) methanol and stored overnight at -20 “C. On the following day, gels containing 3H-labeled proteins were rehy- drated for 1 h in 10% (v/v) glacial acetic acid, soaked (with shaking) for 1 h in 85 ml of EN3HANCE, and further soaked for 1 h in water. All gels were dried on filter paper for 30 min at 80 “C using a Bio- Rad dryer.

Fluorographs and Autoradiographs-Dried gels were exposed to preflashed X-Omat AR-5 x-ray films as previously described (5) for 40 days at -80 “C. Gels containing 14C-proteins were exposed in similar fashion to nonpreflashed X-Omat AR-5 x-ray film for 40 days at 22 “C. Calibration strips prepared with Y-labeled proteins were also exposed to this latter set of films. After exposure, all films were developed in GBX developer (5 min at 20 “C) and fixed in GBX fixer (3 min).

5209

5210 1,25-Dihydroxyuitamin D and Chick Intestine

RESULTS

Puke-Chase Experiment-Two groups of embryonic chick duodena were cultured for 20 h in defined medium containing 380 PM leucine. One group was continuously exposed to 150 nM 1,25-(OH)2D3; the other group was never exposed to hormone and served as a control. After 20 h, both groups were transferred to medium containing 38 PM [3H]leucine for 10 min (pulse) and to a larger volume of nonradiolabeled medium containing 380 PM unlabeled leucine for the next 4 h (chase).

the radiolabeled proteins were visualized by two-dimensional polyacrylamide gel electrophoresis followed by fluorography.

Visual examination of the fluorographs showed that except for vitamin D-dependent calcium binding protein, which was induced by the hormone, all proteins present in extracts from the 1,25-(OH)zD3-treated duodena were qualitatively present in extracts from control duodena. However, the incorporation of [3H]leucine into another protein was quantitatively changed by 1,25-(OH)2D3 treatment in an obvious fashion. This protein, shown in Fig. 1 ( 4 and arrows), was labeled to a much lesser extent in extracts from treated duodena uersus control. Protein 4 has an approximate M, value of 18,000 and an isoelectric point of about 5.1.

Quantitative analysis of the fluorographs revealed that the radiolabeling of protein 4 in treated duodena was reduced to about 15% (p < 0.001) of that observed for control embryos as shown in Fig. 2, while the relative incorporation of [3H] leucine for neighboring polypeptides 1, 2, and 3 was not changed.

, , .' 1

. . . .. ! Cytosolic extracts were prepared from each duodenum, and

""I I r

. ..

4:l 4:2 4:3 1:2 1:3 2:3

polypeptide spot number

FIG. 2. Densitometric analysis of fluorographs produced in the pulse-chase experiment. Film images shown in Fig. 1 were digitized (5) and entered into the McIDAS. The total density of polypeptides 1-4 in Fig. 1 was determined by spot integration after subtraction of background density. For each of the 3 films, the integrated density of spot 4 was divided by the integrated density of spots 1-3. The density of spot 1, divided by the density of spots 2 or 3 and spot 2 divided by 3, is also shown. The figure shows mean density ratios (n = 3 films/group) It: S.D. for duodena cultured for 20 h in the presence of 1,25-(OH)& uersus vitamin D-deficient control duodena. *, p < 0.001 for difference between 1,25-(OH)ZD3-treated and control group.

tissues were electrophoresed in 12% polyacrylamide gels (240 pg of protein and 3.17-4.66 X lo5 dpm/gel) and the radiolabeled proteins were visualized on x-ray films by fluorography. Matching areas of 6

FIG. 1. Detection of a post-translational modification films are shown here. Films A-C display proteins from 3 control caused by 1,25-(OH)zDs. A pulse-chase experiment was completed duodena and films D-F display proteins from 3 duodena cultured in which embryonic chick duodena were cultured with or without with hormone. The protein identified by arrows (4 in A ) is greatly 1,25-(OH)& for 20 h, pulsed with [3H]leucine for 10 min, and reduced in abundance relative to the surrounding proteins (1-3) in cultured for another 4 h. Cytosolic proteins extracted from these tissue exposed to 1,25-(OH),D3 (see Table I).

