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At the beginning of the 20th century, the discovery of vitamin D by Sir EV McCollum allowed a better comprehension of its origin and its role, and made it possible to cure rickets, a largely prevalent disease at that time. The main role of vitamin D3 is to maintain calcium and phosphate homeostasis through the action of 1,25-dihydroxyvitamin D3, its active form. This underlies physiological functions related to calcium and phosphate, such as bone mineralization or muscle function. Progress in basic research for the last 40 years led to the discovery of the main hydroxylation steps that produce and catabolize the active form of vitamin D. It also uncovered the molecular aspects of vitamin D action, from its nuclear receptor, VDR, to the various target genes of this hormone. Recent progress in human genetics pointed out mutations in genes involved in vitamin D metabolism and 1,25-dihydroxyvitamin D3 actions. It also helped to understand the role of the major actors that control vitamin D production and effects, through 1,25-dihydroxyvitamin D3 actions on phosphate and calcium homeostasis, and on bone biology. Genetical engineering targeting the whole animal or defined tissues or cell types have yielded many mouse models in the past decades. When targeted to tissues important for vitamin D metabolism and activity, these models allowed a more detailed comprehension of vitamin effects on calcium and phosphorus homeostasis.

SOST encodes sclerostin, an inhibitor of bone formation that antagonizes canonical Wnt signaling. Variations of SOST expression have an impact on bone mineral density (BMD) and bone strength. We hypothesized that genetic and epigenetic DNA modifications have an impact on SOST gene expression. By analyzing 43 bone samples from women, we found that rs851054 (G/A) is associated with SOST mRNA expression, donors with one or two G allele(s) displaying higher SOST expression (p

Objective To analyze vitamin D metabolism and response to ketoconazole, an imidazole derivative that inhibits the vitamin D-1-hydroxylase, in infants with idiopathic hypercalcemia, and hypercalciuria. Study design Twenty infants (4 days-17 months) with hypercalcemia, severe hypercalciuria, and low parathyroid hormone level, (10 had nephrocalcinosis), including 10 treated with ketoconazole (3-9 mg/kg/day), were followed to the age of 2 to 51 months. Vitamin D receptor expression (VDR), 24-hydroxylase activity, and functional gene polymorphisms of vitamin D metabolism regulators VDR(rs4516035), 1-hydroxylase(rs10877012), 24-hydroxylase(rs2248359), FGF23(rs7955866), Klotho(rs9536314, rs564481, rs648202), were evaluated. Results Serum calcium levels, which occurred faster in the ketoconazole group (0.7 ± 0.2 versus 2.4 ± 0.6 months; P = .0076), and urinary calcium excretion (2.5 ± 0.5 versus 4.2 ± 1.7 months) normalized in all patients. Serum 1,25-(OH)2D levels were high normal and positively correlated to 25-(OH)D levels. Serum 24,25-(OH)2D levels were low normal, and skin fibroblasts from 1 patient showed defective up-regulation of the 24-hydroxylase by 1,25-(OH)2D despite normal VDR binding ability. An abnormally low prevalence of haplotype CC/CC for H589H/A749A in Klotho gene was found in patients and family members. Conclusions Ketoconazole is a potentially useful and safe agent for treatment of infantile hypercalcemia. Abnormal vitamin D metabolism is suggested as the mechanism, possibly involving defective up-regulation of the 24-hydroxylase by 1,25-(OH)2D3, and the klotho-FGF23 axis.

