Association between liver-specific gene polymorphisms and their expression levels with nonalcoholic fatty liver disease.

UNLABELLED: Genetic factors account for a significant proportion of the phenotypic variance of nonalcoholic fatty liver disease (NAFLD); however, very few predisposing genes have been identified. We aimed to (1) identify novel genetic associations with NAFLD by performing a genome-wide association study (GWAS), and (2) examine the biological expression of the strongest genetic associations in a separate cohort. We performed GWAS of a population-based cohort (Raine Study) of 928 adolescents assessed for NAFLD by ultrasound at age 17. Expression of genes with single nucleotide polymorphisms (SNPs) that were associated with NAFLD at a significance level of P < 10(-5) was examined in adults with NAFLD and controls by quantifying hepatic messenger RNA (mRNA) expression and serum levels of protein. After adjustment for sex and degree of adiposity, SNPs in two genes expressed in liver were associated with NAFLD adolescents: group-specific component (GC) (odds ratio [OR], 2.54; P = 1.20 × 10(-6)) and lymphocyte cytosolic protein-1 (LCP1) (OR, 3.29; P = 2.96 × 10(-6)). SNPs in two genes expressed in neurons were also associated with NAFLD: lipid phosphate phosphatase-related protein type 4 (LPPR4) (OR, 2.30; P = 4.82 × 10(-6)) and solute carrier family 38 member 8 (SLC38A8) (OR, 3.14; P = 1.86 × 10(-6) ). Hepatic GC mRNA was significantly reduced (by 83%) and LCP1 mRNA was increased (by 300%) in liver biopsy samples from patients with NAFLD compared to controls (P < 0.05). Mean serum levels of GC protein were significantly lower in patients with NAFLD than controls (250 ± 90 versus 298 ± 90, respectively; P = 0.004); GC protein levels decreased with increasing severity of hepatic steatosis (P < 0.01). CONCLUSION: The association between GC and LCP1 SNPs and NAFLD as well as altered biological expression implicate these genes in the pathogenesis of NAFLD.

Expressions of vitamin D metabolic components VDBP, CYP2R1, CYP27B1, CYP24A1, and VDR in placentas from normal and preeclamptic pregnancies.

Vitamin D insufficiency/deficiency during pregnancy has been linked to increased risk of preeclampsia. Placenta dysfunction plays an important role in the pathogenesis of this pregnancy disorder. In this study, we tested the hypothesis that disturbed vitamin D metabolism takes place in preeclamptic placentas. Protein expressions of vitamin D binding protein (VDBP), 25-hydroxylase (CYP2R1), 1α-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1), and vitamin D receptor (VDR) were examined in placentas from normotensive and preeclamptic pregnancies. By immunostaining we found that in normal placenta VDBP, CYP24A1, and VDR expressions are localized mainly in trophoblasts, whereas CYP2R1 and CYP27B1 expressions are localized mainly in villous core fetal vessel endothelium. Protein expressions of CYP2R1 and VDR are reduced, but CYP27B1 and CYP24A1 expressions are elevated, in preeclamptic compared with normotensive placentas. Because increased oxidative stress is an underlying pathophysiology in placental trophoblasts in preeclampsia, we further determined whether oxidative stress contributes to altered vitamin D metabolic system in placental trophoblasts. Trophoblasts isolated from normal-term placentas were treated with hypoxic-inducing agent CoCl(2), and protein expressions of VDBP, CYP2R1, CYP27B1, CYP24A1, and VDR were determined. We found that hypoxia-induced downregulation of VDBP, CYP2R1, and VDR and upregulation of CYP27B1 and CYP24A1 expressions were consistent with that seen in preeclamptic placentas. CuZnSOD expression was also downregulated in trophoblasts treated with CoCl(2). These results provide direct evidence of disrupted vitamin D metabolic homeostasis in the preeclamptic placenta and suggest that increased oxidative stress could be a causative factor of altered vitamin D metabolism in preeclamptic placentas.

