also detected a band at higher mass (,100 kDa) that is unaffected by siRNA expression, suggesting that GalNT7 is not involved in the modification of this band. We reasoned that over-expressing the 39UTR-containing nephronectin mRNA resulted in greater binding capacity of nephronectin 39UTR to miR-378 and lesser concentrations than usual of free miR-378 that would be available for targeting and binding to GalNT7. This would free more GalNT7 mRNA and up-regulate GalNT7 protein levels for enhancing glycosylation of nephronectin. As a consequence, due to more than usual glycosylation of the core protein, secretion of nephronectin product would be promoted. To test this, competition assays were performed. The luciferase reporter vector containing the 39UTR of GalNT7 was co-transfected with Npnt+39 or Npnt+39-mu. Analysis of luciferase activities indicated that luc-Gal-UTR expressed greater activities when co-transfected with Npnt+39 than when co-transfected with Npnt+39-mu (Fig. 9A). As shown in the diagram (Fig. 9B), Npnt 39UTR was able to bind miR-378 and free GalNT7 39UTR. As a result, luc-Gal-UTR activities increased. On the other hand, Npnt 39UTR-mu could no longer bind miR-378, nor up-regulate luc-Gal-UTR activities. To confirm these results, we prepared cell lysate from cells transfected with nephronectin or Npnt+39 and subjected them to western blot analysis, probing them with anti-GalNT7 antibody (Abnova, Taipei). The results indicated that cells transfected with Npnt+39 expressed higher levels of GalNT7 than cells transfected with nephronectin (Fig. 9C). The experiments concluded that the presence of nephronectin 39UTR arrested miR-378 and freed GalNT7 mRNA for GalNT7 protein expression. Discussion The clonal osteoblastic cell line MC3T3-E1 that is derived from newborn mouse calvaria has been used extensively as an in vitro model system for studying bone development. These cells undergo an orderly, time-dependent developmental sequence over three stages that are analogous to the respective stages of in vivo bone development. Nephronectin is an extracellular matrix protein known to be highly expressed in the long bone. This study was initiated by examination of the role of nephronectin in osteoblast differentiation and we discovered that ectopic expression of nephronectin promoted osteoblast differentiation. In the in vitro model system for bone development, ALP activity was used as a marker for osteoblast differentiation. Only the differentiated osteoblasts would stain pink/red for ALP expression. There was a significant difference in ALP expression between MC3T3-E1 cells transfected with the empty vector and those expressing full- length nephronectin. The function of endogenous nephronectin was confirmed by knocking down nephronectin expression with the siRNA approach. We then investigated the mechanisms that could be involved in the regulation of nephronectin-mediated action. MiRNAs are known to bind to the 39UTRs of genes to regulate their expression post-transcriptionally. Since nephronectin bears a long 39UTR, we hypothesized that nephronectin expression is regulated by miRNAs. To test this hypothesis, we engineered a fragment of the nephronectin 39UTR into the nephronectin expression construct. As expected, MC3T3-E1 cells stably transfected with nephronectin bearing the 39UTR fragment (Npnt+39) differenti- ated in lower levels as compared with those transfected with nephronectin alone in the early stages of differentiation. These results suggested that some endogenous miRNAs might have interacted with the 39UTR fragment and repressed nephronectin expression resulting in a lower level of osteoblast differentiation. miR-378 in Differentiation Figure 9. Npnt 39UTR competes with GalNT7 39UTR for miR-378 binding. (A) U343 cells were transiently transfected with luciferase reporter vector harboring the GalNT7 39UTR (lu-Gal-UTR) and different constructs at the amounts indicated on the figure (ng). Luciferase activities were normalized using the control as 100%. The luciferase activities were higher when co-transfected with Npnt+39 as compared with Npnt+39-mu, suggesting that increased supplies of Npnt+39 absorbed some endogenous miRNAs freeing luciferase translation. (B) Diagram showing competition of Npnt+39, but not Npnt+39-mu, with GalNT7 for miR-378 binding. As a result, luc-Gal-UTR was freed from miR-378 binding and thereby having higher luciferase activities. (C) Western blot showing increased expression of GalNT7 in the Npnt+39- transfected cells compared with the Npnt-transfected cells. doi:10.1371/journal.pone.0007535.g009 Nevertheless, Npnt+39-transfected cells still differentiated at higher levels than the mock-transfected cells suggesting incomplete repression of ectopically transfected nephronectin. Unexpectedly, on examining the Npnt+39-transfected cells over the three stages of bone development, we observed that the initial delay in osteoblast differentiation was followed by an equal rate of differentiation, that finally resulted in the Npnt+39-transfected cells exhibiting significantly higher levels of osteoblast differentiation. This pattern PLoS ONE | www.plosone.org 10 October 2009 | Volume 4 | Issue 10 | e7535