Newborn rat brain astrocytes, cultured in a serum-free medium, were exposed for 30 min to two types of reactive oxygen species. Cells were either treated with the xanthine/xanthine oxidase (X/XOD) system, which generates both H2O2 and the O2.- radical, or to H2O2 alone. Both treatments induced a dose-dependent accumulation of nerve growth factor (NGF) transcripts, 6 h after the exposure. Maximal effect was obtained with 6 mU/ml XOD, or 10(-4) M H2O2. A rapid expression of protooncogenes of the jun and fos families was also noticed in X/XOD- or H2O2-treated cells. This phenomenon was transient in cells exposed to X/XOD. However, in the case of H2O2-treated cells, the accumulation of c-fos or c-jun mRNAs was still pronounced 6 h after the end of the treatment and the levels of cell-secreted NGF appeared relatively reduced, when compared with those obtained after a shock with the X/XOD system. This raised the possibility that H2O2 at 10(-4) M could depress protein synthesis. Measurements of the incorporation of radiolabeled amino acids into trichloroacetic acid-precipitable material supported this assumption. Level of radioactivity associated with cellular material was dramatically reduced in H2O2-treated cells, when it was compared with control or X/XOD-treated cells. Furthermore, treatment of cells with the protein synthesis inhibitor anisomycin had an effect similar to that of H2O2 because it caused an accumulation of c-fos, c-jun, and NGF transcripts after 6 h of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The human vitamin D receptor (hVDR) gene encompasses eight exons (2-9) in the so-called coding region and six more exons (1a-1f) in the so-called regulatory region, which contains several reported promoters. Evolutionary comparison performed on the VDR promoter sequences of a dozen of mammalian species shows a very high conservation of numerous regions around and in the 1a promoter, including exons 1e, 1a and 1d, and the Sp1 site region. This suggests that the so-called 1a promoter is well conserved among mammals. Homology among mammals also concerns three functional SNP site regions of the hVDR 1a promoter, the 1e-G-1739A SNP region (a Cdx-2 binding site), and both 1a-G-1521C and 1a-A-1012G sites, the 1a-1012A being located within a GATA site. Interestingly, the 1521G and 1012A nucleotides are being evolutionary conserved, suggesting that the 1521C/1012G haplotype, which is found in human chromosomes (43% of Caucasians), is a human specificity.

We report here the first association between vitamin D–resistant rickets, alopecia, and type 1 diabetes in a child with compound heterozygous mutations in the VDR gene. Transfection studies suggest dissociated effects of VDR gene mutations on the regulation of genes involved in vitamin D metabolism and dendritic cell maturation. Introduction: Whereas vitamin D may play a role in the immune tolerance process, no patient has been reported to associate hereditary vitamin D–resistant rickets (HVDRR) and an autoimmune disease, and no attempt has been made to delineate the outcome of mutations of the vitamin D receptor (VDR) on the transcription of genes controlling immune tolerance. Materials and Methods: The VDR gene was analyzed in a child with vitamin D–resistant rickets, total alopecia, and early childhood–onset type 1 diabetes. Patient's fibroblasts and COS-7 cells transfected with wildtype or mutant VDRs were studied for ligand-binding capacity, transactivation activity using two gene promoters [CYP-24, a classical 1,25(OH)2D3-responsive gene, and relB, a critical NF-κB component for regulation of dendritic cell differentiation], VDR-RXR heterodimers association to CYP 24 VDREs by gel mobility shift assays, and co-activator binding by Glutathione-S-transferase pull-down assays. Results: Two novel compound heterozygous mutations (L263R and R391S) were identified in the VDR ligand-binding domain in this child. Both mutations significantly impaired VDR ligand-binding capacity but had dissociated effects on CYP-24 and RelB promoter responses to vitamin D. CYP 24 response binding to SRC-1 and RXR-heterodimer binding to CYP24 VDREs were abolished in L263R mutants but normal or partially altered in R391S mutants. In the opposite, RelB responses to vitamin D were close to normal in L263R mutants but abolished in R391S mutants. Conclusions: We report the first clinical association between HVDRR, total alopecia, and early childhood–onset type 1 diabetes. Mutations in the VDR ligand-binding domain may hamper the 1,25(OH)2D3–mediated relB responses, an effect that depends on the site of the VDR mutation and cannot be anticipated from VDR ligand-binding ability or CYP-24 response. Based on these results, we propose to survey the immune function in patients with HVDRR, including those with moderate features of rickets.

Polymorphisms of the vitamin D receptor gene (VDR) have been shown to be associated with several complex diseases, including osteoporosis, but the mechanisms are unknown and study results have been inconsistent. We therefore determined sequence variation across the major relevant parts of VDR, including construction of linkage disequilibrium blocks and identification of haplotype alleles. We analyzed 15 haplotype-tagging SNPs in relation to 937 clinical fractures recorded in 6,148 elderly whites over a follow-up period of 7.4 years. Haplotype alleles of the 5' 1a/1e, 1b promoter region and of the 3' untranslated region (UTR) were strongly associated with increased fracture risk. For the 16% of subjects who had risk genotypes at both regions, their risk increased 48% for clinical fractures (P = .0002), independent of age, sex, height, weight, and bone mineral density. The population-attributable risk varied between 1% and 12% for each block and was 4% for the combined VDR risk genotypes. Functional analysis of the variants demonstrated 53% lower expression of a reporter construct with the 1e/1a promoter risk haplotype (P = 5 x 10(-7)) in two cell lines and 15% lower mRNA level of VDR expression constructs carrying 3'-UTR risk haplotype 1 in five cell lines (P = 2 x 10(-6)). In a further analysis, we showed 30% increased mRNA decay in an osteoblast cell line for the construct carrying the 3'-UTR risk haplotype (P = .02). This comprehensive candidate-gene analysis demonstrates that the risk allele of multiple VDR polymorphisms results in lower VDR mRNA levels. This could impact the vitamin D signaling efficiency and might contribute to the increased fracture risk we observed for these risk haplotype alleles.

