STIMULATION OF INTESTINAL MUCOSAL PROLIFERATION BY VITAMIN D

Transcription

1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.5 Printed in U.S.A. STIMULATION OF INTESTINAL MUCOSAL PROLIFERATION BY VITAMIN D STANLEY J. BIRGE, M.D., AND DAVID H. ALPERS, M.D. Endocrinology Division, Washington University School of Medicine, The Jewish Hospital of St. Louis and Gastrointestinal Division, Washington University School of Medicine, St. Louis, Missouri Intestinal mucosal cell turnover was studied in vitamin D-deficient rats in response to vitamin D3 repletion. Four hours after a single oral dose of 100 U of vitamin D3, 3H-thymidine incorporation into deoxyribonucleic acid was enhanced. Mucosal cell migration rate of the vitamin D-repleted animals was 1.5 times that of the deficient animals. The accelerated mucosal cell turnover was associated with an increase of approximately 20% in villus height. This stimulation of mucosal cell proliferation preceded enhanced calcium transport, and, therefore, may be an important component of action of vitamin D on the intestine. The principal action of vitamin D in the maintenance of calcium homeostasis and skeletal growth has been considered to be the promotion of intestinal calcium absorption. 1, 2 However, the alterations of the intestine which result in the enhanced absorption of calcium induced by the vitamin and its metabolites remains in dispute. There is general agreement that vitamin D and certain hydroxylated metabolites of vitamin D induce in vivo the synthesis of an intestinal calcium-binding protein, 3, 4 a brush border alkaline phosphatase,5-7 and a calcium-dependent phosphatase of the brush border.8 This vitamin D-induced increase in protein synthesis is associated with accelerated ribonucleic acid synthesis which can be Received June 12, Accepted December 14, Address requests for reprints to: Dr. Stanley J. Birge, The Jewish Hospital of St. Louis, 216 S. Kingshighway, St. Louis, Missouri This investigation was supported by Grants AM-14571, AM-14038, and AM-0528 from the National Institutes of Health. The authors wish to thank Carol Goodwin, Helen Gilbert, and Sandra Switzer for excellent technical assistance. blocked by the prior administration of actinomycin. 9 Although the increase in activity of these proteins correlates in time with the increase in intestinal calcium absorption, the role of these proteins in the transport process remains obscure. These studies were initiated to elucidate further the action of vitamin D on intestinal mucosal cell metabolism. In view of the well recognized growth-promoting effect of vitamin D in the treatment of the vitamin-deficient animal, the influence of vitamin D on intestinal mucosal cell proliferation was examined. D was found to accelerate deoxyribonucleic acid (DNA) synthesis and mucosal cell migration along the villus and to increase villus height. This trophic influence of vitamin D on the intestinal mucosa is evident in time before the reported increases occur in the vitamin-induced enzymes of the brush border and the calcium-binding protein. Methods The influence of vitamin Da on calcium absorption and thymidine incorporation into DNA was evaluated in 7-week-old rachitic Wistar rats. The animals were fed a rachitogenic diet (Nutritional Biochemical Co., Cleve- 977

