The E1A oncogene induces resistance to the effects of 1,25-dihydroxyvitamin D3 on inhibition of growth of mouse keratinocytes

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited DNA synthesis in transformed mouse keratinocytes (Pam212) in a time- and dose-dependent manner as measured by [3H]thymidine incorporation. To investigate the mechanism through which 1,25-(OH)2D3 acts, we examined its effects on Pam212 cells further transformed with the E1A oncogene. Here, we show that transformation of the cells with the E1A oncogene induced resistance to the effects of 1,25-(OH)2D3 on inhibition of growth of Pam212 cells. While 1,25-(OH)2D3 treatment increased the level of expression of vitamin D receptor mRNA 20-fold in parental cells, the E1A-transformed cells failed to express vitamin D receptor mRNA even after treatment with 1,25-(OH)2D3. Transfection of the E1A-transformed cell line with an expression construct encoding the vitamin D receptor restored receptor expression as well as the inhibition of growth by 1,25-(OH)2D3. These results suggest that one of the mechanisms for acquisition of 1,25-(OH)2D3 resistance induced by E1A may involve loss of vitamin D receptor inducibility by 1,25-(OH)2D3.     

1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation

1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation-dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation.     

Microperoxidases catalytically degrade reactive oxygen species and may be anti-cataract agents

1,25-(OH)2D3 and 24,25-(OH)2D3 mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)2D3 which have been modified on the A-ring and C,D-ring side chain (1 alpha-(hydroxymethyl)-3 beta-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YA = 3a) and 1 beta-(hydroxymethyl)-3 alpha-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)2D3 and 24,25-(OH)2D3. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)2D3. Chondrocyte proliferation ([3H]-thymidine incorporation), proteoglycan production ([35S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)2D3, 24,25-(OH)2D3, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)2D3 and 24,25-(OH)2D3. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)2D3 and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)2D3 having a greater effect. 24,25-(OH)2D3 caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)2D3, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation-dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites.     

1,25-Dihydroxy vitamin D3 inhibits adipocyte differentiation and gene expression in murine bone marrow stromal cell clones and primary cultures

Bone marrow stromal stem cells differentiate into adipocytes and osteoblasts. These two lineages are thought to be reciprocally related, in part due to the observation that the osteoblast-inducing factor, 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], inhibited adipogenesis of rat femoral-derived stromal cell cultures. However, the literature is divided concerning the adipogenic effects of this steroid hormone. This work examined the effect of 1,25(OH)2D3 (10(-12)-10(-8) M) on murine femoral-derived bone marrow stromal cell differentiation in response to adipogenic agonists employing two different classes of nuclear hormone receptors: the glucocorticoid receptor (hydrocortisone) or peroxisome proliferator-activated receptors (thiazolidinediones). Experiments used the multipotent murine bone marrow stromal cell line, BMS2, and its subclones, as well as primary-derived murine bone marrow stromal cell cultures. In all systems examined, 1,25(OH)2D3 blocked adipogenesis induced by hydrocortisone, methylisobutylxanthine, and indomethacin based on flow cytometric analysis of lipid accumulation. This correlated with reduced messenger RNA levels of the late adipocyte gene markers, aP2 and adipsin. In the BMS2 subclone no. 24, the 1,25(OH)2D3 actions were concentration dependent. Whereas 1,25(OH)2D3 partially inhibited thiazolidinedione-induced adipogenesis in the parental BMS2 cell line, it had minimal effect on the thiazolidinedione-induced differentiation of the BMS2 subclone and primary cultures. These findings indicate that 1,25(OH)2D3, at nanomolar concentrations, completely inhibits murine bone marrow stromal cell differentiation in response to glucocorticoid-based adipogenic agonists but is a less effective adipogenic antagonist following induction with thiazolidinediones. This work supports the conclusion that 1,25(OH)2D3 inhibits murine femoral-derived bone marrow stromal cell adipogenesis.     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC. Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC. When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2. These effects are cell maturation- and time-dependent and metabolite-specific.     

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.     

Studies on the bioactive constituents of Aurantii Fructus Immaturus

This study investigated the regulatory activity of 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) on phagocytic cells obtained from normal human peripheral blood. Flow cytometric analysis enabled identification of two discrete populations of cells, one predominantly monocytes ("monocyte" gate) and one containing primarily lymphoid and other cell types ("lymphoid" gate). The monocyte-associated antigens CD13 and CD33 were highly expressed by cells in this monocyte gate and used to monitor this population. Following 5 days of culture, cells in the monocyte gate manifested high phagocytic activity as determined by ingestion of fluorescent carboxylmicrospheres and exhibited high expression of class II HLA-DR products. 1,25-(OH)2D3 profoundly upregulated phagocytic activity while downregulating HLA-DR antigen expression on the cells in the monocyte gate. Moreover, 1,25-(OH)2D3 also reduced cell surface CD13 expression on the cells with low but not high phagocytic activity in this gate. Proportional activities by the 1,24-(OH)2D3 and 24,25-(OH)2D3 metabolites indicated the regulatory effects are likely mediated by the 1,25-(OH)2D3 receptor (VDR). Prostaglandin E2 (PGE2), a known modulator of monocyte/macrophage activity also markedly inhibited HLA-DR expression while enhancing the phagocytic activity of cells in the monocyte gate. In contrast to 1,25-(OH)2D3, PGE2 clearly upregulated CD13 expression in cells with high phagocyte activity. Since indomethacin, an inhibitor of PGE2 synthesis, failed to reverse the 1,25-(OH)2D3 induced inhibitory effect on HLA-DR expression, this effect is apparently not mediated through endogenous PGE2 synthesis. Based on these findings we speculate that 1,25-(OH)2D3 may be capable of acting as both an upregulating agent during natural immunity via the enhancement of phagocytosis by monocyte/macrophage populations and as a "downregulator" during acquired immune responses via an inhibitory effect on MHC class II antigen expression by professional antigen-presenting cells.     

