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.