It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate and calcium homeostasis through its membrane receptor termed the 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. I determined the various PKC isotypes involved in the rapid regulation of phosphate uptake and calcium extrusion in chick intestinal cells. 1,25(OH)2D3-mediated phosphate uptake was stimulated within 1 min after addition of the hormone. Western blot analyses on isolated intestinal cells treated with steroid hormone resulted in dose-dependent increases in PKC alpha and PKC beta in postnuclear centrifugation fractions, but not in the low speed centrifugation fractions. The highest immunoreactivity of PKC alpha was found after treatment of the cells with 300 pM 1,25(OH)2D3 and declined at 650 pM hormone, relative to corresponding controls, while the highest immunoreactivity of PKC beta was found in cells treated with either 300 pM or 650 pM 1,25(OH)2D3. Therefore, PKC alpha and PKC beta redistribution are likely to relate to the dose-response curve for both phosphate uptake and calcium efflux, respectively. Using transfection of primary cultures of intestinal cells with siRNA for these two isotypes, I found decreased 32P uptake in cells transfected with siRNA to either PKC alpha or PKC beta in both controls (relative to untransfected controls), and hormone-treated cells. Further study of the effect of chemical blockers for PKC alpha or PKC beta on phosphate uptake was conducted in suspensions of isolated intestinal cells. The results from these experiments also confirmed the findings from the siRNA experiments and demonstrated decreased 32P uptake in cells treated with 1,25(OH)2D3 plus blockers in comparison with cells treated with 1,25(OH)2D3 alone. The effects of PKC alpha and PKC beta in steroid-mediated calcium extrusion were further investigated using siRNA for PKC alpha or PKC beta. We found the siRNA to PKC beta alone caused decreased calcium extrusion. We also found that the inhibitors of PKC beta, but not PKC alpha caused significantly enhanced calcium uptake by decreasing calcium efflux from the cells. This result suggested that PKC beta might be involved in the rapid response of 1,25(OH)2D3-stimulated calcium extrusion. I used confocal microscopy to study the redistribution of PKC alpha and PKC beta in cells exposed to steroid hormone for 30 sec. PKC alpha was found to increase significantly in the apical membrane after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH)2D3. By comparison, anti-PKC beta immunofluorescence was found to increase significantly in the basal region of cells, relative to controls, following exposure of cells to 300 pM seco-steroid. These combined results, lead me to conclude the involvement of both PKC alpha and PKC beta in the signal transduction mechanism of 1,25(OH)2D3-mediated phosphate uptake while PKC beta is involved in the mechanism of 1,25(OH)2D3-mediated calcium efflux in chick intestinal epithelial cells.

Over 200,000 cases of breast cancer are diagnosed every year. Nearly 20% of these patients supplement their diets with some form of vitamin D. This high frequency of vitamin D supplement use may be due in part to research suggesting that cancer patients with higher serum vitamin D3 levels have better prognoses than patients with low serum vitamin D3. However, double-blind clinical trials on the efficacy of vitamin D3 supplementation in breast cancer have been inconclusive. A recent meta-analysis showed evidence of reduced cancer recurrence in patients taking vitamin D3 supplements who had ‘estrogen receptor positive’ (ERα66+) breast cancer, but not those who had estrogen receptor negative’ (ERα66-) breast cancer. Once ingested, vitamin D3 is metabolized in the liver into the circulating pre-hormone 25(OH)D3, which is then further metabolized into 1a,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 has been shown to activate a number of membrane signaling pathways, some of which overlap with 17b-estradiol (E2) signaling through ERα36, a membrane isoform of ERα66. The central hypothesis of this thesis was that 24R,25(OH)2D3 is tumorigenic in certain cancers and that this tumorigenicity is mediated in part by ERa isoforms. E2 signaling through ERa36 has been described in the ERa66-, ERa36+ breast cancer cell line HCC381. Specific aim 1 determined whether E2 signaling through ERa36 was tumorigenic other cancers with different ERa profiles. Specific aim 2 determined how 24R,25(OH)2D3 affected tumorigenicity in breast cancer using the common breast cancer cell line MCF7 (ERa66+, ERa36+) as a model. Specific aim 3 investigated the role of ERa isoforms in 24R,25(OH)2D3 signaling in breast cancer cell lines by comparing the tumorigenic effects of 24R,25(OH)2D3 in MCF7 cells (ERa66+, ERa36+) and HCC38 cells (ERa66-, ERa36+). To determine whether ERa66 regulates the effects of 24R,25(OH)2D3, ERa66 was expressed in two ERα66- cell lines. The effect of 24R,25(OH)2D3 on apoptosis was assessed in wild-type and ERa-expressing cell lines.

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