Seasonal variation in expression of markers in the vitamin D pathway in prostate tissue.

PURPOSE: Recent studies suggest variation in genes along the vitamin D pathway, as well as vitamin D receptor (VDR) protein levels, may be associated with prostate cancer. As serum vitamin D levels vary by season, we sought to determine whether the expression of genes on the vitamin D pathway, assessed in prostate tumor tissue, do the same. METHODS: Our study incorporates mRNA expression data from 362 men in the Swedish Watchful Waiting cohort, diagnosed between 1977 and 1999, and 106 men enrolled in the US Physicians' Health Study (PHS) diagnosed between 1983 and 2004. We also assayed for VDR protein expression among 832 men in the PHS and Health Professionals Follow-up Study cohorts. Season was characterized by date of initial tissue specimen collection categorically and by average monthly ultraviolet radiation levels. One-way analysis of variance was used to examine variation in the expression levels of six genes on the vitamin D pathway-VDR, GC, CYP27A1, CYP27B1, RXRα, CYP24A1-and VDR protein by season, adjusted for age at diagnosis and Gleason grade. Variation was also examined separately among lethal and nonlethal cases. RESULTS: Tumor expression levels of the six genes did not vary significantly by season of tissue collection. No consistent patterns emerged from subgroup analyses by lethal versus nonlethal cases. CONCLUSIONS: Unlike circulating levels of 25(OH) vitamin D, expression levels of genes on the vitamin D pathway and VDR protein did not vary overall by season of tissue collection. Epidemiological analyses of vitamin D gene expression may not be biased by seasonality.

Inverse association between serum concentrations of actin-free vitamin D-binding protein and the histopathological extent of fibrogenic liver disease or hepatocellular carcinoma.

BACKGROUND AND AIM: Next to its role as a carrier protein for vitamin D and its plasma metabolites, the primary function of vitamin D-binding protein (DBP; Gc-globulin) is to serve and to bind and neutralize extracellular monomeric actin (G-actin) released from necrotic cells, which in its long filamentous form (F-actin) triggers coagulation. We, therefore, investigated the kinetics of serum concentrations of actin-free DBP during the pathogenetic sequence from liver fibrosis to hepatocellular carcinoma (HCC) to initiate a discussion on a hypothetical association of serum concentrations of actin-free DBP and, thus, altered actin clearance in the circulation, and an increased risk of thrombosis in patients with functional liver insufficiency. RESULTS: Our data show that serum levels of actin-free DBP are decreased in patients with liver fibrosis (n=288) and HCC (n=102) compared with healthy controls (n=120 and n=63) and nonliver disease sick (n=312) and that the level of decrease correlates with the severity of disease as determined according to the score by the French METAVIR Cooperative Study Group staging system for hepatic fibrosis or the Edmondson-Steiner's grading system for the assessment of HCC. CONCLUSION: Although further, in particular, longitudinal studies are still necessary to back up our data, the presented findings suggest that decreasing levels of the actin-scavenger DBP could potentially contribute to the thrombophilic predisposition frequently observed in patients with chronic fibrogenic liver disease and HCC.

Questioning the role of actinfree Gc-Globulin as actin scavenger in neurodegenerative central nervous system disease: relationship to S-100B levels and blood-brain barrier function.

