Recent studies have associated mutations in lamin A/C, a component of the nuclear lamina, with premature aging and severe bone loss. In this study, we hypothesized that reduced expression of lamin A/C has a negative impact on osteoblastogenesis and bone formation in vitro. We inhibited lamin A/C using increasing doses of lamin A/C siRNA in normal human osteoblasts and differentiating mesenchymal stem cells (MSCs). Untreated cells and cells treated with vehicle but without the siRNA-oligo were used as control. The level of effectiveness of siRNA was determined by RT-PCR, Western blot, and immunofluorescence. Nuclear blebbing, a typical finding of lamin A/C inhibition, was quantified using propidium iodine staining, and its effect on cell survival was determined using MTS-formazan. Furthermore, alizarin red and alkaline phosphatase staining were correlated with osteocalcin secretion and levels of expression of osteocalcin, osterix, bone sialoprotein, and Runx2. Finally, the nuclear binding activity of Runx2, an essential transcription factor for osteoblast differentiation, was assessed using ELISA and EMSA. A successful inhibitory effect on the lamin A/C gene at doses of 400-800 nM oligo was obtained without affecting cell survival. Whereas osteoblast function was significantly affected by lamin A/C inhibition, siRNA-treated MSC showed a higher incidence of nuclear changes, lower osteoblast differentiation, and enhanced adipocyte differentiation. Finally, lamin A/C knockdown reduced Runx2 nuclear binding activity without affecting Runx2 expression. In summary, our results indicate that lamin A/C is a new factor needed for osteoblast differentiation that plays an important role in the cellular mechanisms of age-related bone loss.

Based on remarkable success of PTH as an anabolic drug for osteoporosis, case reports of off-label use of teriparatide (1-34 PTH) in patients with complicated fractures and non-unions are emerging. We investigated the mechanisms underlying PTH accelerated fracture repair. Bone marrow cells from 7 days 40cg/kg of teriparatide treated or saline control mice were cultured and Osx and osteoblast phenotypic gene expression assessed by real time RT-PCR in these cells. Fractured animals injected daily with either saline or 40cg/kg of teriparatide for up to 21 days were X-rayed and histological assessment performed, as well as immunohistochemical analyses of the Osx expression in the fracture callus. Osx, Runx2 and osteoblast or chondrocyte phenotypic gene expression was also assessed in fracture calluses. Our data shows that Osx and Runx2 are up-regulated in marrow-derived MSCs isolated from mice systemically treated with teriparatide. Furthermore, these MSCs undergo accelerated osteoblast maturation compared to saline injected controls. Systemic teriparatide treatments also accelerated fracture healing in these mice concomitantly with increased Osx expression in the PTH treated fracture calluses compared to controls. Collectively, these data suggest a mechanism for teriparatide mediated fracture healing possibly via Osx induction in MSCs.

Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.