At the beginning of the 20th century, the discovery of vitamin D by Sir EV McCollum allowed a better comprehension of its origin and its role, and made it possible to cure rickets, a largely prevalent disease at that time. The main role of vitamin D3 is to maintain calcium and phosphate homeostasis through the action of 1,25-dihydroxyvitamin D3, its active form. This underlies physiological functions related to calcium and phosphate, such as bone mineralization or muscle function. Progress in basic research for the last 40 years led to the discovery of the main hydroxylation steps that produce and catabolize the active form of vitamin D. It also uncovered the molecular aspects of vitamin D action, from its nuclear receptor, VDR, to the various target genes of this hormone. Recent progress in human genetics pointed out mutations in genes involved in vitamin D metabolism and 1,25-dihydroxyvitamin D3 actions. It also helped to understand the role of the major actors that control vitamin D production and effects, through 1,25-dihydroxyvitamin D3 actions on phosphate and calcium homeostasis, and on bone biology. Genetical engineering targeting the whole animal or defined tissues or cell types have yielded many mouse models in the past decades. When targeted to tissues important for vitamin D metabolism and activity, these models allowed a more detailed comprehension of vitamin effects on calcium and phosphorus homeostasis.

Objective. Chronic HCV infection is associated with extra-hepatic manifestations. Recent studies have suggested an immunomodulatory role for vitamin D during HCV infection. We investigated the association between serum vitamin D status and the presence of HCV extra-hepatic manifestations. Methods. 25(OH)D serum levels were assessed in 94 HCV + RNA + patients [including 48 patients with mixed cryoglobulinaemia (MC) vasculitis]. Correlations between serum 25(OH)D levels and the presence of extra-hepatic manifestations of HCV infection were analysed. Results. Overall, 84 of 94 patients (89%) had hypovitaminosis D (430 ng/ml). Patients with vitamin D deficiency vs insufficiency vs sufficiency more frequently had systemic vasculitis (P=0.02), in particular purpura (P=0.006), detectable MC (P=0.008) and low C4 serum levels (P=0.006). Serum levels of 25(OH)D were also correlated with cryoglobulin and C4 levels and with marginal zone B cells and regulatory T cells. In multivariate analysis, the presence of MC and systemic vasculitis remained independently associated with low 25(OH)D levels. Conclusion. In chronic HCV infection, low 25(OH)D levels correlate with the presence of mixed cryoglobulinaemia and systemic vasculitis in chronic HCV infection. These findings suggest the potential multifaceted benefits of vitamin D supplementation in HCV-infected patients with extra-hepatic manifestations, but interventional studies are needed to confirm these data.

Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as α-thrombin or collagenase induce a continuous, dose-dependent enhancement of the levels of cell-secreted NGF. Incubation of astrocytes for a 24-h period with 300 ng/ml of α-thrombin (∼9 nM, or 1 U/ml) results in an increase of the levels of cell-secreted NGF by a factor of three- to fourfold, and at doses 10 times higher, stimulation by a factor of up to four-to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP-1 family, such as c-fos, c-jun or junB. Trypsin, plasmin, α-chymotrypsin, or elastase also enhanced, to different extents, the levels of cell-secreted NGF. However, unlike α-thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose-response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell-substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of α-thrombin-indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of α-thrombin with D-phenyl-alanyl-L-propyl-L-arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of α-thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of α-thrombin require binding to the cell surface. We found that γ-thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as α-thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity. Part of this effect may be due to the cleavage of a transmembrane receptor for a heptapeptide that acts as an agonist of this receptor and displays a limited but significant action on the levels of cell-secreted NGF. These data suggest that proteolytic enzymes might induce astrocytes to produce NGF in vivo.

ObjectiveThe prevalence of lower limb deformities physiologically decreases after 5 years of age. It remains high in some tropical and subtropical regions where it has been associated with severe vitamin D deficiency, low calcium/milk intakes, malnutrition, and/or fluoride overexposure. Very little data is available in apparently healthy Caucasian children and adolescents.DesignWe evaluated the prevalence of genu varum/valgum and other clinical symptoms, and assessed vitamin D status and markers of calcium metabolism in 226 apparently healthy European full-time boarders (7–16 years) seen during winter–spring and fed a cereal-based diet with little access to meat, milk, and dairy products. A cohort of 71 white children and adolescents hospitalized for acute illness served as age-matched controls.ResultsAssociation studies showed a high prevalence of lower limb deformities (36%) and higher alkaline phosphate activities in the 21% of children and adolescent full-time boarders with serum 25-(OH)D levels ≤30 nmol/l, and low serum calcium in the 74% of boarders with 25-(OH)D levels ≤50 nmol/l, compared with boarders with higher vitamin D status. No such anomalies were found in the control cohort despite lower serum 25-(OH)D levels.ConclusionsLow 25-(OH)D levels, at least during winter–spring, combined with additional risk factors such as very low calcium/milk intakes and possibly digestive disorders, are associated with an increased risk of genu varum/valgum in European children and adolescents. Thus, dietary fortification, or supplementation with vitamin D, may be recommended, at least during the winter, to European children and adolescents with either none or insufficient calcium/dairy product intakes.

Circulating concentrations of corticosterone and 1,25-(OH)2D3 have been reported to be respectively increased and decreased in the streptozotocin-treated rats. Using the cell line L929 cultured in a steroid-free medium, we show that the alteration in the levels of corticosterone and 1,25-(OH)2D3 found in vivo in experimental diabetes is able to decrease the synthesis of NGF by these cells. This finding raises a possible relationship between the balance in the concentration of these steroids and some aspects of the neuropathic complications found in experimental diabetes.

Calcium homeostasis and bone metabolism require the vitamin D receptor (VDR) to function properly, as evidenced in patients and transgenic mice with VDR mutations. We have shown that (A/G) polymorphism at the -1012 locus of the VDR promoter (rs4516035) is frequent in European populations, may influence VDR expression, is associated with height in French adolescent girls, and is associated with their lumbar spine mineral density in case of insufficient milk intake. Here, an association study was performed in a cohort of Moldovan children and adolescents, living at latitude similar to the first cohort but receiving a cereal-based diet with very low milk/dairy product intakes. Children and adolescents in this cohort had similar 25-(OH) D levels, but a short stature and low serum calcium levels, compared to the first cohort. Their height remained associated with the A-1012G VDRp genotype. In addition, their serum calcium levels were associated with VDRp polymorphism, excepted when their 25-(OH) D levels were low (below 33 nmol/L). In conclusion, the -1012 VDRp genotype appears to be associated with height in European children whatever their calcium/dairy product intakes, and may modulate their calcium homeostasis in conditions of low calcium/milk intakes when vitamin D status is sufficient.

Low milk intakes hamper bone mineral acquisition during adolescence, especially in European girls. We hypothesized that ethnic-specific polymorphisms of the vitamin D receptor gene promoter (VDRp) influence this milk/bone association. We evaluated lumbar spine BMC and BMD, milk/dairy products and calcium intakes, markers of P-Ca metabolism, and VDRp polymorphisms at the Cdx-2 binding (rs11568820) and -1012 (rs4516035) loci in 117 healthy European peri- and postmenarcheal girls (14.9 +/- 1.6 yr) during a 4-yr follow-up. Calcium intakes from milk, nonmilk dairy products, and nondairy products averaged 199, 243, and 443 mg/d at the initiation of the study. Results show no association between milk intakes and bone mass accrual in girls bearing an A/A genotype at the -1012 VDRp locus (30% of the cohort). In contrast, A/G or G/G girls had lower spine BMC (-13%, p = 0.031), BMD (-10%, p = 0.004), and BMD Z-score (-0.84 SD, p = 0.0003) when their milk intakes were260 ml/d compared with genotype-matched girls with higher milk intakes and with girls with an A/A genotype. The negative impact of low milk intake persisted up to 19.0 +/- 1.7 yr. These findings suggest that European girls bearing a -1012 A/G or G/G VDRp genotype should have higher milk/calcium intakes for optimal vertebral mass accrual during adolescence than girls bearing an A/A genotype, a genotype found in 30% of European and 98% of Asian and Sub-Saharan African populations. VDRp genotype diversity may contribute to the ethnic differences observed in milk requirements for bone health during adolescence.