INTRODUCTION: Preliminary studies report on significantly higher levels of the major cytoskeleton protein actin in CSF of patients with neurodegenerative conditions and that the dynamics of these levels obviously correlates with disease progression and clinical disability. One of the primary functions of actinfree Gc-Globulin is to bind and neutralize extracellular monomeric actin, released into the circulation by necrotic or ruptured cells, and thus ameliorating the clinical outcome in situations of severe organ damage. AIM AND METHODS: This is the first study to investigate actinfree Gc-Globulin and S100-B levels (as reliable marker of neurodegeneration) in paired CSF and serum samples of patients with multietiological CNS diseases. RESULTS: 42% of all patients with CNS disease displayed serum concentrations of actinfree Gc-Globulin above the established reference range. CSF concentrations of actinfree Gc-Globulin and S100-B were positively correlated with the severity of blood-brain barrier (BBB) dysfunction. Furthermore, patients with severe BBB dysfunction presented a higher percentage of intrathecal synthesis of actinfree Gc-Globulin compared to patients with mild to moderate dysfunction and to patients with normal BBB function. Representative longitudinal data from selected patients demonstrated an inverse behaviour of actinfree Gc-Globulin and S100-B CSF concentrations, suggesting a consumption of the actin scavenger capacity of Gc-Globulin in times of increased neuronal damage. This presumption was supported by the fact that those conditions associated with a severe neuronal damage, in particular CNS trauma, and highest S100-B concentrations simultaneously displayed lowest actinfree Gc-Globulin levels, and thus residual actin binding capacity of Gc-Globulin. CONCLUSION: In summary, our data propose a function of actinfree Gc-Globulin also in the clearance of actin filaments from CSF of patients with neuronal damage. However, active recruitment of hepatic derived actinfree Gc-Globulin to the site of CNS injury is not observed. Much more, BBB leakage enables extraneuronally synthesized actinfree Gc-Globulin to extent its scavenger capacity for actin also to the subarachnoidal space. Furthermore, intrathecal synthesis of actinfree Gc-Globulin seems to be increased in patients with severe neurodegeneration.

2-D DIGE and MS/MS analysis of protein serum expression in rats housed in concrete and clay cages in winter.

In a previous study, we examined the physiological responses of male Sprague-Dawley rats over a 4-week exposure to concrete and clay cages. No general toxicological changes were observed in rats exposed to either of the two cage types in summer. Under winter conditions, however, various general toxicological effects were detected in rats housed in concrete cages, although rats housed in clay cages showed no such effects. The infrared thermographic examination indicated that skin temperature in the concrete-housed rats was abnormally low, but not so in the clay-housed rats. We examined proteomic changes in the serum of rats housed in winter in concrete and clay cages using two-dimensional differential in-gel electrophoresis and mass spectrometry/mass spectrometry. Five proteins were identified and quantitatively validated; all were cold stress-induced, acute phase proteins that were either up-regulated (haptoglobin) or down-regulated (alpha-1-inhibitor III, alpha-2u globulin, complement component 3, and vitamin D-binding protein) in the concrete-housed rats. These results suggest that the 4-week exposure to a concrete cage in winter elicited a typical systemic inflammatory reaction (i.e. acute phase response) in the exposed rats.

Gc-globulin (vitamin D binding protein) is synthesized and secreted by hepatocytes and internalized by hepatic stellate cells through Ca(2+)-dependent interaction with the megalin/gp330 receptor.

BACKGROUND: Gc-globulin or vitamin D binding protein is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D metabolites in the circulation. The present study was performed to analyze the interaction between gc-globulin of hepatocytes and hepatic stellate cells, the most important fat-/retinol-storing cell type in the liver, which spontaneously transdifferentiates to myofibroblasts in culture. METHODS: Hepatic stellate cells and hepatocytes were isolated by the pronase/collagenase reperfusion method, hepatocytes by collagenase reperfusion of the organ. Gc-globulin expression was monitored by immunocytochemistry, immunoblotting, RT-PCR, metabolic labelling with [(35)S]-methionine, and its intracellular binding to alpha-smooth-muscle actin was investigated by co-immunoprecipitation. Cytoskeletal stainings of gc-globulin and alpha-smooth-muscle actin in hepatic stellate cells and the identification of the receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2 were performed with confocal immunocytochemistry, immunoblotting and/or FACS-analysis. RESULTS: Hepatocytes synthesize and secrete gc-globulin as shown by RT-PCR and [(35)S]-methionine labelling, which could be suppressed by cycloheximide. Also, a strong signal for gc-globulin was detected in the immunoblot of native hepatic stellate cell lysates. However, no mRNA for gc-globulin was found in this cell type, which suggests no active synthesis by hepatic stellate cells. Hepatic stellate cells were tested positively for the presence of known gc-globulin interacting receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2. Inhibition of the megalin/gp330 receptor by a competitive, neutralizing antibody resulted in decreased intracellular availability of gc-globulin in hepatic stellate cells. The latter effect was enhanced by additional incubation of hepatic stellate cells with EDTA for complexing Ca(2+), suggesting a Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells via the megalin/gp300 receptor. This was supported by confocal microscopy which showed a co-localization of gc-globulin with the multifunctional megalin/gp330 receptor on this cell type. Inside hepatic stellate cells, a linkage between gc-globulin and alpha-smooth muscle actin filaments of hepatic stellate cells was detected by immunocytochemistry. Intracellular binding of gc-globulin to alpha-smooth-muscle actin filaments was confirmed by co-immunoprecipitation. CONCLUSION: We give evidence to the expression of the megalin/gp330 receptor on hepatic stellate cells and that this receptor is involved in the Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells, a protein synthesized and secreted into the extracellular space and circulation by hepatocytes. Inside hepatic stellate cells, it co-localizes with and binds to alpha-smooth muscle actin filaments. Under consideration of the available literature, these findings propose a participation of gc-globulin in hepatic vitamin D metabolism as well as in hepatic stellate cell stability and apoptosis as important mechanisms of liver regeneration.

The human vitamin D-binding protein gene contains locus control determinants sufficient for autonomous activation in hepatic chromatin.

The human vitamin D-binding protein (hDBP) gene is a member of a cluster that includes albumin, alpha-fetoprotein and alpha-albumin genes. The common origin, physical linkage and hepatic expression of these four genes predict shared regulatory element(s). However, separation of hDBP from the other three genes by 1.5 Mb argues that hDBP may be under autonomous control. To test for hDBP autonomy, mouse lines were generated with a transgene containing the hDBP gene along with extensive flanking sequences. Expression of this transgene was hepatic, robust and proportional to transgene copy number. DNase I hypersensitive site (HS) mapping revealed five liver-specific HS at the hDBP locus: HSI and HSIII at -2.1 kb and -0.13 kb upstream of the transcription initiation site, HSIV and HSV within intron 1 and HSVII located 3' to the poly(A) site. A second transgene with minimal flanking sequences confirmed the sufficiency of these gene-proximal determinants for hepatic activation. The hepatic-specific HS aligned with segments of phylogenetically conserved non-coding sequences. These data demonstrate the autonomy of the hDBP locus and suggest that this control is mediated by chromatin-based locus control determinants in close proximity to, and within the transcription unit.

Vitamin D binding protein in endometriosis.

OBJECTIVE: Two-dimensional gel electrophoresis is a powerful method for identifying post-translationally modified molecules in biological fluids. We examined the presence and expression of vitamin D binding protein (DBP) in the peritoneal fluid (PF) and plasma (PL) of women with endometriosis. METHODS: PL and PF samples were obtained from 36 women with untreated mild endometriosis (revised classification of the American Fertility Society [rAFS] stage I-II), 52 women with untreated severe endometriosis (rAFS stage III-IV), 17 women with endometriosis treated with the oral contraceptive (OC), and 40 controls (infertility, n = 23; tubal sterilization, n = 12; pelvic pain, n = 5). PF and PL samples were analyzed by quantitative, high-resolution 2-dimensional gel electrophoresis. RESULTS: The expression of one DBP isoform (DBPE) in the PF of patients with untreated endometriosis was significantly lower than in the control group (P <.05). The levels of PF DBPE in patients with endometriosis using OC were significantly higher than in women with untreated endometriosis (P <.05). No significant difference was observed in PL DBPE expression between women with and without endometriosis, while it was significantly increased in patients with endometriosis using OC (P <.05). DBP expression was not correlated with the stage of endometriosis (rAFS classification) or the phase of the menstrual cycle. CONCLUSION: The decreased level of DBPE in the PF but not in PL of women with untreated endometriosis suggests that this molecule may be relevant in the pathogenesis of this disease.

Recombinant human interleukin-1alpha increases serum albumin, Gc-globulin, and alpha1-antitrypsin levels in burned mice.

The response to thermal injury is a complex physiologic process requiring communication between sites of injury and distant target organs. The liver, one of these target organs, synthesizes a family of secretory proteins, the acute phase proteins, that carries out specific immunoprotective functions. In this study we investigated the effects of daily recombinant human interleukin-1alpha (rhIL-1alpha) administration on the serum levels of negatively regulated, i.e., albumin and Gc-globulin and positively regulated, i.e., alpha1-antitrypsin, acute phase proteins in a murine model of thermal injury. Adult CF-1 female mice underwent a 6.5-seconds, 20% total burn surface area, full thickness steam injury, and received either intraperitoneal rhIL-1alpha (20 microg x kg(-1) x day(-1)) or diluent for 10 days. Seven and 14 days after injury, mice were sacrificed, and serum albumin, Gc-globulin and alpha1-antitrypsin levels were measured by crossed immunoelectrophoresis technique. Thermal injury significantly lowered serum albumin levels, tended to decrease Gc-globulin levels, and increased serum alpha1-antitrypsin levels. Daily rhIL-1alpha administration after burn injury prevented hypoalbuminemia, and increased serum levels of Gc-globulin and alpha1-antitrypsin. IL-1 therapy might be helpful to maintain the homeostasis and immunity of the host after thermal injury.

Trauma stimulates the synthesis of Gc-globulin.

OBJECTIVE: Actin is the dominating intracellular protein and is released to the circulation after tissue injury. Gc-globulin is one of the plasma proteins responsible for removal of actin from the circulation. Recent studies have shown that the level of Gc-globulin is reduced shortly after trauma. Serial changes in Gc-globulin after severe injury have not been studied so far and could provide additional information about the role of Gc-globulin in the pathophysiological response to trauma. DESIGN: Prospective, observational. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Thirty-eight patients were included in the study: 12 women and 26 men with a median age of 38 years (range 19-86) and a median Injury Severity Score (ISS) of 18 (range 6-45). Seven patients died, on day 5, 8, 8, 10, 10, 13 and 21, respectively. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The serum concentration of Gc-globulin (Gctotal) and the percentage of Gc-globulin bound to actin (Gc%complexed) were measured daily for 1 week using rocket immunoelectrophoresis. Concentrations of free Gc-globulin (Gcfree) and Gc-globulin bound to actin (Gcbound) were calculated from these analytical results. The concentration of Gctotal and Gccomplexed correlated significantly (r = -0.99, p < 0.001) throughout the time period. After day 3 levels of Gc%complexed normalised, whereas levels of Gctotal continued to increase above control values. The concentrations of Gctotal and Gcfree were significantly lower in non-survivors compared to survivors; p = 0.005 and p = 0.03, respectively. This was combined with an inverse correlation of Gcbound between these two groups (r = -0.73; p = 0.04). CONCLUSIONS: Severe injury results in a prolonged load on the extracellular actin scavenger system; more pronounced in patients who do not survive. Gc-globulin displays characteristics of an acute phase reactant, with supra-normal serum levels 1 week after severe injury. Serial measurements of Gc-globulin after trauma could prove to be a method of early identification of patients with increased risk of mortality.

Reconstitution of the actin-scavenger system after orthotopic liver transplantation for end-stage liver disease: a prospective and longitudinal study.

Serum levels of the actin scavenger Gc-globulin (group-specific component, vitamin D-binding protein), a member of the albumin multigene family, are decreased in severe liver disease but have not been evaluated in relation to liver transplantation. We measured Gc-globulin and Gc-globulin-actin complex ratio daily for 2 weeks after transplantation in 17 patients with end-stage liver disease. Before transplantation, Gc-globulin levels were significantly less in the patients than in healthy controls (235 +/- 106 v 340 +/- 35 mg/L, respectively; P<.001), whereas complex ratio level was in the normal range. Five patients (group N) had pretransplantation Gc-globulin values within the normal range (mean +/- 2 SD), and 12 patients had subnormal values (group S). In group N, mean Gc-globulin levels posttransplantation remained stable at a lower level than before transplantation but still within normal range. In this group, cold ischemia time correlated inversely with Gc-globulin levels on day 2 (r = -0.88; P <.05). In group S, normal mean levels were reached at a mean of 11 days after transplantation. However, almost half these patients had subnormal Gc-globulin levels at day 14. Complex ratio levels remained normal in the study period in both groups. Prothrombin index levels (plasma coagulation factors II, VII, and X) were identical in both groups and returned to normal 7 days posttransplantation, whereas plasma albumin levels were less than normal in both groups and further decreased after transplantation. In conclusion, the maintenance (group N) or reestablishment (group S) of serum Gc-globulin to normal levels occurred in the early posttransplantation course in the same time frame as the prothrombin index. Gc-globulin synthesis seems unrelated to albumin synthesis. A prolonged cold ischemia time may cause reduced Gc-globulin levels early after transplantation.

Vitamin D-binding protein gene transcription is regulated by the relative abundance of hepatocyte nuclear factors 1alpha and 1beta.

Vitamin D-binding protein (DBP)/Gc-globulin, the major carrier of vitamin D and its metabolites in blood, is synthesized predominantly in the liver in a developmentally regulated fashion. By transient transfection analysis, we identified three regions in the 5'-flanking region of the rat DBP gene, segments F-2, B, and A, that contain tissue-specific transcriptional determinants. Gel mobility shift and DNase I footprinting analyses showed that all three regions contained binding sites for the hepatocyte nuclear factor 1 (HNF1), a transcriptional regulator composed of HNF1alpha and HNF1beta hetero- and homodimers. The activity of the most proximal segment A (coordinates -141 to -43) was DBP promoter-specific, position-dependent, and positively controlled by HNF1alpha. In contrast, the two more distal determinants (segments F-2 and B; coordinates -1844 to -1621 and -254 to -140, respectively) acted as classical enhancers in transfected hepatocyte-derived HepG2 cells; their activities were promoter- and orientation-independent, and disruption of their respective HNF1-binding sites resulted in marked loss of DBP gene expression. Remarkably, the activities of these two distal elements depended upon the relative levels of HNF1alpha and HNF1beta; HNF1alpha had a major stimulatory effect, whereas HNF1beta acted as a trans-dominant inhibitor of HNF1alpha-mediated enhancer activity. These results suggested that the net expression of the DBP gene reflected a balance between the two major HNF1 species; the relative abundance of HNF1alpha and HNF1beta proteins in a cell may thus play a critical role in determining the pattern of gene expression.

Abnormal expression and regulation of vitamin D receptor in experimental uremia.