Human and murine osteocalcin gene expression: conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo

Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6+/-3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2+/-0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2+/-7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5+/-0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly upregulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.     

Triiodothyronine restricts myofibrillar growth and enhances beating frequency in cultured adult rat cardiomyocytes

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.     

Changes in 1,25-(OH)2D3 synthesis and its receptor expression in spleen cell subpopulations of mice infected with LPBM5 retrovirus

This study examines the influence of chronic retroviral infection of mice with a LPBM5 virus mixture on the paracrine system involving immune cells and 1,25-(OH)2D3 in the spleen. Plasma ionized calcium, 25-(OH)D and 1,25-(OH)2D of infected mice were unchanged. In contrast, the specific binding of 1,25-(OH)2D3 to spleen cytosol and the number of monocyte/macrophages expressing 1,25-(OH)2D3 receptors (VDR) were markedly increased. The retroviral infection also influenced the local production of 1,25-(OH)2D3 in the spleen. It did not alter this production in monocyte/macrophages but increased that in isolated T cells. Isolated B cells in control mice did not produce 1,25-(OH)2D3, but they increased the ability of isolated T cells to produce this metabolite during coculture incubations. Infection altered this cell interaction as 1,25-(OH)2D3 production in infected T cells decreased when these cells were cocultured with infected B cells. Thus, chronic retroviral infection alters both the local vitamin D metabolism and VDR expression by immune cells in mice. These findings suggest close local interactions between 1,25-(OH)2D3 and immune system activation during retroviral infection.     

Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.     

Combinational effects of vitamin D3 and retinoic acid (all trans and 9 cis) on proliferation, differentiation, and programmed cell death in two small cell lung carcinoma cell lines

The effects of a combination of vitamin D3 [1,25(OH)2D3] and retinoic acid (RA) on proliferation, differentiation, and apoptosis of the human small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H209 were evaluated. Cell proliferation was inhibited by 1,25(OH)2D3 and RA alone. The combination of 1,25(OH)2D3 and the cis form of retinoic acid resulted in an additive decrease in cell proliferation and the induction of apoptosis in various concentrations. Moreover, 3H-thymidine incorporation was inhibited and the number of viable cells was decreased. The characteristics of the apoptotic cells were examined and confirmed by morphologic analysis, light and electron microscopy, and fluorescence detection. It was concluded that 1,25(OH)2D3 and RA exert additive effects on the inhibition of proliferation and the induction of apoptosis in both the NCI-H82 and the NCI-H209 SCLC cell lines. This finding has important implications for the use of retinoids and 1,25(OH)2D3 in cancer prevention and in the therapy of small cell lung carcinoma.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

In vivo regulation of chromogranin A messenger ribonucleic acid in the parathyroid by 1,25-dihydroxyvitamin D: studies in normal rats and in chronic renal insufficiency

Chromogranin-A (CgA) and PTH are the two major secretory products of the parathyroid gland. In vitro, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases CgA, but decreases PTH messenger RNA (mRNA) levels. We investigated the physiological significance of the induced changes in CgA expression by examining the effects of 1,25-(OH)2D3 on parathyroid CgA mRNA levels in vivo. Normal rats were injected with 1,25-(OH)2D3 at 48 and 24 h before blood sampling and isolation of both parathyroid glands. Parathyroid total RNA was extracted and CgA and PTH mRNA quantified by Northern blot analysis. CgA mRNA levels increased 1.6-, 3.2- and 5.6-fold, whereas PTH mRNA levels decreased by 37, 63 and 97%, respectively, with 1,25-(OH)2D3 doses of 10, 50, and 250 pmol/100 g BW. Parathyroid gland CgA expression also was examined in rats with mild chronic renal insufficiency, induced by a 5/6 nephrectomy 5 weeks earlier. Chronic renal insufficiency rats, fed normal chow, had elevated serum urea, creatinine, and PTH levels and reduced 1,25-(OH)2D3 but normal serum levels of calcium and phosphate. PTH mRNA levels were elevated 4-fold and CgA mRNA levels were 50% lower in the uremic animals. This indicates that the regulation of CgA expression in normocalcemic rats occurs at physiological 1,25-(OH)2D3 concentrations. In summary, increases and decreases in serum 1,25-(OH)2D3 levels are associated with corresponding increases and decreases in CgA mRNA levels in the parathyroid glands of rats. Therefore, this study is the first to demonstrate the physiological relevance of the earlier in vitro observations.