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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We identified cellular targets of canonical Wnt signaling within the skeleton, which included chondrocytes, osteoblasts, and osteocytes in growing bone, but only osteocytes and chondrocytes in the mature skeleton. Mechanical deformation induced Wnt signaling in osteoblasts in vitro.Introduction: Genetic evidence in mice and humans has implicated the canonical Wnt signaling pathway in the control of skeletal development and bone mass. However, little is known of the details of Wnt signaling in the skeleton in vivo. We used Wnt indicator TOPGAL mice to identify which cells activated this pathway during bone development and in the mature skeleton. Materials and Methods:We examined canonical Wnt signaling during embryonic and neonatal bone development in TOPGAL mice. The TOPGAL transgene consists of a ␤-galactosidase gene driven by a T cell factor (TCF)␤-catenin responsive promoter so that canonical Wnt activity can be detected by X-gal staining. Expression of Wnt signaling components was examined in primary calvarial cell cultures by RT-PCR. The effect of mechanical deformation on Wnt signaling was examined in primary calvarial cells grown on collagen I and stretched using Flexercell Tension Plus System FX-4000T. Immunohistochemistry was used to examine the localization of ␤-catenin in cartilage, bone, and cultured calvarial cells exposed to physical deformation. Results and Conclusions: Canonical Wnt signaling was active in several cell types in the fetal and neonatal skeleton, including chondrocytes, osteoblasts, and osteocytes. With age, activation of Wnt signaling became less prominent but persisted in chondrocytes and osteocytes. Although osteoblasts in culture expressed many different individual Wnt's and Wnt receptors, the TOPGAL transgene was not active in these cells at baseline. However, Wnt signaling was activated in these cells by physical deformation. Together with the activation of canonical Wnt signaling in osteocytes seen in vivo, these data suggest that Wnt signaling may be involved in the coupling of mechanical force to anabolic activity in the skeleton.

TP. Nox4 mediates TGF-␤1-induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells.

Cytokines help mediate the acute and chronic inflammation and associated destruction of connective tissue in arthritic temporomandibular joints (TMJ). The proinflammatory cytokines TNFα, IL-1β, IL-6, IL-8, and IFN-γ are associated with inflammation in synovial joints and connective tissue destruction. Therefore, the increased levels of these cytokines in the synovial fluid of temporomandibular disorder (TMD) patients would be expected. Conversely, IL-1ra and IL-10 acts as inhibitors to these proinflammatory cytokines. Thus, in TMD patients, low levels of IL-1ra and IL-10 might be expected. A review of studies from multiple investigators confirms that proinflammatory cytokine levels increase in TMD patients, IL-1ra levels are also increased, and IL-10 levels remain unchanged. Because IL-10 can inhibit TNFα, IL-1, IL-6, and IL-8, the lack of IL-10 in the TMJ in the face of the other studies showing increases in TNFα, IL-1β, IL-6, and IL-8 could partially explain the exacerbation of the associated osteoarthritis. In addition, although IL-1ra levels are elevated in most of the TMD patients, the increases do not appear to be sufficient to inhibit the inflammation and connective tissue degradation associated with IL-1β. Thus, it appears that treatment of TMD requires a delicate balance between proinflammatory cytokines and cytokine inhibitors such as IL-1ra and IL-10.