The low concentration of the biologically active metabolite of vitamin D, namely 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), is critical to the pathogenesis of secondary hyperparathyroidism in chronic renal failure. The actions of 1,25(OH)2D3 are mediated through binding to a cellular receptor protein, the vitamin D receptor (VDR). In order to further investigate expression and regulation of VDR in uremia, we measured specific [3H]-1,25(OH)2D3 binding capacity and VDR mRNA concentration in intestinal mucosa and in parathyroid glands of subtotally nephrectomized rats (Nx) and compared Nx to sham-operated rats with normal kidney function (Intact). Intestinal [3H]-1,25(OH)2D3 binding capacity in short-term Nx (6-10 days after nephrectomy) was 663 +/- 114 fmol/mg protein; it was 517 +/- 34 in Intact (p = 0.06, n = 6 experiments). Intestinal VDR mRNA concentration was comparable between Nx and Intact. Specific 1,25(OH)2D3 binding capacity in parathyroid glands was higher in Nx (195 +/- 9 fmol/mg protein) than in Intact (116 +/- 14 fmol/mg protein, n = 5, p < 0.05). The affinity of the VDR for 1,25(OH)2D3 (KD) did not change in Nx. The 1,25(OH)2D3 binding capacity in intestinal mucosa of more long-term uremic animals (14-16 weeks after subtotal Nx) was 519 +/- 32 fmol/mg protein versus 349 +/- 31 in Intact (n = 3, p < 0.01). Parathyroid VDR was 171 +/- 9 fmol/mg protein in long-term Nx and 125 +/- 3 in Intact (p < 0.01). These results were confirmed when 1,25(OH)2D3 binding capacity in uremic rats with hereditary polycystic kidney disease was compared to control rats with normal kidney function (757 +/- 54 fmol/mg protein versus 495 +/- 59 in intestinal mucosa, p < 0.05; 273 +/- 48 versus 104 +/- 27 in parathyroid glands, p < 0.05). In parallel to changes in intestinal 1,25(OH)2D3 binding capacity, 1,25(OH)2D3-mediated stimulation of intestinal 25(OH)D3-24-hydroxylase activity was significantly higher in long-term subtotally Nx (1.43 +/- 0.06 pmol 24,25-dihydroxyvitamin D3/mg protein) than in sham-operated normal rats (1.04 +/- 0.10, p < 0.05). Administration of 1,25(OH)2D3 to sham-operated normal rats resulted in an increase of 1,25(OH)2D3 binding capacity by 20-40% in intestinal mucosa and by 40-50% in parathyroid glands. In contrast, 1,25(OH)2D3 caused down-regulation of mean 1,25(OH)2D3 binding capacity in short-term Nx by 38% in intestinal mucosa (p < 0.01) and by 43% in parathyroid glands (p < 0.01). In long-term Nx, mean 1,25(OH)2D3 binding capacity was reduced by 20% in intestinal mucosa (p < 0.05) and by 22% in parathyroid glands (p < 0.01). After prolonged exposure to 1,25(OH)2D3 for 6 weeks, intestinal 1,25(OH)2D3 binding capacity was markedly down-regulated in uremic rats (43% versus vehicle-treated animals p < 0.05). Taken together, our results provide evidence for abnormal expression and regulation of VDR in experimental uremia. This may be relevant for responsiveness to 1,25(OH)2D3 in renal insufficiency.

Regulation of human Gc (vitamin D--binding) protein levels: hormonal and cytokine control of gene expression in vitro.

Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group-specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting from treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin. We particularly focused on compounds known to be prototypic stimulants during the acute phase response. Interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGF beta) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants. In addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGF beta suggest that high basal levels of Gc synthesis may be maintained during the acute phase response.

Vitamin D binding protein is produced by human monocytes.

The expression of the DBP (vitamin D binding protein) gene was investigated in monocytes and in peripheral blood lymphocytes. DBP message was amplified through 35 cycles of PCR amplification using specific oligonucleotide primers. PCR products of the expected size were further identified by Southern blotting using a specific DBP probe. No expression of the DBP gene could be detected in peripheral blood lymphocytes, nor in the monocyte-derived U 937 cell line. In contrast, message for DBP was identified in monocytes activated with lipopolysaccharide when analyzed between 6 and 10 h following stimulation. These results suggest that the temporal expression of the DBP gene could play a major role in the activation of monocytes by 1-25(OH)2D3.

Hormonal regulation of vitamin D-binding protein production by a human hepatoma cell line.

Regulation of vitamin D-binding protein production by various hormones was studied in an established human hepatoma cell line, HuH-7. Among all the hormones studied, the maximal production of the binding protein was obtained by an extra-cellular addition of triamcinolone or epidermal growth factor. Cell numbers were not so changed except for the addition of insulin or glucagon. Our data indicate that the protein production may be regulated by insulin, estradiol, triamcinolone, dihydrotestosterone or epidermal growth factor.