2 978 BIRGE AND ALPERS Vol. 64, No.5 land, 0.) for 4 weeks prior to use. After oral administration of 0.1 cc of cottonseed oil or 0.1 cc of cottonseed oil containing 100 U of vitamin Ds (Philips Duphar), calcium transport was studied by use of the everted duodenal sac preparation of Wilson and Wiseman 10 as modified by Schachter et a!. 11 The incubation buffer contained 0.4 mm Ca, 2 mm fructose, 140 mm NaCl, 0.2 mm phosphate buffer at ph 7.4, and "Ca (New England Nuclear Corp., Boston, Mass.) at a final concentration of 0.2 J.Lc per m!. The rate of thymidine incorporation in DNA was determined in similar rachitic animals treated with a single dose of 100 U of vitamin Ds orally. SH-thymidine (New England Nuclear Corp., 20 c per mm) in saline was injected by tail \l"ein at 4, 8, 12, and 24 hr after vitamin Ds administration at a dose of 10 J.Lc per kg of body weight. A minimum of 12 animals were used at each time point. One hour after the SH-thymidine injection, the animals were killed and two 1-cm segments of duodenum were analyzed for total DNA content and specific activity. Segments were quick frozen on dry ice, homogenized in 10% ice-cold trichloroacetic acid, centrifuged at 1500 x g, and the supernatant fluid discarded. The pellet was washed twice with 10% trichloroacetic acid and dissolved in 0.3 N KOH. After diluting 1: 1 with water, aliquots were removed for DNA analyses and for counting. DNA was measured by the method of Ceriotti 12 as modified by Keck IS and Bonting and Jones.14 This technique enables one to measure DNA with virtually no interference from protein or ribonucleic acid, and to extract only once with chloroform, so that small volumes may be handled. SH-thymidine in DNA was analyzed by counting in Bray's solution 15 in a Packard model 3320 liquid spectrometer. Student's t-test was applied to the analysis of the differences between the vitamin Ds-treated and rachitic control groups. The rate of mucosal cell migration was studied by intravenous administration of sh_ thymidine 8 hr after the animals received 100 U of vitamin Ds orally. At 8,12,16,24,36, and 48 hr after administration of the thymidine label, the animals were killed, and histologic sections of mucosa were prepared for autoradiography as described by Messier and Leblond. IS Tissue sections were incubated in 4% formaldehyde containing 0.09 M calcium chloride and 1 mg per ml of thymidine,17 then embedded in paraffin, and sections 4 J.L thick were cut. Radioautograms were prepared by dipping sections in NTB2 emulsion (Eastman Kodak Co., Rochester, N. Y.) and exposing them at room temperature for 3 to 4 weeks. The radioautograms were developed with Kodak D-19 developer and stained with hematoxylin and eosin. Twenty well oriented crypts and villi were examined in the proximal duodenum and proximal jejunum from each of three sections taken from 2 or 4 animals at 8, 16, 24, and 36 hr after administration of SH-thymidine. Villus height was determined by counting the number of epithelial nuclei in complete villus columns. Villus cell migration rate was measured according to Cairnie and Bentley.18 Labeled nuclei began to appear on the villus by 10 hr after thymidine injection. Thus, cell migration was estimated at 16, 24, and 36 hr. The method of scanning for labeled cells on the villus was similar to that described for crypt cells. 19 The labeling index was determined at the base of the villus, and then, on the same section, it was calculated how far from the top ofthe villus the index had fallen to half. These results were recorded as percentage of villus height. This method avoids some of the problems encountered by Quastler and Sherman 20 who found that there was variation in the leading edge of radioactivity, even within the same villi. Results were plotted as described previously20 as percentage of villus height of labeled cells versus time. The slope of the line (rate of migration) was determined by calculating a regression line by the least square method. The difference between the two regression coefficients of the line describing the response of the control and vitamin D-treated animals was analyzed by an F test of the coefficient of variation of the two lines. 21 Results Oral administration of 100 U of vitamin Da enhanced the absorption of 45Ca by everted duodenal sacs as compared with untreated rachitic control animals. This stimulation of 45Ca accumulation by duodenal sacs was not apparent until more than 12 hr had elapsed after administration of the vitamin, reaching a peak response 48 hr after a single dose (fig. 1). No effect on calcium absorption or change in serum calcium and phosphorus was detectable at 4 and 8 hr after vitamin Da treatment in agreement with the temporal response to comparable doses of vitamin Da reported by others , 23 In light of the total body growth promot-

