IL-17 derived from juxta-articular bone and synovium contributes to joint degradation in rheumatoid arthritis

The origin and role of IL-17, a T-cell derived cytokine, in cartilage and bone destruction during rheumatoid arthritis (RA) remain to be clarified. In human ex vivo models, addition of IL-17 enhanced IL-6 production and collagen destruction, and inhibited collagen synthesis by RA synovium explants. On mouse cartilage, IL-17 enhanced cartilage proteoglycan loss and inhibited its synthesis. On human RA bone explants, IL-17 also increased bone resorption and decreased formation. Addition of IL-1 in these conditions increased the effect of IL-17. Blocking of bone-derived endogenous IL-17 with specific inhibitors resulted in a protective inhibition of bone destruction. Conversely, intra-articular administration of IL-17 into a normal mouse joint induced cartilage degradation. In conclusion, the contribution of IL-17 derived from synovium and bone marrow T cells to joint destruction suggests the control of IL-17 for the treatment of RA.

Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta

Leptin is a 167-aa protein that is secreted from adipose tissue and is important in the regulation of energy balance. It also functions in hematopoiesis and reproduction. To assess whether leptin is involved in fetal growth and development we have examined the distribution of mRNAs encoding leptin and the leptin receptor (which has at least six splice variants) in the 14.5-day postcoitus mouse fetus and in the placenta using reverse transcription–PCR and in situ hybridization. High levels of gene expression for leptin, the leptin receptor, and the long splice variant of the leptin receptor with an intracellular signaling domain were observed in the placenta, fetal cartilage/bone, and hair follicles. Receptor expression also was detected in the lung, as well as the leptomeninges and choroid plexus of the fetal brain. Western blotting and immunocytochemistry, using specific antibodies, demonstrated the presence of leptin and leptin receptor protein in these tissues. These results suggest that leptin may play a role in the growth and development of the fetus, both through placental and fetal expression of the leptin and leptin receptor genes. In the fetus, leptin may be multifunctional and have both paracrine and endocrine effects.

Calcium isotope fractionation between soft and mineralized tissues as a monitor of calcium use in vertebrates

Calcium from bone and shell is isotopically lighter than calcium of soft tissue from the same organism and isotopically lighter than source (dietary) calcium. When measured as the 44Ca/40Ca isotopic ratio, the total range of variation observed is 5.5‰, and as much as 4‰ variation is found in a single organism. The observed intraorganismal calcium isotopic variations and the isotopic differences between tissues and diet indicate that isotopic fractionation occurs mainly as a result of mineralization. Soft tissue calcium becomes heavier or lighter than source calcium during periods when there is net gain or loss of mineral mass, respectively. These results suggest that variations of natural calcium isotope ratios in tissues may be useful for assessing the calcium and mineral balance of organisms without introducing isotopic tracers.

Identification of Osteopontin as a Novel Ligand for the Integrin α8β1 and Potential Roles for This Integrin–Ligand Interaction in Kidney Morphogenesis

Epithelio–mesenchymal interactions during kidney organogenesis are disrupted in integrin α8β1-deficient mice. However, the known ligands for integrin α8β1—fibronectin, vitronectin, and tenascin-C—are not appropriately localized to mediate all α8β1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for α8β1. We have coexpressed the extracellular domains of the mouse α8 and β1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the α8β1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with α8β1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In “far Western” blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to α8β1-AP. Cell adhesion assays using K562 cells expressing α8β1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin α8β1 may help regulate kidney development and other morphogenetic processes.

Generation and reproductive phenotypes of mice lacking estrogen receptor β

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERα and ERβ. We previously generated and studied knockout mice lacking estrogen receptor α and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor β (ERβ −/−) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERβ −/− mice lack normal ERβ RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERβ is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERβ in bone and cardiovascular homeostasis.

Paleoclimate and Amerindians: Evidence from stable isotopes and atmospheric circulation

Two Amerindian demographic shifts are attributed to climate change in the northwest plains of North America: at ≈11,000 calendar years before present (yr BP), Amerindian culture apparently split into foothills–mountains vs. plains biomes; and from 8,000–5,000 yr BP, scarce archaeological sites on the open plains suggest emigration during xeric “Altithermal” conditions. We reconstructed paleoclimates from stable isotopes in prehistoric bison bone and relations between weather and fractions of C4 plants in forage. Further, we developed a climate-change model that synthesized stable isotope, existing qualitative evidence (e.g., palynological, erosional), and global climate mechanisms affecting this midlatitude region. Our isotope data indicate significant warming from ≈12,400 to 11,900 yr BP, supporting climate-driven cultural separation. However, isotope evidence of apparently wet, warm conditions at 7,300 yr BP refutes emigration to avoid xeric conditions. Scarcity of archaeological sites is best explained by rapid climate fluctuations after catastrophic draining of the Laurentide Lakes, which disrupted North Atlantic Deep Water production and subsequently altered monsoonal inputs to the open plains.

Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.

Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways

Extraembryonic ectoderm-derived factors instruct the pluripotent epiblast cells to develop toward a restricted primordial germ cell (PGC) fate during murine gastrulation. Genes encoding Bmp4 of the Dpp class and Bmp8b of the 60A class are expressed in the extraembryonic ectoderm and targeted mutation of either results in severe defects in PGC formation. It has been shown that heterodimers of DPP and 60A classes of bone morphogenetic proteins (BMPs) are more potent than each homodimers in bone and mesoderm induction in vitro, suggesting that BMP4 and BMP8B may form heterodimers to induce PGCs. To investigate how BMP4 and BMP8B interact and signal for PGC induction, we cocultured epiblasts of embryonic day 6.0–6.25 embryos with BMP4 and BMP8B proteins produced by COS cells. Our data show that BMP4 or BMP8B homodimers alone cannot induce PGCs whereas they can in combination, providing evidence that two BMP pathways are simultaneously required for the generation of a given cell type in mammals and also providing a prototype method for PGC induction in vitro. Furthermore, the PGC defects of Bmp8b mutants can be rescued by BMP8B homodimers whereas BMP4 homodimers cannot mitigate the PGC defects of Bmp4 null mutants, suggesting that BMP4 proteins are also required for epiblast cells to gain germ-line competency before the synergistic action of BMP4 and BMP8B.