Expression of the rat arginine vasopressin gene in transgenic mice

A line of mice has been developed which are transgenic for an 8.2-kilobase (kb) genomic clone of the rat vasopressin (VP) gene. Using a polymerase chain reaction technique, the rat VP (rVP) transgene was shown to have tissue-specific mRNA expression in the hypothalamus, temporal lobe, parietal cerebral cortex, cerebellum, and posterior pituitary, similar to the tissue distribution of endogenous mouse and rat VP expression. Expression of transgenic rVP mRNA was also found in the lung and pancreas of the transgenic mice, sites of known ectopic expression of VP. Using two methods, Northern blot analysis with species-specific cRNA probes and a quantitative polymerase chain reaction technique, the quantity of rVP transgene mRNA was shown to regulate appropriately in response to an osmotic stimulus. After 72 h of water deprivation, the quantity of transgenic rVP mRNA increased 6.8 +/- 3.0-fold. This was not significantly different than the fold increase in mouse VP mRNA quantity seen in nontransgenic mice (4.8 +/- 1.5) but was significantly different (P < 0.05) than the 1.2 +/- 0.03-fold increase in rat VP mRNA seen in normal rats after water deprivation. In the rat hypothalamus, VP mRNA poly(A) tail length increases with osmotic stimulation, while in the mouse it does not. The poly(A) tail of transgenic rVP mRNA expressed in mouse hypothalamus did not increase in length after osmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pharmacological and biochemical evidence for the regulation of osteocalcin secretion by potassium channels in human osteoblast-like MG-63 cells

Previous reports have suggested the involvement of voltage-activated calcium (Ca2+) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast-like cells. We now report that potassium (K+) channels can also modulate the secretion of osteocalcin by MG-63 cells, a human osteosarcoma cell line. When 1,25-dihydroxyvitamin D3(1,25(OH)2D3)-treated MG-63 cells were depolarized by step increases of the extracellular K+ concentration ([K+]out) from 5-30 mM, osteocalcin (OC) secretion increased from a control value of 218 +/- 13 to 369 +/- 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance). In contrast, in the absence of 1,25(OH)2D3, there is no osteocalcin secretion nor any effect of cell depolarization on this activity. The depolarization-induced increase in 1,25(OH)2D3-dependent osteocalcin secretion was totally inhibited in the presence of 10 microM Nitrendipine (a Ca2+ channel blocker, p < 0.005) without affecting cellular alkaline phosphatase nor cell growth. Charybdotoxin, a selective blocker of Ca2+-dependent K+ channels (maxi-K) present in MG-63 cells, stimulated 1,25(OH)2D3-induced osteocalcin synthesis about 2-fold (p < 0.005) after either 30, 60, or 120 minutes of treatment. However, Charybdotoxin was without effect on basal release of osteocalcin in the absence of 1,25(OH)2D3 pretreatment. Using patch clamp technique, we occasionally observed the presence of a small conductance K+ channel, compatible with an ATP-dependent K+ channel (GK[ATP]) in nonstimulated cells, whereas multiple channel openings were observed when cells were treated with Diazoxide, a sulfonamide derivative which opens GK(ATP). Western blot analysis revealed the presence of the N-terminal peptide of GK(ATP) in MG-63 cells, and its expression was regulated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle. Glipizide and Glybenclamide, selective sulfonylureas which can block GK(ATP), dose-dependently enhanced 1,25(OH)2D3-induced OC secretion (p < 0.005). Reducing the extracellular calcium concentration with EGTA (microM range) totally inhibited the effect of Glipizide and Glybenclamide on osteocalcin secretion (p < 0.005), which remained at the same levels as controls. Diazoxide totally prevented the effect of these sulfonylureas. These results suggest that voltage-activated Ca2+ channels triggered via cell depolarization can enhance 1,25(OH)2D3-induced OC release by MG-63 cells. In addition, OC secretion is increased by blocking two types of K+ channels: maxi-K channels, which normally hyperpolarize cells and close Ca2+ channels, and GK(ATP) channels. The role of these channels is closely linked to the extracellular Ca2+ concentration.     

The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro

Effects of dexamethasone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in cultures of adult human marrow stromal cells. In primary culture, dexamethasone (10(-8) M) increased the number of fibroblast colonies formed but decreased their average size. The number of colonies expressing alkaline phosphatase activity was increased, consistent with the enhancement of osteogenic differentiation by this glucocorticoid. In secondary culture, osteogenic differentiation was assessed by measurement of the steady-state levels of particular mRNAs that are characteristic of cells of the osteoblast lineage. The mRNAs for alpha 1(I)-procollagen, alkaline phosphatase, osteopontin and bone sialoprotein were expressed under all culture conditions used. In contrast, osteocalcin mRNA expression was detectable only in cultures treated with 1,25(OH)2D3 (10(-8) M). Addition of 1,25(OH)2D3 to control increased the expression of the mRNAs for alkaline phosphatase and osteopontin but had no significant effect on bone sialoprotein expression. The highest levels of expression of the mRNAs for alkaline phosphatase, bone sialoprotein and osteocalcin were observed in dexamethasone-treated cultures to which 1,25(OH)2D3 had been added. These results demonstrate that, as earlier found in other species, dexamethasone and 1,25(OH)2D3 promote the osteogenic differentiation of human marrow stromal cells as measured by expression of these osteogenic markers.     