Reduction of vitamin D hormone receptor mRNA levels in Alzheimer as compared to Huntington hippocampus: correlation with calbindin-28k mRNA levels.

Receptors for vitamin D hormone (VDR) and the calcium binding protein, calbindin-28k, have been localized in many tissues, including brain. In brain, VDR and calbindin-28k were reported to colocalize in hippocampal CA1 cells. We have shown that mRNA pool size for calbindin-28k was reduced, on average, by 35% in Alzheimer hippocampal CA1 cells, as compared to Huntington control (manuscript in preparation). In the present study, in situ hybridization with tritiated antisense RNA probes was used to examine VDR expression in paired Alzheimer and Huntington brain tissue. Message levels for VDR were reduced, on average, by 34% and 31%, respectively, in Alzheimer hippocampal CA1 and CA2 pyramidal cells, as compared to Huntington control. However, VDR message levels were not significantly different from control in Alzheimer temporal cortex or cerebellum. There was no correlation between VDR message levels and brain weight, autopsy interval, patient age or the extent of neurofibrillary degeneration. Instead, VDR mRNA pool size in hippocampal CA1 cells correlated significantly with calbindin-28k message levels (r = 0.52, P less than 0.001). Decreased message levels for VDR and calbindin-28k in these cells were due to an increased percentage of cells expressing lower message levels for these proteins. These results show that in Alzheimer hippocampal CA1 cells, VDR mRNA pool size is downregulated and that this downregulation may play a role in the reduction of calbindin-28k expression.

Plasma protein synthesis and secretion in the visceral yolk sac of the fetal rat: gene expression, protein synthesis and secretion.

This report compares the relative levels of messenger RNA species coding for plasma proteins in rat visceral yolk sac and fetal liver from 12.5 days to 21.5 days gestation. Transthyretin, retinol-binding protein, transferrin and alpha 1-fetoprotein mRNAs were detected in both tissues, although relative levels were much higher in the yolk sac compared to fetal liver, in early gestation. Messenger RNA coding for the positive acute phase proteins thiostatin, fibrinogen, alpha 2-macroglobulin and alpha 1-antitrypsin were detected at a low but significant level in yolk sac, while the levels in fetal liver steadily increased from 16.5 days gestation and, with the exception of alpha 1-antitrypsin, reached levels higher than those found in adult liver just prior to birth. Albumin, inter-alpha 1-trypsin inhibitor, alpha 1-acid glycoprotein, haptoglobin, vitamin D-binding protein and ceruloplasmin messenger RNA levels were either very low or undetectable in yolk sac and fetal liver. Secretion of proteins by yolk sac endoderm occurred largely across the basolateral surface, i.e. towards the fetal compartment. These data support the hypothesis that one function of the yolk sac in the rat is the synthesis and secretion of a select group of plasma proteins to maintain homeostasis in the fetal compartment in the period before the fetal liver has matured sufficiently to carry out this function.

Expression of transferrin and vitamin D-binding protein genes in an osteogenic sarcoma cell line.

Expression of genes encoding transferrin and the vitamin D-binding protein is described in a cell line, U-2 OS, derived from a human osteogenic sarcoma. The mRNA transcripts of transferrin and vitamin D-binding protein were shown to be the lengths of those found in normal human liver. The cells synthesize and secrete the transferrin and vitamin D-binding proteins, in addition to human albumin and ceruloplasmin. The U-2 OS cells were successfully transfected with chimeric genes carrying 670 bp of the 5' regulatory sequence of the human transferrin gene fused to a reporter chloramphenicol acetyltransferase gene. These data indicate that the appropriate transcriptional factors required for expression of four plasma proteins are produced by U-2 OS nuclei and that the U-2 OS cell line will be useful for studies analyzing regulation of these genes.