Bone Vol. 16, No. 3 Abstracts 395 March 1995:391410 9 IS THE ANTI-MITOGENIC ACTION OF VITAMIN D COMPOUNDS IN HUMAN COLON ADENOCARCINOMA- DERIVED CACO-2 CELLS DEPENDENT UPON APPROPRIATE RECEPTOR EXPRESSION? W=-M. Tona. M. Peterllk. H.S. Cross. Institute of General and Experimental Pathology, University of Vienna, Austria The active metabolite of vitamin D, 1,25(OH)2D3, and some of Its nonhypercalcemic analogs have been shown by us to reduce proliferation and Increase differentiation of human colon cancer cells (J. Natl. Cancer Inst. 84: 1355, 1992). We were also able to demonstrate that treatment of Caco-2 cells with epidermal growth factor (EGF) substantially Increases the responsiveness of the tumor cells to the anti-mitogenlc action of vitamin D compounds (manuscript in preparation). We therefore posed the question whether changes In vitamin D receptor (VDR) expression by the growth factor are responsible for this Increased efficiency of vitamin D. VDR per DNA unit was quantified by immunofluorescence methods on a Millipore CytoFluor-2350. In addition, a radioactive whole cell binding assay was used for quantification of VDR per mg protein. Caco-2 cells cultured for up to 14 days with only EGF (50ng/ml) showed a significant reduction In VDR fluorescence units by up to 50%. Identical results were obtained with the radlollgand binding assay. When Caco-2 cells were exposed to 10 nM of either 1,25(OH)2D 3 or the analog 1,25(OH)2-16ene-23yne-D 3 Ior the same time period, no change In VDR fluorescence was observed. Co-culture, however, with the vitamin D sterols and EGF resulted in a significant Increase of VDR fluorescence units by 30 %. These results indicate that increased levels of VDR as a result of the combined treatment with a growth factor could cause the increased antl-mitogenie efficiency of vitamin D compounds. In addition, down-regulation of the VDR by EGF Indicates the existence of a growth regulatory loop which, In the presence of subthreshold amounts of vitamin D, could lead to Increased proliferation and possibly to malignant growth of colonoeytes. II INTEGRIN EXPRESSION IN NORMAL AND MALIGNANT INTESTINAL CELLS AFTER VITAMIN D AND THYROID HORMONE TREATMENT. G. Blses. W.-M. Tono. M. Peterllk. H.S. Cross. Institute of General and Experimental Pathology, University of Vienna Medical School, Austria Integrlns are transmembrane proteins that mediate cell-cell and cell-substratum adhesion. In Intestinal epithelial cells, ilntegrln expression appears to depend on the level of cellular !maturation since It changes during enterocyte differentiation !along the crypt to villus axis, or, conversely, during progression of colonocytes towards a malignant state. We addressed the question whether 1,25-dlhydroxyvltamln D3 (1,25(OH)2D3) and 3,3',5-t rllodot hyro nine (T3), two dlflerentlatlon-promotlng hormones In enterocytes, could change the Integrln pattern In the normal rat fetal Intestinal cell line FRIC and In the human colon carcinoma cell line Caco-2. Cells were cultured with and without 10 nM of 1,25(OH)2D3 and/or T3. Integrln pattern was monitored by Immunohlstochemleal methods and was quantified as Integrln fluorescence per unit DNA on a Mllllpore CytoFluor 2350. 1,25(OH)2D3, particularly In combination with T3, slgnflcantly Inhibted [3H]thymidine incorporation Into FRIC DNA (by up to 30%) and at the same time increased expression of c~3, an Integrln component of mature cells at the top of the villus, and of ¢¢1 (normally located laterally on differentiating enterocytes). In subconfluent highly proliferative Caco-2 cells, ct2 (which is mainly expressed by normal crypt cells) was Increased 25% by 1,25(OH)D3, whereas In confluent cells no change was observed. In both subconfluent and confluent Caco-2 cells, 1,25(OH)2D3 decreased ¢¢v~,3 (by 30%), an Integrln functionally Important on a wide range of both normal and malignant cells, which Is overexpressed, e. g., during melanoma cell Invasion. Our data demonstrate that 1,25(OH)2D3 and T3 alter, in parallel to their antlprollferatlve capacity, the Integrln pattern of normal and tumor cells towards a higher level of differentiation. The reduction of ¢¢v~3 by 1,25(OH)2D3 Is of particular Interest In view of a possibly protective role of vitamin D compounds against metastasis of colon cancer cells. 10 EFFECTS OF iNSUUN UKE GROWTH FACTORS I & II ON CELL ACTIVITY AND CALCITRIOLRECEPTORLEVELS IN HUMANBONECELLS IN VITRO. A. Battrnann*, T. Dreyer*, =3. Mohan, 2D.J. Baylink and A. Schulz, Inst of Pathology, Jusfus-Liebig-Univ, D-35385Giessen;2MineralMetabolism,J.L. Pettis Memorial Hospital, Loma Linda, CA 92357, USA. Recent studies suggest that vitamin D receptor (VDR) function is connected with the onset of postmenopausalosteoporosis. Therefore the regulationof these receptors takes on special significance. To test the hypothesis, that Insulin-like growth factors (IGF) are involved in the regulation of the VDR, we studied the effect of IGF pretreatment on alkaline phosphatase (ALP) activity as well as on osteocalcin (OC) production. Furthermore, VDR mRNA and VDR binding was determined. Serum free cultures of SaOS 2 or normal human bone cells were treated with 1, 10 or 100 ng IGF I or IGF II for 1-24 hours prior to the addition of 10"8M 1,25 D for 48 hours. ALP was determined in cell extracts and OC in levels in the conditioned medium by RIA.6 hours of pretreatment with either IGF t or IGF II increased ALP activity in a dose dependent manner significantly by 40% and 60% compared to 1,25 D treated control. OC production was elevated by 150% and 140% of 1,25 D treated control (p < 0,001). Treatment with IGF's alone decreased ALP activity,the OC productionwas not affected. Since receptor number is an important determinant of receptor function, we determined VDR mRNA as well as changes in VDR binding under influence of IGF I and IGF I1.Treatmentwith 100 ng/ml IGF I or II showed no increase of VDR mRNA after 1 hour, after 6 hours VDR mRNAwas increased significantly by 40% and 50 %. After 24 hours, levels were increased by 29% and 34%. Consistent with these findings, an increase of VDR of 65% respectively 60% (p< 0.05) as determined by radiolabelled 1,25Dcould be found. In summary: 1) pretreatmentwith IGF I or IGF II increased in a dose dependent manner ALP activity and OC production by 1,25 D. 2) IGF I or IGF II significantly increased at 10ng/ml VDR mRNA by 40% or 50% over control. 3) VDR levels were increased after IGF pretreatmentby 65% and 60% comparedto control. In conclusion, our results dearly show that IGF I or IGF I] potentiate the differentiation promoting action of 1,25 D on human bone cells, partly through an upregulatJon of the VDR. Thus, IGF's could be important regulaors of 1,25 D action, 12 T3 AND CALCITRIOL DIFFERENTIALLY INFLUENCE ALKALINE PHOSPHATASE ACTIVITY AND EGF INDUCED EXPRESSION OF EARLY GENES IN MC3T3-E1 CELLS. F. Varaafll. M. RumDler[l]. H. Glantschniafl]. M. Peterlik[2]. K. Kla0shoferrl]. [1]l_udwigBoltzmann Institute of Osteology, 4th Med. Dept., Hanusch Hospital and [2]Inst. of General and Experimental Pathology, University of Vienna Medical School,Vienna, Austria. Thyroid hormones and calcitriol act on their target cells via nuclear receptors. They influence bo'ne cell growth and differentiation (1). In transgenic mice, deregulated expression of the cellular oncogene c-fos was reported to result in severe disturbance of skeletal growth and development (2). Therefore c-fos is considered to be the regulatory master switch for osteobiastic proliferation and differentiation. At the molecular level c-fos interacts with other early genes like c-jun to form heterodimers which bind to the AP-1 bindig sites (3). In this study we investigated the influence of T3 and calcitriol on the EGF stimulated expression of these genes in MC3T3-E1 cells. The cells were cultured in aMEM supplemented with 5% FCS with or without hormones for 11 days. DNA content and alkaline phosphatase activity were measured every two days. For study of c-fos expression the cells were cultured for 4 days in the respective media. Thereafter c-fos expression was stimulated with EGF under low serum (0.1% BSA) conditions and analyzed by Northern hybridization. Both T3 and calci.triol stopped proliferation. Calcitriol had no effect on alkaline phosphatase activity whereas T3 treatment resulted in significant increase of the activity of this marker enzyme. No influence of the two hormones on basal c-fos expression could be detected. Interestingly, T3 treatment signifi- cantly reduced EGF stimulated c-fos as well as c-jun expression, while calcitriol had no effect. Our data reveal a possible mechanism by which thyroid hormones modulate important intracellular signal transduction pathways. We speculate that T3 acts at the switch between proliferation and differentiation, whereas calcitriol could be a regulator of more differentiated osteoblastic functions. I 1) Perry III HM (1989) in Bone and Mineral Res 6:113-137 2) nether U et al (1987) Nature 325:412-416 (3)Angel P Karin M (1991) Biochem Biophys Acta 1072:129-157