In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKC␦) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKC␦ translocates to the nucleus, PKC␦ and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKC␦ and Runx2 function. INTRODUCTIONBone formation and remodeling is a tightly organized and dynamic process that requires the coordinated action of osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. It is hypothesized that osteoblasts and osteocytes coordinate their activities, at least in part, through direct cell-to-cell communication through gap junctions. Gap junctions are composed of connexins, a family of transmembrane proteins that constitute the intercellular gap junction channels (Beyer et al., 1990). Six connexins assemble to make up the gap junction hemichannel on the plasma membrane of one cell, which docks with a hemichannel on an adjacent cell to form an aqueous gap junction channel. The resultant gap junctions provide direct conduits for the passage of ions and other low molecular weight molecules, including second messengers, among cells.The gap junction protein connexin43 (Cx43) is abundantly expressed in both osteoblasts and osteocytes, where it has been hypothesized to transmit hormonal signals, mechanical load, and growth factor cues among cells in order to coordinate the synthesis of new bone (reviewed in Stains and Civitelli, 2005a;Jiang et al., 2007). Genetic ablation of gja1, the gene encoding Cx43, in mice leads to a severe delay in the ossification of both intramembranous and endochondral derived skeletal elements during embryonic development (Lecanda et al., 2000). The bones of these animals are remarkably brittle, and the osteoblasts isolated from the Cx43 null animals are dysfunctional, with reduced osteogenic and mineralizing capacity (Lecanda et al., 2000). These Cx43-deficient mice die at birth because of a defect in cardiac f...

Astronaut intestinal health may be impacted by microgravity, radiation, and diet. The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function. Three independent experiments were conducted to achieve these goals: 1) fractionated low LET γ radiation (137Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET γ radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET 38Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET38Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity may affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFβ gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health.

Objective Accumulating experimental evidence implicates β-catenin signaling and enzyme transglutaminase 2 (TG2) in the progression of vascular calcification, and our previous studies have shown that TG2 can activate β-catenin signaling in vascular smooth muscle cells (VSMCs). Here we investigated the role of the TG2/β-catenin signaling axis in vascular calcification induced by warfarin. Methods and Results Warfarin-induced calcification in rat A10 VSMCs is associated with the activation of β-catenin signaling and is independent from oxidative stress. The canonical β-catenin inhibitor Dkk1, but not the Wnt antagonist Wif-1,prevents warfarin-induced activation of β-catenin, calcification, and osteogenic trans-differentiation in VSMCs. TG2 expression and activity are increased in warfarin-treated cells, in contrast to canonical Wnt ligands. Vascular cells with genetically or pharmacologically reduced TG2 activity fail to activate β-catenin in response to warfarin. Moreover, warfarin-induced calcification is significantly reduced on the background of attenuated TG2 both in vitro and in vivo. Conclusions TG2 is a critical mediator of warfarin-induced vascular calcification that acts through the activation of β-catenin signaling in VSMCs. Inhibition of canonical β-catenin pathway or TG2 activity prevents warfarin-regulated calcification, identifying the TG2/β-catenin axis as a novel therapeutic target in vascular calcification.

IGF-I plays an important anabolic role in stimulating bone formation and maintaining bone mass. We show that the pro-proliferative, anti-apoptotic, and functional responses to IGF-I in bone and BMSCs decrease with aging. These changes are associated with impaired receptor activation and signal transduction through the MAPK and PI3K pathways.Introduction: IGF-I is a potent anabolic agent having effects across diverse tissues and cell types. With aging, bone becomes resistant to the anabolic actions of IGF-I. To examine the effects of aging on bone responsiveness to IGF-I, we measured the pro-proliferative, anti-apoptotic, and functional responses of bone and bone marrow stromal cells (BMSCs) to IGF-I and evaluated IGF-I signal transduction in young, adult, and old mice. Materials and Methods: Male C57BL/6 mice 6 wk (young), 6 mo (adult), and 24 mo (old) were treated with IGF-I for 2 wk using osmotic minipumps, and osteoblast proliferation (BrdU labeling) in vivo, and osteoprogenitor number (BMSC culture and calcium nodule formation) were measured. Proliferation, apoptosis, and expression of key osteoblast factors (alkaline phosphatase, collagen, osteocalcin, RANKL, osteoprotegerin (OPG), macrophage-colony stimulating factor [M-CSF]) and IGF-I signaling elements and their activation in IGF-I-treated cells were studied using QRT-PCR and Western blot analysis. Data were analyzed using ANOVA. Results: Aging decreased the basal and IGF-I-stimulated number of BrdU-labeled osteoblasts and reduced the ability of IGF-I to stimulate osteoprogenitor formation (calcium nodule number) by 50%. The proproliferative and anti-apoptotic actions of IGF-I were blunted in cells from old animals. These changes were accompanied by age-related alterations in the ability of IGF-I to regulate alkaline phosphatase, collagen, osteocalcin, RANKL, OPG, and M-CSF expression. IGF-I binding was normal, but IGF-I receptor mRNA and protein expression was increased in aged animals by 2-and 10-fold, respectively. The age-related changes in proliferation, apoptosis, and function were accompanied by loss of IGF-I-induced signaling at the receptor level and at key regulatory sites along the MAPK (ERK1/2) and PI3K (AKT) pathways. Conclusions: Our data show that aging is accompanied by loss of bone and BMSC/osteoblast responsiveness to IGF-I and that these changes are associated with resistance to IGF-I signaling that involve receptor activation and downstream signaling events.