Dexamethasone is known to decrease the pool of nerve growth factor (NGF) mRNA in various experimental systems. The negative regulatory effect of the glucocorticoid was first observed in mouse fibroblast-like L929 cells, and was subsequently reported to take place in many experimental systems, including in vivo following sciatic nerve injury. Conversely, another steroid hormone, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) was recently reported to promote NGF synthesis in mouse L929 cells. The present work was undertaken to investigate the effect of the concomitant addition of both steroids to L929 cells. Measurements of NGF mRNA and assays of the mature protein secreted by the cells provide evidence that the negative regulation exerted by dexamethasone may be counteracted in a dose-dependent manner by the positive action of 1,25-(OH) 2 D 3 , and vice versa. Therefore, the expression of the NGF gene can be regulated in a subtle way by the balance between the two steroids. It may be expected on the basis of these observations that in tissues that are responsive to both hormones, administration of 1,25-(OH) 2 D 3 should be able to reverse the down-regulation of NGF synthesis elicited by glucocorticoids.

Familial hypophosphatemic rickets is a rare disease, which is mostly transmitted as an X-linked dominant trait, and mutations on the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) gene are responsible for the disease in most familial cases. In this study we analyzed PHEX in a large cohort of 118 pedigrees representing 56 familial cases and 62 sporadic cases. The high-resolution melting curves technique was tested as a screening method, along with classical sequencing. PHEX mutations have been found in 87% of familial cases but also in 72% of sporadic cases. Missense mutations were found in 16 probands, two of which being associated with other PHEX mutations resulting into truncated proteins. By plotting missense mutations described so far on a 3D model of PHEX we observed that these mutations focus on two regions located in the inner part of the PHEX protein. Family members of 13 sporadic cases were analyzed and a PHEX mutation was detected in one of the apparently healthy mother. These results highlight the major role of PHEX in X-linked dominant hypophosphatemic rickets, and give new clues regarding the genetic analysis of the disease. A screening of the different family members should be mandatory when a PHEX mutation is assessed in a sporadic case and the search for another PHEX mutation should be systematically proceed when facing a missense mutation.

Familial hypophosphatemic rickets is transmitted in most cases as an X-linked dominant trait and results from the mutation of the PHEX gene predominantly expressed in osteoblast and odontoblast. Patients with rickets have been reported to display important dentin defects. Our purpose was to explore the structure, composition and distribution of noncollagenous proteins (NCPs) of hypophosphatemic dentin. We collected teeth from 10 hypophosphatemic patients whose mineralization occurred either in a hypophosphatemic environment or in a corrected phosphate and vitamin environment. Teeth were examined by scanning electron microscopy, immunohistochemistry and Western blot analysis. An abnormal distribution (accumulation in interglobular spaces) and cleavage of the NCPs and particularly of matrix extracellular phosphoglycoprotein were observed in deciduous dentin. In contrast, it was close to normal in permanent dentin mineralized under corrected conditions. In conclusion, dentin mineralization in a corrected phosphate and vitamin D environment compensates the adverse effect of PHEX mutation.

Low calcium intake hampers bone mineral acquisition in adolescent girls. This study explores dietary calcium sources and nutrients possibly associated with vertebral mass. Milk intake is not influenced by genetic variants of the lactase gene and is positively associated with serum IGF-1 and with lumbar vertebrae mineral content and density. Low calcium intake hampers bone mineral acquisition during adolescence. We identified calcium sources and nutrients possibly associated with lumbar bone mineralization and calcium metabolism in adolescent girls and evaluated the possible influence of a genetic polymorphic trait associated with adult-type hypolactasia. Lumbar bone mineral content (BMC), bone mineral density (BMD), and area, circulating IGF-1, markers of bone metabolism, and −13910 LCT (lactase gene) polymorphism; and intakes of milk, dairy products, calcium, phosphorus, magnesium, proteins, and energy were evaluated in 192 healthy adolescent girls. After menarche, BMC, BMD, serum IGF-1, and serum PTH were tightly associated with milk consumption, but not with other calcium sources. All four parameters were also associated with phosphorus, magnesium, protein, and energy from milk, but not from other sources. Girls with milk intakes below 55 mL/day have significantly lower BMD, BMC, and IGF-1 and higher PTH compared to girls consuming over 260 mL/day. Neither BMC, BMD, calcium intakes, nor milk consumption were associated with −13910 LCT polymorphism. Milk consumption, preferably to other calcium sources, is associated with lumbar BMC and BMD in postmenarcheal girls. Aside from being a major source of calcium, milk provides phosphates, magnesium, proteins, and as yet unidentified nutrients likely to favor bone health.

