(Fig. 1B). We analyzed the activity of alkaline phosphatase (ALP), a well known marker of commitment to the osteoblast lineage, in the nephronectin-transfected MC3T3-E1 cells and observed a signif- icantly higher level of ALP expression in the cells over-expressing nephronectin as compared with the vector-transfected cells (Fig. 1C). Typical micrographs are provided to show cell morphology after staining (ALP activity seen as the red colour in the wells, Fig. 1D, upper panel). Using a different phase on the microscope, we could focus on only the cells exhibiting ALP activity due to their red staining (Fig. 1D, lower panel). This allowed quantification of the stained cells. Our results indicated that over-expression of nephronectin promotes osteoblast differentiation. To determine the effect of endogenous nephronectin on MC3T3-E1 cell differentiation, we analyzed the effect of knocking down nephronectin expression on osteoblast differentiation. Cell lines stably transfected with a small interfering RNA (siRNA) plasmid targeting nephronectin (Npnt-siRNA), or with the GFP vector alone, were generated. The GFP- and Npnt-siRNA- expressing cells were cultured under nodule-inducing conditions and then stained for ALP expression to identify differentiating cells. We observed minimal ALP expression in the MC3T3-E1 cells over-expressing the siRNA as compared with the vector control (Fig. 1E). Our results indicated that down-regulation of endogenous nephronectin expression inhibited osteoblast differentiation. The effect of nephronectin 39UTR on osteoblast differentiation We then studied regulation of nephronectin expression by miRNAs, as miRNAs have been reported to play essential roles in cell differentiation. We generated a nephronectin expression construct with a fragment of the nephronectin 39UTR (Fig. 2A, Npnt+39). MC3T3-E1 cells were transfected with the construct Npnt+39 to generate stable cell lines. Conditioned medium was examined for expression and secretion of the ectopic nephronectin by western blot (Fig. 2A). MC3T3-E1 cells transfected with either the Npnt construct, the Npnt+39 construct, or the GFP vector control were cultured for 6 days under nodule-inducing condi- tions, followed by staining for ALP activity to identify differenti- ating osteoblasts. Again, over-expression of nephronectin promot- ed osteoblast differentiation as seen by the significantly higher ALP staining as compared with the control cells (Fig. 2B and C). A much lower rate of osteoblast differentiation was observed in the cells expressing the Npnt+39 construct (Npnt+39-1 and Npnt+39- 2), thereby providing further evidence towards the post-transcrip- tional repression of nephronectin by endogenous miRNAs during osteoblast differentiation. We then investigated the effect of over-expressing the 39UTR of nephronectin over the full course of osteoblast development, including mineralization and nodule formation. The transfected cells were grown in parallel in nodule inducing conditions (regular growth medium supplemented with ascorbic acid, beta-glycero- phosphate, and dexamethasone) over the three phases of bone development (proliferation, differentiation, and mineralization/ nodule formation). ALP expression and nodule formation were analyzed on different days over the full course of osteogenesis. Consistent with the above results, the Npnt-transfected cells showed a significantly higher rate of differentiation as compared with the vector controls (Fig. 3A). The Npnt+39-transfected cells exhibited a lower rate of differentiation than the Npnt-transfected cells during the early stages of osteogenesis until day 13. The rates of differentiation became similar between the Npnt- and Npnt+39- transfected cells by day 17, and finally, during the later stages of miR-378 in Differentiation osteogenesis (after day 27) the Npnt+39-transfected cells displayed much higher levels of ALP activity than the Npnt-transfected cells. Photographs of the whole wells are provided in Supplementary (Fig. S1A). Statistical analysis indicated that all differences were significant (Fig. 3B). Once we observed nodule formation, the cells were stained with silver nitrate for visualization of bone nodules. Multiple nodules were first seen in the nephronectin-transfected cells on day 52 after plating, indicating that the faster and greater rates of differenti- ation in these cells led to faster and more pronounced nodule formation as compared with the controls. The Npnt+39-transfect- ed cells exhibited mineralization with minimal nodule formation on day 52 (Fig. 3C). Interestingly, although the Npnt+39- transfected cells exhibited delayed nodule formation compared with the nephronectin-transfected cells, by day 56, the Npnt+39- transfected cells exhibited fully formed nodules in a significantly higher density than the nephronectin-transfected cells (Fig. 3D, upper panel, Fig. S1B). By day 60, the Npnt+39 plates were almost fully filled with bone nodules (Fig. 3D, lower panel). The effect of 39UTR on nephronectin glycosylation We then investigated whether or not the increase in differen- tiation and nodule formation seen in the Npnt+39-transfected cells was associated with nephronectin production. Npnt- and Npnt+39- transfected cell lines were cultured in parallel under nodule inducing conditions. One set of cells was lysed and the protein was extracted after 48 hours (just before confluence), while the other set was lysed for protein extraction after 10 days. Both sets were analyzed for nephronectin levels by western blot. Forty-eight hours after induction, the level of glycosylated nephronectin was found to be much lower than the level of core protein in the Npnt- transfected cells, while the Npnt+39-transfected cells containing the 39UTR showed much greater levels of glycosylated protein than core protein (Fig. 4A). Ten days after induction, the level of secreted glycosylated protein was significantly greater in the Npnt+39-transfected cells. It has been predicted that a high degree of glycosylation occurs in the region of nephronectin containing the MAM domain [55]. Therefore, to confirm glycosylation of nephronectin, a truncated nephronectin construct was generated containing only the MAM domain. MC3T3-E1 cells were transfected separately with either the construct containing only the MAM domain or the full-length nephronectin. The conditioned medium obtained from the MAM and the Npnt-transfected cells was dialyzed and subjected to treatments with PNGase F, sialidase, O-glycanase, and a mix of sialidase and O-glycanase. A reduction in molecular size was obtained with PNGase F treatment, and treatment with a mix of sialidase and O-glycanase (Fig. 4B). De-glycosylation of nephro- nectin by various enzymes indicated its potential N- and O- glycosylation. The major band shift by co-treatment with sialidase and O-glycanase suggested its glycoform as sialic acid-capped O- glycosylation. Effect of miR-378 on osteoblast differentiation The above results indicate that the 39UTR of nephronectin played a role in modulating nephronectin-mediated osteoblast differentiation. We then searched for potential miRNAs that might regulate nephronectin functioning. Multiple computational ap- proaches were used to analyze the 39UTR of nephronectin mRNA for miRNA binding sites/sequences. The miRNA that was predicted to be of great potential for nephronectin binding was miR-378. MiR-378 base pairs well with the 39UTR of nephro- nectin from nucleotides 1893–1913 and 1903–1923 (Fig. 5A), and was therefore selected for further analyses as a putative regulator PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7535