Catabolic and anabolic actions of parathyroid hormone on the skeleton.

PTH, an 84-amino acid peptide hormone synthesized by the parathyroid glands, is essential for the maintenance of calcium homeostasis.While in its traditional metabolic role, PTH helps to maintain the serum calcium concentration within narrow, normal limits and participates as a determinant of bone remodeling, more specific actions, described as catabolic and anabolic are also well known. Clinically, the catabolic effect of PTH is best represented by primary hyperparathyroidism (PHPT), while the osteoanabolic effect of PTH is best seen when PTH or its biological amino-terminal fragment [PTH(1-34)] is used as a therapy for osteoporosis. These dual functions of PTH are unmasked under very specific pathological (PHPT) or therapeutic conditions. At the cellular level, PTH favors bone resorption, mostly by affecting the receptor activator of nuclear factor κ-B (RANK) ligand (RANKL)-osteoprotegerin- RANK system, leading to an increase in osteoclast formation and activity. Increased bone formation due to PTH therapy is explained best by its ability to enhance osteoblastogenesis and/or osteoblast survival. The PTH-induced bone formation is mediated, in part, by a decrease in SOST/sclerostin expression in osteocytes. This review focuses on the dual anabolic and catabolic actions of PTH on bone, situations where one is enhanced over the other, and the cellular and molecular mechanisms by which these actions are mediated.

Classical genotropic versus kinase-initiated regulation of gene transcription by the estrogen receptor alpha.

Elucidation of kinase-initiated routes by which the estrogen receptors alpha and beta (ERalpha and ERbeta) control gene transcription, along with evidence of distinct biologic outcomes in response to ligands that can selectively activate nongenotropic signaling of the ERs or the androgen receptor, suggest that the ERs control a range of genes wider than that regulated by their direct association with DNA. To ascertain the extent and significance of nongenotropic ER-mediated transcription, we employed transduced HeLa cells expressing wild-type ERalpha or the ligand binding domain of ERalpha localized to the cell membrane (E-Mem), the OB-6 osteoblastic cell line, MCF-7 breast carcinoma cells and uteri from mice treated with 17beta-estradiol (E(2)), or the nongenotropic signaling activator 4-estren-3alpha,17beta-diol (estren). E(2) and estren induced ERK1/2 and Akt phosphorylation in ERalpha or E-Mem stably transfected HeLa cells; however, the phosphorylation kinetics differed between the two cell lines. In all four models, nongenotropic ER actions regulated a population of genes distinct from those regulated by genotropic ER actions. Specifically, the expression of Wnt2, Frizzled10, Egr-1, and c-Fos was strongly up-regulated in E-Mem-containing HeLa cells treated with E(2) or estren, or in ERalpha-containing HeLa cells treated with estren. Up-regulation of Frizzled10 by estren was reproduced in MCF-7 cells. Egr-1 was up-regulated by both estren and E(2); but complement 3, only by E(2) in the uteri. Estren had no effect on complement 3, cathepsin D, progesterone receptor, bcl-2, and cyclin D1 in MCF-7 cells, whereas E(2) up-regulated all these estrogen response element or activating protein-1-containing genes. These results support an extensive divergence in gene expression depending on the mode of ER activation.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein alpha gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1.

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPalpha genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPalpha genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPalpha gene (xC/EBPalpha). The -1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPalpha or xC/EBPss. Deletion analysis showed that the -321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPalpha promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPalpha promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPalpha expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

FoxO1-dependent induction of acute myeloid leukemia by osteoblasts in mice.

Osteoblasts, the bone forming cells, affect self-renewal and expansion of hematopoietic stem cells (HSCs), as well as homing of healthy hematopoietic cells and tumor cells into the bone marrow. Constitutive activation of ß-catenin in osteoblasts is sufficient to alter the differentiation potential of myeloid and lymphoid progenitors and to initiate the development of acute myeloid leukemia (AML) in mice. We show here that Notch1 is the receptor mediating the leukemogenic properties of osteoblast-activated ß-catenin in HSCs. Moreover, using cell-specific gene inactivation mouse models, we show that FoxO1 expression in osteoblasts is required for and mediates the leukemogenic properties of ß-catenin. At the molecular level, FoxO1 interacts with ß-catenin in osteoblasts to induce expression of the Notch ligand, Jagged-1. Subsequent activation of Notch signaling in long-term repopulating HSC progenitors induces the leukemogenic transformation of HSCs and ultimately leads to the development of AML. These findings identify FoxO1 expressed in osteoblasts as a factor affecting hematopoiesis and provide a molecular mechanism whereby the FoxO1/activated ß-catenin interaction results in AML. These observations support the notion that the bone marrow niche is an instigator of leukemia and raise the prospect that FoxO1 oncogenic properties may occur in other tissues.