3 May 1973 VITAMIN D STIMULATION OF INTESTINAL CELL PROLIFERATION A o TIME (hrs) AFTER VITAMIN D FIG. 1. Absorption of "ea by eyerted duodenal sacs as a function of time after oral administration of 100 U of vitamin D3. The data are expressed as a ratio of the concentration of "ea inside the sac (serosal) and the concentration of "ea outside the sac (mucosal) at the end of a go-min incubation. The vertical bars indicate the standard errors of the mean. 50 t' I, 4 8 I B ing response to vitamin D repletion of the rachitic animal, the rate of mucosal cell multiplication was studied by measuring the incorporation of 3H-thymidine into duodenal mucosal DNA (fig. 2). D significantly (P < 0.05) enhanced the incorporation of the thymidine label into mucosal DNA 4 hr after oral administration of the vitamin. The difference in the 3H-thymidine incorporation into DNA between the vitamin-treated and rachitic controls progressively decreased at the 8th and 12th hr after vitamin D repletion when expressed as dpm per mg of DNA. The apparent decrease in rate of incorporation per microgram of DNA with time in the vitamin D-treated animals may reflect the progressive increase with time in total DNA prior to administration of the intravenous pulse label of thymidine. Accordingly, the incorporation of radioacitivty into DNA was expressed as a function of intestinal length (disintegrations per minute per centimeter) in order to obtain a measurement which would be less dependent on changes in cell mass or DNA. When expressed as disintegrations per minute per centimeter of intestine, 3H-thymidine incorporation was significantly enhanced at 4 hr and at 24 hr (P < 0.05) after vitamin D treatment. The effect of vitamin D on villus cell number and mucosal cell migration rate HOURS AFTER VITAMIN 0 TREATMENT FIG. 2. Effect of oral administration of 100 U of vitamin D3 on 3H-thymidine incorporation into deoxyribonucleic acid (DNA) as a function of time. The rats were given intravenously 0.1 p,c of 3H-thymidine per g of body weight and killed 1 hr later. In A, the radioactivity [disintegrations per minute (dpm) 1 is expressed per microgram of DNA; in B, the radioactivity (dpm) is expressed per centimeter of intestine. The vertical bars indicate the standard errors of the mean. The response of the vitamin D,-treated animals are indicated by.--.; the rachitic control animals by on the villus was studied after a single intravenous pulse of 3H-thymidine given 8 hr after administration of the vitamin. Table 1 demonstrates that, in the duodenum, villus cell number increases in response to vitamin D, being first apparent at 24 hr after administration of vitamin D, and reaching an apparent plateau by 32 hr. Changes in the upper jejunum were less striking, but cell number was increased by 44 hr. Sections were taken from the animals in the experiment outlined in table 1, and the rate of migration of labeled cells along the villus was ascertained as a function of vitamin D treatment. In both treated and untreated groups, labeled cells began to appear on the villus by 10 to 12 hr after injection of 3H-thymidine. Table 2 demon-

4 980 BIRGE AND ALPERS Vol. 64, No.5 TABLE 1. Effect of vitamin D on villus cell numbera Tissue Time after vitamin D hr D-deficient D-treated P'value Duodenum ± 6 73 ± 3 NS ± 4 75 ± 4 < ±6 85 ± 6 < ± 3 84 ± 3 <0.005 Jejunum ± 3 71 ± 6 NS ± 4 73 ± 6 NS ± 7 70 ± 6 NS ± 6 77 ± 3 <0.005 a Four animals in each group and at each time point were killed after administration of vitamin D, 100 U, or cottonseed oil. Tissue was removed, prepared, and analyzed as described in "Methods." The results are combined from two experiments with 2 animals in each group and are recorded as the mean ± 1 SD. The 20 villi counted in each microscopic section were recorded as one observation, thus making three observations per animal. NS, not significant. strates that at 16, 24, and 36 hr, the migration of a zone of cells corresponding to one-half the labeling index at the villus base was significantly higher up the villus after giving vitamin D. The slope of the line defined by location of the thymidine lable along the duodenum at 16, 24, and 36 hr was 1.39 for control animals and 2.13 for the vitamin D-treated animals (P < 0.01), and 1.74 and 2.02 respectively for jejunum. The latter difference in slope or rate of migration was not significant. Discussion The physiologic role of vitamin D has been attributed to the maintenance of calcium homeostasis primarily through the enhancement of intestinal calcium absorption. In addition, it has been recognized that vitamin D repletion of rachitic animals results in a marked acceleration of growth 24 and increase in weight and dimensions of the intestine. 25 These changes have been attributed to increased availability of calcium. This concept is supported by the observation in vitro that calcium stimulates DNA synthesis. 26 TABLE 2. Effect of vitamin D on the leading edge of labeled cells on the villus a Tissue Time after 'H- TdR Fractional distance from top of villus D-deficient D-treated hr % Pvalue Duodenum ± ± 6.2 < ± ± 7.8 < ± ± 7.8 <0.001 b = 1.39 b = 2.13 <0.01 Jejunum ± ± 3.81 < ± ± 3.6 < ± ± 6.86 <0.005 b = 1.74 b = 2.02 NS a Experimental conditions are as described in "Methods" and table 1. 3H-thymidine (3H-TdR) was given intravenously to rats 8 hr after oral vitamin D treatment, or administration of cottonseed oil as control. Twelve observations were recorded for each recorded result, which correspond to the mean ± 1 SD. Linear regression lines were plotted assuming that labeled cells leave the crypts 10 hr after 3H_ thymidine was given. Thus, the times used for calculation of the slope of the regression line (b) were 6, 14, and 26 hr, corresponding to the time after label l,cgins to appear on the villus. The P value was derived from an F test of the coefficient of variation of the regression lines. NS, not significant. These studies were designed to test whether vitamin D3 may have a primary effect on mucosal cell proliferation. During the first 12 hr after the initiation of vitamin D replacement, no significant alteration of intestinal calcium absorption or serum calcium and phosphorus concentrations could be detected. However, mucosal DNA synthesis was observed to be enhanced at 4 hr, preceding the earliest response in calcium absorption to the vitamin by at least 8 to 20 hr and preceding by 36 to 48 hr the maximal response to the vitamin. Stimulation of DNA synthesis and villus height (as reflected by cell number) was greater in the duodenum than in the jejunum. Similarly, it has been shown by others that the proximal portion of the rat intestinal mucosa manifests the greatest increases in the vitamin-dependent proteins and calcium absorption in