Background & Aims: The Cdx2 homeobox gene exerts multiple functions including trophectoderm specification, antero-posterior patterning, and determination of intestinal identity. The aim of this study was to map genomic regions that regulate the transcription of Cdx2, with a particular interest in the gut. Methods: Genomic fragments covering 13 kilobase (kb) of the mouse Cdx2 locus were analyzed in transgenic mice and in cell assays. Results: No fragment was active in the trophectoderm. Fragments containing the first intron and extending up to −5-kb upstream of the transcription start site became active posteriorly at gastrulation and then inactive at midgestation in every tissue including the endoderm. Specific persistence of activity in the intestinal endoderm/epithelium beyond midgestation requires extending the genomic fragment up to −9 kb. We identified a 250-base pair segment around −8.5-kb binding and responding to endodermal factors, with a stimulatory effect exerted synergistically by HNF4α, GATA6, Tcf4, and β-catenin. These factors were able to activate endogenous expression of Cdx2 in nonintestinal Hela cells. Conclusions: Multiple regulatory regions control the complex developmental pattern of Cdx2, including far upstream sequences required for the persistence of gene expression specifically in the gut epithelium throughout life. Cooperation between HNF4α, GATA6, β-catenin, and Tcf4 contributes to the intestine-specific expression of Cdx2.

International audience; The homeobox gene CDX2 plays a major role in development, especially in the gut, and it also acts as a tumor suppressor in the adult colon. Using orthotopic and heterotopic xenografts of human primary colorectal tumor cells and cell lines in nude mice, we addressed the effect of the microenvironment on CDX2 expression. In cells expressing CDX2 at a high level in culture, this level was maintained in subcutaneous grafts but was reduced when implanted into the cecum wall. Reciprocally, in cells with low CDX2 expression in culture, the level remained low in grafts into the cecum wall but was stimulated subcutaneously. In vitro co-cultures showed that CDX2 expression was activated in cells grown on layers of skin fibroblasts but not on intestinal fibroblasts. The stimulation was transcriptional, as assessed by transfection experiments with reporter plasmids containing the murine Cdx2 promoter. Together, these data demonstrate experimentally that CDX2 expression is adaptable and strongly dependent on the microenvironment surrounding the tumor cells. We exclude a role of the Notch pathway in this regulation. The regulation of CDX2 by the microenvironment might be relevant during the process of metastatic dissemination when the gene is transiently turned down in invasive cells.

Numerous association studies have dealt with single-nucleotide polymorphisms (SNPs) in coding and intronic regions of the human vitamin D receptor (hVDR) gene. We have hypothesized that phenotypic traits may also be associated with variations in VDR expression due to the presence of SNPs in promoter regions. In this work, we have studied two SNPs located 1521 bp (G/C) and 1012 bp (A/G) upstream of the transcriptional start site of the main human VDR gene promoter. One base-change in any of the two variant sites led to a dramatic change in protein–DNA complex formation using nuclear extracts from HEK293, Caco-2 and COS-7 cells. Genetic analysis of 185 healthy adolescent girls evidenced two major haplotypes: 1521G/1012A and 1521C/1012G and three main genotypes: homozygous for 1521G/1012A (21.1%), homozygous for 1521C/1012G (17.3%) and heterozygous 1521CG/1012GA (57.3%). On the basis of transfection data, promoter activity was nearly 2-fold higher with the 1521G/1012A haplotype, when compared with the 1521C/1012G haplotype. Clinical and biological association study in the adolescent cohort showed that girls with a CC/GG genotype had (i) lower circulating levels of 25-dihydroxyvitamin D, with no detectable consequence on calcium metabolism, (ii) lower serum IGF-1 levels and (iii) smaller height from 11 years of age up to adult height.