Characterisation and developmental regulation of the Xenopus laevis CCAAT-enhancer binding protein beta gene.

We report here the cloning, characterisation and developmental expression profile of the Xenopus laevis CCAAT-enhancer binding protein beta (xC/EBPbeta) gene. The protein synthesised from the xC/EBPbeta gene interacts specifically with a C/EBP-recognition sequence and acts as a transcriptional activator. Several conserved regions are present in the xC/EBPbeta sequence, including the basic region, leucine zipper, activation domains, three in-frame AUG codons, and a consensus site for mitogen activated protein kinase. The corresponding mRNA is present at high levels in the kidney, liver, lung, muscle and adipose tissue, and at low levels in the ovary, brain and heart. Although the xC/EBPbeta mRNA and protein are present throughout embryogenesis, there is a biphasic increase in their expression levels during development. Whole-mount in situ hybridisation shows a restricted spatial expression profile of the xC/EBPbeta gene during early embryogenesis, with transcripts present around the blastopore lip and in the endodermal cells at the mid-gastrula stage, and, the whole dorsal side at the neurula and early tailbud stage. The expression domain becomes almost ubiquitous during later embryonic development, and includes the brain, spinal cord, somites and regions that give rise to the liver and the heart.

Sequence and expression analysis of a novel Xenopus laevis cDNA that encodes a protein similar to bacterial and chloroplast ribosomal protein L24.

We report here the cloning and the characterization of a Xenopus laevis cDNA that encodes a basic protein of 276 amino acids with a central core region, which shows a substantial degree of homology to bacterial and chloroplast ribosomal protein L24, and additional diverged N- and C-terminal polypeptide extensions. The N-terminal extension displays similarities to the mitochondrial targetting sequence, thereby suggesting that the cDNA probably codes for a mitochondrial ribosomal protein. Although the gene was expressed ubiquitously, at fairly constant levels, during embryogenesis, the abundance of the transcripts in the different tissues varies with the mRNA levels in the kidney, adipose tissue, muscle and liver being greater than that present in the brain, heart, ovary and lung.

Sequence and expression analysis of a Xenopus laevis cDNA which encodes a homologue of mammalian 14-3-3 zeta protein.

We report the cloning and characterisation of a cDNA that encodes a novel member of the Xenopus laevis 14-3-3 protein family. Sequence analysis reveals that the cDNA-encoded protein shares 84% identity with the rat, human or sheep 14-3-3zeta isoform, and between 66% and 77% identity with bovine, human or rat beta, bovine gamma, human tau, Drosophila 14-3-3 and a previously isolated Xenopus member. The corresponding mRNA is present in all adult tissues examined with the highest levels in the brain. Although the gene is expressed throughout embryogenesis, higher levels of mRNA accumulate after gastrulation. Whole-mount in situ hybridisation on tailbud stage embryo reveals strong expression of the gene in the head, optic vesicles, spinal cord and branchial arches with weaker expression in the somites. In addition, expression along the notochord is observed at stage 45 (tadpole). This spatial and temporal expression profile along with recent studies implicating the importance of 14-3-3 proteins in the regulation of signal transduction pathways argues for a key role of this isoform in embryonic development.

Circulating osteogenic precursor cells in type 2 diabetes mellitus.

CONTEXT: Type 2 diabetes mellitus (T2D) is associated with an increased risk of fractures and low bone formation. However, the mechanism for the low bone formation is not well understood. Recently, circulating osteogenic precursor (COP) cells, which contribute to bone formation, have been characterized in the peripheral circulation. OBJECTIVE: Our objective was to characterize the number and maturity of COP cells in T2D. PATIENTS, DESIGN, AND SETTING: Eighteen postmenopausal women with T2D and 27 controls participated in this cross-sectional study at a clinical research center. MAIN OUTCOME MEASURES: COP cells were characterized using flow cytometry and antibodies against osteocalcin (OCN) and early stem cell markers. Histomorphometric (n = 9) and molecular (n=14) indices of bone turnover and oxidative stress were also measured. RESULTS: The percentage of OCN(+) cells in peripheral blood mononuclear cells was lower in T2D (0.8 ± 0.2 vs. 1.6 ± 0.4%; P < 0.0001), whereas the percentage of OCN(+) cells coexpressing the early marker CD146 was increased (OCN(+)/CD146(+): 33.3 ± 7 vs. 12.0 ± 4%; P < 0.0001). Reduced histomorphometric indices of bone formation were observed in T2D subjects, including mineralizing surface (2.65 ± 1.9 vs. 7.58 ± 2.4%, P = 0.02), bone formation rate (0.01 ± 0.1 vs. 0.05 ±0.2 µm(3)/um(2) · d, P = 0.02), and osteoblast surface (1.23 ±0.9 vs. 4.60 ± 2.5%, P = 0.03). T2D subjects also had reduced molecular expression of the osteoblast regulator gene Runx2 but increased expression of the oxidative stress markers p66(Shc) and SOD2. CONCLUSIONS: Circulating OCN(+) cells were decreased in T2D, whereas OCN(+)/CD146(+) cells were increased. Histomorphometric indices of bone formation were decreased in T2D, as was molecular expression of osteoblastic activity. Stimulation of bone formation may have beneficial therapeutic skeletal consequences in T2D.

Parathyroid hormone stimulates circulating osteogenic cells in hypoparathyroidism.

CONTEXT: The osteoanabolic properties of PTH may be due to increases in the number and maturity of circulating osteogenic cells. Hypoparathyroidism is a useful clinical model because this hypothesis can be tested by administering PTH. OBJECTIVE: The objective of the study was to characterize circulating osteogenic cells in hypoparathyroid subjects during 12 months of PTH (1-84) administration. DESIGN: Osteogenic cells were characterized using flow cytometry and antibodies against osteocalcin, an osteoblast-specific protein product, and stem cell markers CD34 and CD146. Changes in bone formation from biochemical markers and quadruple-labeled transiliac crest bone biopsies (0 and 3 month time points) were correlated with measurements of circulating osteogenic cells. SETTING: The study was conducted at a clinical research center. PATIENTS: Nineteen control and 19 hypoparathyroid patients were included in the study. INTERVENTION: Intervention included the administration of PTH (1-84). RESULTS: Osteocalcin-positive cells were lower in hypoparathyroid subjects than controls (0.7 ± 0.1 vs. 2.0 ± 0.1%; P < 0.0001), with greater coexpression of the early cell markers CD34 and CD146 among the osteocalcin-positive cells in the hypoparathyroid subjects (11.0 ± 1.0 vs. 5.6 ± 0.7%; P < 0.001). With PTH (1-84) administration, the number of osteogenic cells increased 3-fold (P < 0.0001), whereas the coexpression of the early cell markers CD34 and CD146 decreased. Increases in osteogenic cells correlated with circulating and histomorphometric indices of osteoblast function: N-terminal propeptide of type I procollagen (R(2) = 0.4, P ≤ 0.001), bone-specific alkaline phosphatase (R(2) = 0.3, P < 0.001), osteocalcin (R(2) = 0.4, P < 0.001), mineralized perimeter (R(2) = 0.5, P < 0.001), mineral apposition rate (R(2) = 0.4, P = 0.003), and bone formation rate (R(2) = 0.5, P < 0.001). CONCLUSIONS: It is likely that PTH stimulates bone formation by stimulating osteoblast development and maturation. Correlations between circulating osteogenic cells and histomorphometric indices of bone formation establish that osteoblast activity is being identified by this methodology.

Molecular characterization of the Xenopus CCAAT-enhancer binding protein beta gene promoter.

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family play key roles in the regulation of genes implicated in the control of growth, differentiation, metabolism, and inflammation. The recent limited studies on the promoter regions of C/EBP genes, particularly C/EBPalpha, have indicated the potential existence of species-specific regulatory mechanisms. It is therefore essential that the promoter regions of different C/EBP genes from a wide range of species are investigated in detail. As an important step toward this goal, we report here the characterization of the Xenopus laevis C/EBPbeta gene promoter. Sequence analysis showed that the 1.6-kb promoter region contained putative binding sites for several transcription factors that have previously been implicated in the regulation of the C/EBPs, including C/EBP, CREB, Myb, STAT, and USF. The -288/+91 promoter region was capable of directing high levels of expression in the hepatoma Hep3B cell line. In addition, this minimal promoter could be autoregulated by both C/EBPalpha and C/EBPbeta and activated by lipopolysaccharide, interleukin-6 and CREB. These results therefore demonstrate that several aspects of C/EBPbeta regulation in mammals have been highly conserved in amphibians. However, a comparison of C/EBPbeta gene promoters characterized to date does indicate the existence of species-specific differences in autoregulation.

Tumor necrosis factor and the p55TNF receptor are required for optimal development of the marginal sinus and for migration of follicular dendritic cell precursors into splenic follicles.

The development and function of secondary lymphoid tissue require signaling by tumor necrosis factor and lymphotoxins. Mice deficient in LTbetaR show defective organogenesis of lymph nodes and Peyer's patches and a severely disturbed splenic architecture. In contrast, TNF or p55TNF-R deficiency does not affect the organogenesis of peripheral lymphoid organs but interferes with the formation of B cell follicles and the appearance of FDC networks and germinal centers in all secondary lymphoid organs. Based on these differences, we have previously hypothesized that the role of TNF in lymphoid structure is distinct from that of LT and restricted in regulating cellular interactions that allow the differentiation and/or correct positioning of FDCs. In the present study we show that, in addition to the defects in follicular structure, TNF or p55TNF-R knockout mice exhibit defects in the formation of the macrophage populations and of the sinus lining cells of the splenic marginal zone. Interestingly, a large number of dendritic-shaped cells stained with FDC-specific markers and able to trap immune complexes are retained within the defective marginal zone of TNF and p55TNF-R knockout spleens. We conclude that the primary defect in the lymphoid phenotype of TNF or p55TNF-R knockout mice is the failure of FDC precursors to migrate through the disorganized marginal sinus and to home properly into the splenic follicular areas where they would promote the formation of B cell follicles and germinal centers.

Sex steroids and bone.

The adult skeleton is periodically remodeled by temporary anatomic structures that comprise juxtaposed osteoclast and osteoblast teams and replace old bone with new. Estrogens and androgens slow the rate of bone remodeling and protect against bone loss. Conversely, loss of estrogen leads to increased rate of remodeling and tilts the balance between bone resorption and formation in favor of the former. Studies from our group during the last 10 years have elucidated that estrogens and androgens decrease the number of remodeling cycles by attenuating the birth rate of osteoclasts and osteoblasts from their respective progenitors. These effects result, in part, from the transcriptional regulation of genes responsible for osteoclastogenesis and mesenchymal cell replication and/or differentiation and are exerted through interactions of the ligand-activated receptors with other transcription factors. However, increased remodeling alone cannot explain why loss of sex steroids tilts the balance of resorption and formation in favor of the former. Estrogens and androgens also exert effects on the lifespan of mature bone cells: pro-apoptotic effects on osteoclasts but anti-apoptotic effects on osteoblasts and osteocytes. These latter effects stem from a heretofore unexpected function of the classical "nuclear" sex steroid receptors outside the nucleus and result from activation of a Src/Shc/extracellular signal-regulated kinase signal transduction pathway probably within preassembled scaffolds called caveolae. Strikingly, estrogen receptor (ER) alpha or beta or the androgen receptor can transmit anti-apoptotic signals with similar efficiency, irrespective of whether the ligand is an estrogen or an androgen. More importantly, these nongenotropic, sex-nonspecific actions are mediated by the ligand-binding domain of the receptor and can be functionally dissociated from transcriptional activity with synthetic ligands. Taken together, these lines of evidence strongly suggest that, in sex steroid deficiency, loss of transcriptional effects may be responsible for the increased osteoclastogenesis and osteoblastogenesis and thereby the increased rate of bone remodeling. Loss of nongenotropic anti-apoptotic effects on mature osteoblasts and osteocytes, in combination with an opposite effect on the lifespan of mature osteoclasts, may be responsible for the imbalance between formation and resorption and the progressive loss of bone mass and strength. Elucidation of the dual function of sex steroid receptors has important pathophysiologic and pharmacologic implications. Specifically, synthetic ligands of the ER that can evoke the nongenotropic but not the genotropic signal may be bone anabolic agents, as opposed to natural estrogens or selective estrogen receptor modulators that are antiresorptive agents. The same ligands may also circumvent the side effects associated with conventional hormone replacement therapy.

Reversal of bone loss in mice by nongenotropic signaling of sex steroids.

We show that sex steroids protect the adult murine skeleton through a mechanism that is distinct from that used to preserve the mass and function of reproductive organs. The classical genotropic actions of sex steroid receptors are dispensable for their bone protective effects, but essential for their effects on reproductive tissues. A synthetic ligand (4-estren-3alpha,17beta-diol) that reproduces the nongenotropic effects of sex steroids, without affecting classical transcription, increases bone mass and strength in ovariectomized females above the level of the estrogen-replete state and is at least as effective as dihydrotestosterone in orchidectomized males, without affecting reproductive organs. Such ligands merit investigation as potential therapeutic alternatives to hormone replacement for osteoporosis in both women and men [corrected].

Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity.

The relationship of the classical receptors and their transcriptional activity to nongenotropic effects of steroid hormones is unknown. We demonstrate herein a novel paradigm of sex steroid action on osteoblasts, osteocytes, embryonic fibroblasts, and HeLa cells involving activation of a Src/Shc/ERK signaling pathway and attenuating apoptosis. This action is mediated by the ligand binding domain and eliminated by nuclear targeting of the receptor protein; ERalpha, ERbeta, or AR can transmit it with similar efficiency irrespective of whether the ligand is an estrogen or an androgen. This antiapoptotic action can be dissociated from the transcriptional activity of the receptor with synthetic ligands, providing proof of principle for the development of function-specific-as opposed to tissue-selective-and gender-neutral pharmacotherapeutics.