5 May 1973 VITAMIN D STIMULATION OF INTESTINAL CELL PROLIFERATION 981 response to vitamin D It is of interest that in the vitamin D-treated animal the time required to replace the entire villus structure with a new population of cells generated after administration of the vitamin was extrapolated from the observed migration rates to be 46.2 hr. This compares with the maximum response by the intestine observed at 48 hr both with respect to calcium absorption and the various proteins reported to be specifically induced by the vitamin. 3-8 These observations suggest that the vitamin's stimulation of calcium absorption may be mediated in part by new cell formation and cell differentiation occurring at the level of the immature crypt cell. A similar mechanism has been postulated for the induction of disaccharidase activity in the disaccharidase-deficient rat which manifests a similar time course of response. 28 Accordingly, the time required for maximal response is the time required for the differentiated or induced cell population to migrate from the crypt and replace the pre-existing cell population of the villus. Similarly the apparent stimulation of a number of "specific" proteins and enzymes may also reflect in part these changes of cell differentiation. On the other hand, the observed increases in specific activities of the several proteins induced by vitamin D may not be due solely to a specific response to the vitamin. By accelerating cell proliferation and migration, vitamin D is increasing the total number of cells on the villus and, therefore, may be altering the relative proportion of either the mature or undifferentiated cells with respect to the total mucosal cell population. In the vitamin D repleted state, for example, the mucosa may contain a relatively greater number of mature cells than in the vitamin D-deficient animal. Since the enzyme content of the intestinal cell undergoes dramatic evolution with maturation,29 accelerating the mucosal cell migration and increasing mucosal cell number may effect an apparent alteration in specific activity of a particular protein or enzyme with respect to the total mucosal protein content. Experiments are now in progress to ascertain the relative maturity of the vitamin D-repleted villus. Finally, the combination of increased villus height, increased cell production, and increased cell migration rate has also been described in response to lactation, 17 thyroid hormone,30 and cortisol,31 other hormonally mediated responses. The increase in villus height in addition to the increase in mucosal weight previously described 25 might reflect an increase in the absorptive surface area which in itself may facilitate the absorption of calcium. The relative importance of this response of the intestinal mucosa to vitamin D in enhancing calcium absorption remains to be evaluated. REFERENCES 1. Schachter D, Wasserman RH: The Transfer of Calcium and Strontium Across Biological Membranes. New York and London, Academic Press Inc, 1963, p Harrison HE, Harrison HC: Transfer of "Ca across the intestinal wall in vitro in relation to action of vitamin D and cortisol. Am J Physiol 199: , Wasserman RH, Taylor AN: Do-induced calcium binding protein in chick intestinal mucosa. Science 152: , MacGregor RR, Hamilton JW, Cohn DV: The induction of calcium binding protein biosynthesis in intestine by vitamin Do. Biochim Biophys Acta 222: , Haussler JR, Nagode LA, Rasmussen H: Induction of intestinal brush border alkaline phosphatase by vitamin D and identity with Ca-ATPase. Nature 228: , Norman A W, Mircheff AK, Adams TH, et al: Studies on the mechanism of action of calciferol. III. D mediated increase of intestinal brush border alkaline phosphatase activity. Biochim Biophys Acta 215: , Holdsworth ES: The effect of vitamin D on enzyme activities in the mucosal cells of the chick small intestine. J Membr BioI 3:43-53, Melancon MJ, DeLuca HF: D stimulation of calcium-dependent adenosine triphosphatase in chick intestinal brush borders. Biochemistry 9: , Zull JE, Czarnowska-Misztal E, DeLuca HF: On the relationship between vitamin D action and

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