1,25-Dihydroxyvitamin D and Chick Intestine 5211

Time-course Experiment-Nine groups of duodena were cultured for 24 h in defined medium and were transferred to medium containing 150 nM 1,25-(OH)2D3 at 0.5, 0.75, 1, 1.5, 2, 2.5, 4, 6, and 20 h before the end of the culture period. A tenth group (control) was similarly handled but not exposed to the hormone. All groups of duodena were incubated in medium containing [14C]leucine during the final 30 min of the culture period.

Cytosolic proteins extracted from these duodena were re- solved by two-dimensional polyacrylamide gel electrophoresis, and those proteins labeled with [14C]leucine were detected on x-ray film by direct autoradiography. The films were digitized by scanning densitometry and entered into the Man-computer Interactive Data Access System (McIDAS). By reference to standard curves (obtained from acrylamide strips containing known amounts of 14C protein and exposed to the films simultaneously with the two-dimensional gels (5)), the radio- activity contained within protein 4 as well as in 16 arbitrarily selected proteins having similar radioactivity and electropho- retic mobility was measured using the McIDAS system. The positions of these proteins relative to that of protein 4 are shown in Fig. 3.

As summarized in Table I, the radioactivity contained in 15 of these 17 proteins was unchanged from control levels by exposure to 1,25-(OH)2D3 for 0.5 or 20 h. The radioactivity within protein 4 was reduced ( p < 0.05) by approximately 40% after 0.5 h of exposure to hormone. After 20 h, a further 10% reduction was observed ( p < 0.01). Increases in radioac- tivity (p < 0.005) were seen for another protein (protein 6) at both time points. This protein was similar in isoelectric point to protein 4 but had a larger molecular weight. The full time courses of 1,25-(OH)2D3-related changes in proteins 4 and 6 are shown in Fig. 4.

DISCUSSION

The influence of 1,25-(OH)2D3 on the synthesis and intra- cellular processing of cytosolic proteins was examined in a pulse-chase experiment with embryonic chick duodena. In

. . ,.. ."-T-q=,-"":~ ,.e-. 3w ..*p"x - Y

- i

1

FIG. 3. Autoradiograph from the time-course experiment (not pulse-chase) showing the 17 proteins selected for com- puter analysis. Embryonic chick duodena were cultured for 24 h and exposed to 1,25-(OH)2D3 at various times before the end of the culture period. During the final 30 min of incubation, all duodena were pulsed with ['4C]leucine. Cytosolic proteins extracted from these tissues were electrophoresed in 9% polyacrylamide gels (150 pg of protein and 1.2 X lo5 I4C dpm/gel) and the radiolabeled proteins were visualized on x-ray films by direct autoradiography. The protein undergoing 1,25-(OH)2D3-dependent modification (4 above; arrows in Fig. 1) and 16 other proteins having similar charge and size charac- teristics were analyzed by computer. These proteins are numbered in order of increasing image density (see Table I). The relative positions of the proteins seen in this autoradiograph are not identical to those seen in fluorographs from the pulse-chase experiment (Fig. 1) because ( a ) different acrylamide concentrations were used in the gels, and ( b ) protein modifications occurred during the 4-h chase.

TABLE I Incorporation of radiolabel into 17 cytosolic proteins by duodena

exposed to 1,25-(0H)& for 0, 0.5, or 20 h Digitized density values from autoradiographs produced in the

time-course experiment were converted to disintegrations of I 4 C by reference to a standard curve. The radioactivity contained in 17 protein spots (Fig. 3) was determined by integrating the 14C disinte- grations calculated for area within the spot's boundaries.

Disintegrations "C X 106/40-day exposure Protein (11 2 S.E.; n = 3)

spot O h 0.5 h 20 h

1 2

3.26 f 0.71 2.63 f 0.30 2.78 f 0.17 3.92 f 0.88 4.20 f 0.42 3.70 f 0.54

3 4.79 f 0.40 4.38 f 0.71 5.24 f 0.63 4 14.1 f 1.4 8.65 f 1.03" 7.12 f 0.50b 5 6

5.35 f 0.22 5.97 f 0.57 5.09 f 0.38

7 6.18 f 0.43 10.00 f 0.51' 9.68 f 0.86'