The effect of MC903, an analogue of 1,25-dihydroxyvitamin D3, on the expression of the nerve growth factor (NGF) gene has been studied in L cells. MC903 induces an increase in both NGF mRNA and protein with a time course similar to that obtained with 1,25-dihydroxyvitamin D3. This finding points to the potential importance of 1,25-dihydroxyvitamin D3 derivatives in the treatment of NGF-sensitive disorders.

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

Colchicine, nocodazol, and vinblastine, three microtubule-disrupting drugs, were shown to increase the levels of both nerve growth factor (NGF) mRNA and cell-secreted NGF protein in L929 cells, with levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or amyloid precursor protein (APP) mRNAs remaining unaffected. Northern blot analysis demonstrated that colchicine also increased NGF mRNA levels in rat primary astrocytes and mouse skin fibroblasts. The specificity of the effects observed was assessed by the fact that the microtubule-stabilizing agent Taxotere, a semisynthetic compound structurally related to taxol, suppressed the effects of colchicine, whereas lumicolchicine, a colchicine derivative that has no action on the microtubule network, had no influence on NGF expression. Likewise, the disruption of the microfilament network by cytochalasin B did not increase NGF mRNA levels in L929 cells. Furthermore, the increase in NGF gene expression observed following microtubule disruption depended on a cascade of events involving at least one protein kinase, which is not down-regulated by phorbol ester, and on a pertussis toxin sensitive step. These results support the concept that tubulin and/or the microtubule cytoskeleton play an active role in the regulation of the NGF gene.

Primary mouse brain astrocytes were stimulated with phorbol 12-myristate 13-acetate (PMA), serum, forskolin and ionophore A23187, in order to investigate the effect of distinct signalling pathways on the expression of the nerve growth factor (NGF) gene and of proto-oncogenes encoding transcription factors of the Fos and Jun families. PMA, and to a lesser extent serum, induced a marked accumulation of NGF transcripts, in agreement with published observations [Brain Res., 570 (1992) 316-322]. The effect of A23187 was less pronounced and that of forskolin barely detectable. No relationship was observed between the expression of NGF gene and that of c-fos, fos-B, fra-1, jun-B proto-oncogenes. In contrast, changes in the levels of NGF transcripts were associated with corresponding modifications of the levels of c-jun transcripts, a fact which suggests that the c-Jun protein exerts a regulatory role on the expression of the NGF gene. In these cells, however, the regulation of NGF synthesis appears complex, since a pretreatment with forskolin or ionophore A23187 interfered with the promoting effect elicited by PMA or serum in inducing an early decline of the levels of NGF transcripts. This phenomenon was accompanied by a corresponding decrease in the amounts of cell-secreted NGF in cells treated with forskolin and PMA. A23187 had a much more striking effect on the production of mature NGF since this compound maintained the level of cell-secreted NGF to basal values, irrespective of the presence of PMA. A similar inhibitory effect was observed with thapsigargin, another compound able to increase the cytosolic concentration of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

An inflammatory reaction, essential for defence against infection and for wound repair, may also induce irreversible tissue damage. It appears that the central nervous system has developed its own immunosuppressive strategy in order to limit the destructive effects of inflammation. To clarify this point, we have characterized in one unique model of inflammation induced in the rat by intracerebral lipopolysaccharide injection the kinetics of the inflammatory reaction, the participation of immunitary and glial cells and of three growth factors. Among these molecules, brain-derived neurotrophic factor mRNA expression was found decreased following LPS injection. No striking differences were observed in the brain parenchyma after stab lesion or inflammatory lesion apart from an increase in the number of monocytes/macrophages recruited early to the lesion area. Macrophages were later accumulated around the lesion when astroglia and microglia reactions occurred. Some of the macrophages and microglia expressed major histocompatibility complex class II antigens on their surface whereas no T or B lymphocytes were observed in the brain parenchyma. However, a subpopulation of CD3- and CD4-negative CD8-positive cells, likely natural killer cells, was observed around the lesion site; this recruitment was inhibited by the highest dose of LPS. This study therefore supports the hypothesis of a suppression of some aspects of cell-mediated immunity in the brain, mechanisms which need to be further characterized.

Increased expression of the nerve growth factor (NGF) gene may be obtained by treating L929 fibroblasts with serum, phorbol 12-myristate 13-acetate (PMA), or 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3). The possible involvement of GTP-binding proteins (G proteins) in these regulatory events was monitored by exposing the cells to pertussis toxin (PT), a compound known to inactivate several types of G proteins by ADP ribosylation. Measurements of the I pool of NGF mRNA by Northern blot analysis, and quantification of the factor secreted by the cells with a double-site ELISA assay, indicate that pretreatment with PT decreases by about 60% the effect of serum on the levels of NGF transcript and secreted factor. This effect is accompanied by a corresponding decrease of the expression of c-fos gene, which takes place soon after the addition of serum to the cells. In contrast, PT had no effect on the basal level of NGF mRNA found in cells maintained in serum-free medium or in cells stimulated with PMA or 1,25-(OH)2D3. These results indicate that some serum factor(s) acts via plasma membrane receptors able to interact with PT-sensitive G proteins to modulate NGF gene expression. In contrast, 1,25-(OH)2D3 appears to mediate its action through a different signalling pathway, which is likely to require its cytosolic receptor, and is independent of PT-sensitive G protein and c-fos induction. PMA is thought to directly activate enzymes of the protein kinase C family, and would therefore bypass the step involving activation of these enzymes by a G-proteindependent pathway. These results suggest that a dys function in the G protein(s) and c-fos pathway involved in the regulation of NGF gene could lead to neuropathological processes by altering the expression of the NGF gene. In addition, 1,25-(OH)2 D3, in light of its ability to bypass these pathways, could be of potential interest in the treatment of neurodegenerative diseases.

1,25-dihydroxyvitamin D3(1,25-(OH)2D3), a metabolically active form of vitamin D, is shown to increase in a dose-dependent manner the cellular pool of NGF mRNA in murine L-929 fibroblasts cultured in a serum-free medium. This effect can be detected as early as 3 hours after 1,25-(OH)2D3 addition and persists for at least 28 hours. It is accompanied by an enhancement of the amount of NGF-protein secreted in the culture medium. Since the proto-oncogene c-fos appears involved in the regulation of the NGF gene (Mocchetti et al.: Proceedings of the National Academy of Sciences of the United States of America 86:3871-895, 1989; Hengerer et at: Proceedings of the National Academy of Sciences of the United States of America87:3899-3903, 1990), the effect of 1,25-(OH)2D3on c-fos expression was analysed and compared to that elicited by other inducers of the NGF gene, serum (Wion et al: FEBS- Letters 189:37-41, 1985) and phorbol 12-myristate 13-acetate (PMA) (Wion et al: FEBS Letters 262:42-44, 1990). Addition of serum or PMA to L-929 cells was; rapidly followed by a transient activation of the c-fos gene. In contrast,c-fos transcripts remained undetected in the presence of 1,25-(OH)3D3 The failure to find any evidence of c-fos expression suggests that 1,25-(OH)2D3could enhance the pool of NGF mRNA by a mechanism independent of the c-fos pathway. The effective concentration range, as low as 10−10 M raises the possibility that 1,25-(OH)2D3 could represent one of the serum factors that might contribute to the regulation of the NGF gene in vitro and possibly in vivo. This suggests a possible therapeutic interest for 1,25-(OH)2D3in neurodegenerative diseases.

Regulation of the expression of the nerve growth factor (NGF) gene has been reported previously to be mediated via the protooncogene c-fos. Activation of the protein kinase C pathway and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has also been reported to increase the pool of NGF transcripts in L929 fibroblasts. Here we show that activation of the cyclic AMP second messenger pathway antagonized the effect of phorbol 12-myristate 13-acetate (PMA) or serum on NGF synthesis, whereas it enhanced that of 1,25(OH)2D3. A positive effect was also observed when serum, PMA, and 1,25(OH)2D3 were added together, but dexamethasone reduced this enhancement. There was no close correlation between the increase in c-fos mRNA and that in NGF mRNA, suggesting that expression of the c-fos protooncogene is not necessarily followed by induction of the NGF gene. Rather, these two genes are simultaneously, and not sequentially, induced after forskolin treatment. It appears that regulation of the NGF gene depends on a repertoire of multiple regulatory AP-1 complexes arising from activation of the second messenger pathways. This suggests that NGF gene expression is under the control of a complex interplay among second messenger pathways, protooncogenes, and steroid hormones such as 1,25(OH)2D3 and glucocorticoids.