miR-378 in Differentiation Nephronectin was discovered in the developing mouse kidney as a novel ligand for the integrin a8b1. It is a 70–90 kDa secreted extracellular matrix protein that contains a putative signal peptide at the N-terminus, five epidermal growth factor (EGF)-like repeats (amino acids 57–250), an RGD sequence (amino acids 382–384), and a C-terminal MAM domain (amino acids 417–561). As nephronectin is expressed in a variety of tissues in the developing mouse embryo, we studied the role of nephronectin in bone development. We also examined the regulation of nephronectin expression by miR-378 and demonstrated that miR-378 up- regulated nephronectin expression, and enhanced nephronectin glycosylation and secretion via binding to the 39UTR of nephronectin mRNA. Interaction of miR-378 with nephronectin 39UTR arrested this miRNA and freed another miR-378 target GalNT7, an enzyme essential for nephronectin glycosylation. As a consequence, nephronectin glycosylation was enhanced and osteoblast differentiation was promoted. Results Nephronectin promotes osteoblast differentiation To study the role of nephronectin during osteoblast develop- ment, we generated a nephronectin construct (Npnt). A leading peptide (LP) of chicken link protein was tagged at the N-terminal region, which contains the secretion signal and an epitope recognized by the monoclonal antibody 4B6 (Fig. 1A). The osteoblast cell line MC3T3-E1 was stably transfected with the nephronectin construct or an empty vector. Conditioned medium from each stable cell line was examined for expression and secretion of nephronectin by western blot using the 4B6 antibody Figure 1. Alkaline phosphatase expression in MC3T3-E1 cells over-expressing nephronectin. (A) A full-length nephronectin construct was generated containing the 5 EGF-like repeats, RGD domain, and the MAM domain. A leading peptide (LP) was tagged at the N-terminal region of the construct (Panel A). (B) After transfection, conditioned medium from two nephronectin- and one vector-transfected MC3T3-E1 clones was analyzed for stable expression and secretion of the full-length nephronectin protein by western blot, using the monoclonal antibody 4B6 which recognizes an epitope in the leading peptide. (C) Two nephronectin- and two vector-transfected MC3T3-E1 clones were grown for 10 days and stained for ALP expression. Multiple fields were examined for each construct, and stained cells were counted per field for a quantitative graphical representation. Over-expressing nephronectin (Npnt-1 and Npnt-2) enhanced ALP expression. (D) Typical micrographs of ALP expression in the two clones were taken using two different phases (top and bottom panels) of the microscope for better estimation of ALP activity. (E) Left, MC3T3-E1 cells stably transfected with an siRNA construct against nephronectin were grown in parallel with cells transfected with the empty vector for 10 days and then stained for ALP activity. Multiple fields were examined for each construct, and stained cells were counted per field for quantitative analysis. Inset shows the expression level of the nephronectin protein after transfection with the siRNA against nephronectin. Right, typical micrographs of ALP expression are shown. doi:10.1371/journal.pone.0007535.g001 PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7535