8 6.29 f 0.62 5.21 f 0.55 5.65 f 0.63

9 6.84 f 0.32 7.76 f 0.89 7.47 f 0.52 7.06 f 0.21 6.61 f 0.55 6.97 f 0.59

10 11

7.66 ? 0.15 6.98 f 0.48 6.77 f 0.91 8.73 f 1.27 7.02 f 1.25 7.06 f 0.81

12 13

10.2 f 1.2 9.37 f 1.22 11.0 f 0.4

14 12.8 f 0.9 11.2 f 0.5 12.4 f 1.1

15 5.25 f 1.49 3.72 f 0.26 4.51 f 0.56 15.9 f 1.8 13.7 f 2.6 15.1 f 2.7

16 17

18.9 f -d 18.9 f -d 18.9 f -d

92.4 f 16.1 104 f 6 82.5 f 8.5 " p < 0.05 versus 0-h control. b p < 0.01 uersus 0-h control. ' p < 0.001 versus 0-h control. dData from each film were multiplied by a constant so that the

disintegrations in spot 16 were identical for all films. This normali- zation was necessary to correct for animal variability. Spot 16 was selected for this normalization because it appears on the autoradi- ographs as a prominent, well-resolved spot. Its selection, however, was based on the assumption that its synthesis and subsequent modification is unaffected by 1,25-(OH)&. The stability of the normalized data indicate that this assumption is valid.

this experiment, proteins in 1,25-(OH)2D3-treated and control duodena were radiolabeled during a 10-min pulse and exam- ined after 4 h of intracellular processing. One cytosolic protein was found to have a greatly reduced radiolabeled protein in duodena treated with hormone, a result which can be attrib- uted to one of three mechanisms. 1) 1,25-(OH)2D3 did not affect the synthetic rate of the protein but caused the protein to undergo post-translational modification. Under this mech- anism, the protein would be synthesized at identical rates in both groups of duodena but would undergo a major charge/ size modification or intracellular redistribution only in duo- dena treated with hormone. Such a modification could have involved phosphorylation, acetylation, aggregation (or the converse processes), or degradation, thereby producing a pro- tein that no longer resolved in the two-dimensional gel. Intra- cellular redistribution could have involved transfer from the cytosolic fraction to a membrane fraction. 2) 1,25-(OH)& inhibited the protein's synthesis but did not affect subsequent intracellular processing. Under this mechanism, the reduced concentration of label resulted from a decreased synthetic rate. 3) 1,25-(OH)zD3 acted by a combination of both mecha- nisms. The results to date can neither prove nor exclude any of these mechanisms. However, since only a 50% reduction of label in the protein in the presence of 1,25-(OH)& was observed with a 30-min labeling period with no unlabeled chase and an 85% reduction was found with a 10-min pulse followed by a 4-h chase, the more likely explanation is that 1,25-(OH)zD3 directly or indirectly affects a post-transitional event. Certainly a major effort will now be devoted to under- standing the site of action of 1,25-(OH)zD3 in this system. In

5212 1,25-Dihydroxyvitamin D and Chick Intestine

. I . I . I : : I ’ I . ’ hormone administration and then stabilized at approximately A . 150% of the control level. These changes appeared to be

inversely related to the temporal variations in the first protein -

the pulse-chase experiment. However, both proteins respond relate to each other since only the first was affected during (see Fig. 4); however, it is uncertain whether the two proteins

very quickly to 1,25-(OH)&, suggesting that they may be of considerable importance in understanding the mechanism of action of 1,25-(OH)& in the intestine.

I +: - Acknowledgment-We would like to thank Susan Spittle for her

0

* 0 : autoradiographs. rt assistance with the computerized analysis of the fluorographs and

!2 B 1. Spencer, R., Charman, M., Emtage, J. S., and Lawson, D. E. M.

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Radioactivity in protein spots 6 and 4 as a function Bio~hvs. Res. Commun. 102.287-294 of 1,25-(OH)*Ds exposure: The radioactivity in spot 6 ( A ) and spot 4 ( B ) was calculated from films produced in the time-course experiment, as described in Table I. Each time point represents the mean -t S.E. for 3 duodena.

any case, the change seen is early and dramatic enough to warrant further attention.

It is of some interest that 1,25-(OH)2D3 increases radiolabel in a second protein having similar charge and size character- istics. This radiolabeled protein increased 325% by 1 h after

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