Effect of 26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 on the expression of vitamin-D-responsive genes in vitamin-D-deficient mice

26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 (ST-630) is a newly developed agent to maintain the levels of calcium and phosphorus in blood. Herein, we investigated the effect of this compound on the expression of vitamin-D-responsive genes in vitamin-D-deficient mice. ST-630 was more effective than 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3] with respect to the induction of Cyp24 and calbindin-D9k mRNAs in the kidney and in the small intestine. Moreover, the increase in mRNA levels of vitamin-D-responsive genes induced by ST-630 lasted longer than that induced by 1,25(OH)2D3. These results indicate that ST-630 was more effective in inducing Cyp24 and calbindin-D9k gene expression than 1, 25(OH)2D3 when both compounds were injected into vitamin-D-deficient mice.     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation 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.     

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.     

Lack of beta-amyloidosis in transgenic mice expressing low levels of familial Alzheimer''s disease missense mutations

Point mutations within the beta-amyloid precusor protein (beta-APP) gene known to segregate with Alzheimer's disease in certain families were introduced into human beta-APP cDNAs and expressed under the control of a neuron-specific enolase (NSE) promoter in mice. The transgenic animals exhibited transgene expression predominantly in neocortex and hippocampus where the levels were maximally 1.3-fold of those of wild-type mouse beta-APP. Quantitative immunoblot analysis in homozygous mice carrying different missense mutations showed slightly increased alpha-secretory processing. In V7171 mice compared to nontransgenic mice there was more alpha-secretory beta-APP (beta-APPsec) in cortex/hippocampus, less in cerebellum, and no difference in midbrain/brain stem. In none of the transgenic animals tested was a 4 kDa amyloid fragment detected by Western blotting of brain extracts, immunohistochemistry, or by 125I-A beta-binding onto brain sections. No glial reaction was observed. Behavioral analysis of mice carrying the V7171 mutation showed no appreciable deficit in comparison to wild-type mice. Together, these data suggest that low levels of expression of mutated beta-APP in 10-12-month-old transgenic mouse brains result in slightly more beta-APPsec, and are insufficient to induce amyloidogenic processing and AD-like pathology.     

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.     

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.     

Effects of triiodothyronine on DNA synthesis and differentiation markers of normal human osteoblast-like cells in vitro

To study the direct effects of thyroid hormones on human osteoblasts we examined the effects of triiodothyronine (T3) on proliferation and differentiation of human osteoblast-like (hOB) cells in vitro. T3 increased 3H-thymidine incorporation in DNA of hOB cells (p < 0.05, n = 10). Half maximal effects obtained at a T3 concentration of 1-10 nM which lies within the physiological concentration of the hormone. In addition, T3 increased alkaline phosphatase production (p < 0.05, n = 13) and inhibited procollagen type I carboxyterminal propeptide (PICP) production (p < 0.05, n = 13). T3 interaction with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was also studied. 1,25-(OH)2D3 (10(-9)M) alone doubled AP production and induced osteocalcin expression by hOB cells. Concurrent addition of T3 and 1,25-(OH)2D3 did not further increase production of AP, PICP or osteocalcin by hOB cells. In conclusion, T3 exerts significant effects on osteoblast proliferation and differentiation, suggesting that human osteoblasts are targets for thyroid hormones.     

The X-linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene

Mutations in the gene for Bruton's tyrosine kinase result in the B cell differentiation defects X-linked agammaglobulinemia in man and X-linked immunodeficiency in mice. Here we describe the generation of two yeast artificial chromosome (YAC)-transgenic mouse strains in which high-level expression of human Btk is provided by endogenous regulatory cis-acting elements that are present on a 340-kb transgene, Yc340-hBtk. The expression pattern of the transgenic human Btk was found to parallel that of the endogenous murine gene. When the Yc340-hBtk-transgenic mice were mated onto a Btk-deficient background, the xid B cell defects were fully corrected: conventional and CD5+ B-1 B cells were present in normal numbers, serum IgM and IgG3 levels as well as responses to T cell-independent type II antigens were in the normal ranges. In vivo competition experiments in Btk+/- female mice demonstrated that in the conventional B cell population the Yc340-hBtk transgene could fully compensate the absence of expression of endogenous murine Btk. We conclude that in the YAC-transgenic mice Btk is appropriately expressed in the context of native regulatory sequences.     

Comparison of expression of human globin genes transferred into mouse erythroleukemia cells and in transgenic mice

To examine whether transfer of gamma globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of gamma globin gene promoter, Agamma gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (microLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Agamma globin gene expression among MEL cell clones carrying the muLCR(-201)Agamma and muLCR(-382)Agamma gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between the Agamma mRNA levels and the copies of the transgene (r = .28, P = .18). There was significant variation in per copy Agamma globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Agamma globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study of cis elements controlling gamma globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects.