Mice deficient in GATA-1 or NF-E2 have a 200 -300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6-fold when cultured with megakaryocytes. A megakaryocyte-osteoblast interaction plays a role in the increased bone formation in these mice.Introduction: GATA-1 and NF-E2 are transcription factors required for the differentiation of megakaryocytes. Mice deficient in these factors have phenotypes characterized by markedly increased numbers of immature megakaryocytes, a concomitant drastic reduction of platelets, and a striking increased bone mass. The similar bone phenotype in both animal models led us to explore the interaction between osteoblasts and megakaryocytes. Materials and Methods: Histomorphometry, CT, and serum and urine biochemistries were used to assess the bone phenotype in these mice. Wildtype and mutant osteoblasts were examined for differences in proliferation, alkaline phosphatase activity, and osteocalcin secretion. In vitro osteoclast numbers and resorption were measured. Because mutant osteoblasts and osteoclasts were similar to control cells, and because of the similar bone phenotype, we explored the interaction between cells of the osteoblast lineage and megakaryocytes. Results: A marked 2-to 3-fold increase in trabecular bone volume and bone formation indices were observed in these mice. A 20-to 150-fold increase in trabecular bone volume was measured for the entire femoral medullary canal. The increased bone mass phenotype in these animals was not caused by osteoclast defects, because osteoclast number and function were not compromised in vitro or in vivo. In contrast, in vivo osteoblast number and bone formation parameters were significantly elevated. When wildtype or mutant osteoblasts were cultured with megakaryocytes from GATA-1-or NF-E2-deficient mice, osteoblast proliferation increased over 3-to 6-fold by a mechanism that required cell-to-cell contact. Conclusions: These observations show an interaction between megakaryocytes and osteoblasts, which results in osteoblast proliferation and increased bone mass, and may represent heretofore unrecognized anabolic pathways in bone.

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IntroductionHematopoietic stem cells (HSCs) are multipotent progenitor cells that give rise to all types of mature blood cells. HSCs reside in a complex cellular microenvironment containing osteoblasts (OBs), osteoclasts, endothelial cells, stromal cells (SCs), mesenchymal progenitor cells, and adipocytes as well as multiple components of the extracellular matrix. Collectively, these cellular elements and the extracellular matrix constitute the hematopoietic niche, which most probably regulates the size of the stem cell pool and controls HSC fate. 1 OBs play a critical role in HSC function and self-renewal. Primitive HSCs that are in association with the endosteal region have high proliferative and repopulating capacities. 2 OBs can deliver proliferative signals to HSCs during mobilization. 3 Human OBs secrete cytokines, such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and leukemia inhibitory factor, thereby supporting hematopoietic progenitor cell (HPC) function in vitro. [4][5][6] Furthermore, OBs secrete angiopoietin, thrombopoietin, and stromal cell-derived factor-1, all of which regulate HSC maintenance. [7][8][9] Physical and molecular interactions between HPCs and OBs supported in vitro hematopoiesis 5 and survival, 10 whereas cotransplantation of OBs with HSCs improved engraftment. 11 However, others questioned whether OBs contribute to the formation of niches where vascular and perivascular cells play a major role in maintaining HSC function. 12 In addition to stem cell-enhancing activity, microenvironment cells in multiple systems can down-regulate stem cell function.Endothelial cells in the perivascular niche reduce the adipogenic potential of adipose stromal cells by up-regulating inhibitors of adipogenesis. 13 In the hematopoietic system, adipocytes inhibit lineage-specific differentiation 14 and engraftment of transplanted cells. 15 These observations suggest that different cells of the hematopoietic niche mediate both positive and negative effects on stem and progenitor cells.Notch signaling is crucial for HSC formation during embryonic development 16 and is critical for HSC maintenance. 17 Notch signaling regulates differentiation and maintenance of HSCs, and Notch1 activation promotes stem cell self-renewal. 18 Calvi 19 and Weber et al 20 demonstrated the role of the endosteal niche in maintaining HSC self-renewal through the activation of Notch receptors on HSCs by Jagged1 expressed by OBs. However, the role of Notch signaling in HSC homeostasis has been questioned 21,22 because impeding key signaling molecules was ineffective in immediately decreasing HSC numbers or suppressing hematopoiesis.At present, we do not know precisely how different cellular elements of the hematopoietic niche collaborate to promote HSC self-renewal and to maintain the stem cell pool. Similarly, the interplay between different cell types of the hematopoietic niche that promotes or impedes self-renewing signaling pathways is also not well understood. Herein, we investigat...

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Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions. This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary phase of Escherichia coli and Bacillus subtilis. The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic bacterial growth equations for space-flight and control growth curves. Space-flight cultures grew to higher stationary-phase concentrations and had shorter lag-phase durations. Also, evidence was found for increased exponential growth rate in space.

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Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether that response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18α-glycyrrhetinic acid (αGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MK to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation.