Bone density achieved in early adulthood is the major determinant of risk of osteoporotic fracture. Up to 60% of women suffer osteoporotic fractures as a result of low bone density, which is under strong genetic control acting through effects on bone turnover. Here we show that common allelic variants in the gene encoding the vitamin D receptor can be used to predict differences in bone density, accounting for up to 75% of the total genetic effect on bone density in healthy individuals. The genotype associated with lower bone density was overrepresented in postmenopausal women with bone densities more than 2 standard deviations below values in young normal women. The molecular mechanisms by which bone density is regulated by the vitamin D receptor gene are not certain, although allelic differences in the 3' untranslated region may alter messenger RNA levels. These findings could open new avenues to the development and targeting of prophylactic interventions. It follows that other pathophysiological processes considered to be subject to complex multifactorial genetic regulation may also be modulated by a single gene with pleiotropic transcriptional actions.

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Osteocalcin, the most abundant noncollagenous protein in bone, is a marker of bone turnover in normal and disease states. Its synthesis is induced by calcitriol, the active hormonal form of vitamin D, through the vitamin D receptor and a specific vitamin D-responsive element in the osteocalcin gene promoter. Serum concentrations of osteocalcin are under strong genetic influence. To ascertain whether variability in circulating osteocalcin levels may reflect allelic variation in the vitamin D receptor gene, we have analyzed the relationship between frequent restriction fragment length polymorphisms (RFLPs, detected by endonucleases Bsm I, EcoRV, and Apa I) that define human vitamin D receptor alleles and serum osteocalcin in a cohort of normal subjects. In 91 Caucasian subjects, RFLPs in the vitamin D receptor gene predicted circulating osteocalcin levels (P < 0.0001) independent ofage or menopause effects. Since the osteocalcin gene and the vitamin D receptor gene are encoded on different chromosomes, the interaction between these two genes occurs in trans. Thus, common alleles of this trans-acting factor, the vitamin D receptor, are functionally different and contribute to "normal" physiological variability in osteocalcin levels. Preliminary analysis in monozygotic and dizygotic twin pairs indicates that the greater diversity in lumbar spine density between the dizygotic pairs can be explained by divergence in vitamin D receptor alleles. Variations in this receptor and other transacting factor genes may confound physiological studies of regulation of target genes and will need to be considered in future human and animal studies. This approach to genetic analysis provides a paradigm for the study of functional variation in trans-acting factors and the role such variation may play in the generation and evolution of physiological diversity.Vitamin D functions as a potent regulator of bone and calcium homeostasis as well as of cellular differentiation and replication in many target tissues. It acts as its dihydroxylated metabolite (1,25-dihydroxyvitamin D, or (6), and in the androgen receptor, resulting in androgen insensitivity (7), have been reported, and in the estrogen receptor an infrequent natural polymorphism has been correlated with a high rate of spontaneous abortion (8). However, despite a wealth of molecular information, little is known of the potential contribution of natural allelic variation in receptor genes to diversity of response to steroidal hormones in normal physiology and in disease states.Although calcitriol has widespread actions on both cellular differentiation and bone and calcium homeostasis, the osteocalcin gene is the only gene for which a definitive direct interaction between the vitamin D receptor and target gene promoter has been shown. Osteocalcin is an abundant bone protein of uncertain function and is made exclusively by the osteoblast. It binds strongly to mineralized bone but a small proportion of the newly synthesized osteocalcin escapes into the circulation, where it...

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The peribacteroid membrane (pbm) of root nodules is derived from the plant cell plasma membrane but contains in addition several nodule-specific host proteins (nodulins). Antibodies raised against purified pbm of soybean were used to immunoprecipitate polysomes to isolate an RNA fraction that served as a template for the synthesis of a cDNA probe for screening a nodule-specific cDNA library. Clone p1B1 was found to encode a 26.5 kDa polypeptide (nodulin-26) which is immunoprecipitable specifically with the anti-pbm serum. Nodulin-26 has features of a transmembrane protein and its structure differs from that of nodulin-24 which appears to be a surface protein of pbm. The expression of these two pbm nodulins was examined in nodules induced by Bradyrhizobium japonicum Tn5 mutants that arrest nodule development at different stages of pbm biosynthesis. Nodules that do not show release of bacteria from the infection thread express nodulin-24 at a very low level. In contrast, the expression of nodulin-26 occurs fully in nodules that form infection threads only and is not affected by the release of bacteria from the threads.

MCP-1 (monocyte chemotactic protein-1) is a CC

The microarchitecture of bone is regulated by complex interactions between the bone-forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone-forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.

RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology.Introduction: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis. Materials and Methods: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest. Results: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts. Conclusions: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.