Class 3 semaphorins are transcriptionally regulated by 1,25(OH)2D3 in osteoblasts.

The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)2D3 in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)2D3 target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)2D3 in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)2D3 targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)2D3 suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)2D3 signaling in osteoblasts.

Nrp2 deficiency leads to trabecular bone loss and is accompanied by enhanced osteoclast and reduced osteoblast numbers.

Neuropilin 1 (Nrp1) and Nrp2 are transmembrane receptors that can bind class 3 semaphorins (Sema3A-G) in addition to VEGF family members to play important roles in axonal guidance, vascularization and angiogenesis, as well as immune responses. Moreover, recent evidence implicates Sema3A/Nrp-mediated signaling in bone regulation. However, to date the expression of Nrp2 in bone has not been investigated and a possible role for Nrp2 in the maintenance of bone homeostasis in vivo remains unexplored. Here we show that Nrp2, together with its possible coreceptors (Plexin A family members and Plexin D1) and class 3 semaphorin ligands, were expressed during in vitro osteogenic differentiation of bone marrow stromal cells. Moreover, Nrp2 transcript and protein levels were highly induced in hematopoietic bone marrow cell-derived osteoclast cultures. Osteoblastic as well as osteoclastic Nrp2 expression was confirmed by immunohistochemistry of the long bones of mice. Interestingly, Nrp2 knockout mice were characterized by a low bone mass phenotype which was accompanied by an increased number of osteoclasts and a decreased osteoblast count. Collectively, these data point to a physiological role for Nrp2 in bone homeostasis.

The odd-skipped related genes Osr1 and Osr2 are induced by 1,25-dihydroxyvitamin D3.

The odd-skipped related genes Osr1 and Osr2 encode closely related zinc finger containing transcription factors that are expressed in developing limb. However, their role in osteoblast proliferation and differentiation remains controversial and little is known about their regulation. In this study we showed that both Osr1 and Osr2 were expressed in several murine and human osteoblast cell lines as well as in primary osteoblast cultures. Moreover, their transcript levels were regulated by a number of osteogenic stimuli in murine pre-osteoblast MC3T3-E1 cells. The most robust regulation of Osr1 and Osr2 mRNA levels was observed after stimulation with 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 induced transcript levels of Osr1 and Osr2 and this up-regulation was confirmed in other osteoblast cultures, both from murine and human origin. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Vitamin D status at breast cancer diagnosis: correlation with tumor characteristics, disease outcome, and genetic determinants of vitamin D insufficiency.

We correlated serum 25-hydroxyvitamin D(3) (25OHD) levels with tumor characteristics and clinical disease outcome in breast cancer patients and assessed the impact of genetic determinants of vitamin D insufficiency. We collected serum from 1800 early breast cancer patients at diagnosis, measured 25OHD by radioimmunoassay (RIA), and determined genetic variants in vitamin D-related genes by Sequenom. Multivariable regression models were used to correlate 25OHD levels with tumor characteristics. Cox proportional hazard models were used to assess overall survival (OS), disease-specific survival (DSS), and disease-free interval (DFI). Lower 25OHD serum levels significantly correlated with larger tumor size at diagnosis (P = 0.0063) but not with lymph node invasion, receptor status, or tumor grade. Genetic variants in 25-hydroxylase (CYP2R1) and vitamin D-binding (DBP) protein significantly determined serum 25OHD levels but did not affect the observed association between serum 25OHD and tumor size. High serum 25OHD (>30 ng/mL) at diagnosis significantly correlated with improved OS (P = 0.0101) and DSS (P = 0.0192) and additionally had a modest effect on DFI, which only became apparent after at least 3 years of follow-up. When considering menopausal status, serum 25OHD had a strong impact on breast cancer-specific outcome in postmenopausal patients [hazards ratios for 25OHD >30 ng/mL versus ≤30 ng/mL were 0.15 (P = 0.0097) and 0.43 (P = 0.0172) for DSS and DFI, respectively], whereas no association could be demonstrated in premenopausal patients. In conclusion, high vitamin D levels at early breast cancer diagnosis correlate with lower tumor size and better OS, and improve breast cancer-specific outcome, especially in postmenopausal patients.

1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine preosteoblastic MC3T3-E1 cells by direct regulation of cystathionine ß-synthase.

High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY owing to its negative impact on collagen maturation. HCY is cleared by cystathionine ß-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine preosteoblasts treated with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)(2) D(3) . Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)(2) D(3) treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine, and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)(2) D(3) -treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knockout osteoblasts, in which upregulation of cbs in response to 1,25(OH)(2) D(3) was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D(3) levels] and HCY levels. In conclusion, this study showed that cbs is a primary 1,25(OH)(2) D(3) target gene which renders HCY metabolism responsive to 1,25(OH)(2) D(3).

The impact of 1,25(OH)2D3 and its structural analogs on gene expression in cancer cells--a microarray approach.

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], is an important regulator of bone metabolism, calcium and phosphate homeostasis but also has potent antiproliferative and pro-differentiating effects on a wide variety of cell types. To identify key genes that are (directly) regulated by 1,25(OH)2D3, a large number of microarray studies have been performed on different types of cancer cells (prostate, breast, ovarian, colorectal, squamous cell carcinoma and leukemia). The variety of target genes identified through these studies reflects the pleiotropic action of 1,25(OH)2D3. Common cellular processes targeted by 1,25(OH)2D3 in the different cancer cell lines include cell cycle progression, apoptosis, cellular adhesion, oxidative stress, immune function and steroid metabolism. Upon comparison of the lists of genes regulated by 1,25(OH)2D3 in the different microarray studies, only a small set of individual genes were commonly regulated, among which are included 24-hydroxylase, growth arrest and DNA-damage-inducible protein, cathelicidin antimicrobial peptide and multiple cyclins.

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus.

BACKGROUND: The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions. RESULTS: The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants. CONCLUSION: This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.