Deletion of RBPJK in Mesenchymal Stem Cells Enhances Osteogenic Activity by Up-Regulation of BMP Signaling.

Recently we have demonstrated the importance of RBPjk-dependent Notch signaling in the regulation of mesenchymal stem cell (MSC) differentiation during skeletogenesis both in vivo and in vitro. Here we further performed RBPJK loss-of-function experiments to demonstrate for the first time that RBPJK deficient MSC shows enhanced differentiation and osteogenesis acts via up-regulation of the BMP signaling. In the present study, we first compared the spontaneous and osteogenic differentiation in normal and recombination signal binding protein for immunoglobulin kappa J region (RBPJK) deficient human bone marrow-derived mesenchymal stem cells (MSCs). It was found that RBPJK highly expressed in fresh isolated MSCs and its expression was progressing down-regulated during spontaneous differentiation and even greater in osteogenic media inducted differentiation. Deletion of RBPJK in MSCs not only enhances cell spontaneous differentiation, but also significantly accelerates condition media inducted osteogenic differentiation by showing enhanced alkaline phosphatase (ALP) activity, Alizarin red staining, gene expression of Runx2, Osteopontin (OPN), Type I collagen (COL1a1) in culture. Additionally, BMP signaling responsive reporter activity and phosphor-smad1/5/8 expression were also significantly increased upon removal of RBPJK in MSCs. These data proved that inhibition of Notch signaling in MSCs promotes cell osteogenic differentiation by up-regulation of BMP signaling, and RBPJK deficient MSC maybe a better cell population for cell-based bone tissue engineering

Human MSC isolation and characterization.

<p>Newly isolated human Mesenchymal stem cells (MSCs) were cultured in stem cell growth media for up to 80% confluence before being harvested for Flow Cytometry. (A) Representative Flow Cytometry histograms showing CD105 expression in 6 different isolations of MSCs. (B) Representative Flow Cytometry histograms showing CD73 expression in 6 different isolations of MSCs. (C) Representative Flow Cytometry histograms showing CD90 expression in 6 different isolations of MSCs. (D) Representative Flow Cytometry histograms showing CD45 and CD34 in 6 different isolations of MSCs. (E, F) Representative Flow Cytometry histograms showing isotype controls, FITC versus APC and PE versus APC, in 6 different isolations of MSCs. (G) Quantification of the CD105, CD90, CD73 subpopulations in total MSCs.</p

Enhanced BMP signaling in RBPJK deficient MSCs.

<p>(A) Real time RT-PCR analysis reveals that expression of RBPJK was significantly inhibited by 90% in shRBPJK lentivirus infected MSCs (shRBPJK) at day 2 when compared to shRNA-lentivirus control MSCs (Co). (B) Luciferase assays showed a significant increase of BMP responsive reporter activity in shRBPJK MSCs and this transcriptional up-regulation is further enhanced by BMP-2 treatment. Data are means ± s.d. of three independent experiments performed in duplicate and all the results were normalized to internal control (*, p < 0.05 compared with control MSCs (Co) without BMP-2 treatment). (C) Western Blot shows phosphor- smad1/5/8 (P-smad1/5/8) protein levels were significantly enhanced by deletion of RBPJK in MSC culture at day 2. β-actin was used as a loading control. (D) Quantification of P-smad1/5/8 protein level in Western blots was determined by measuring band intensity with ImageJ. (E) Western Blot shows deletion of RBPJK did not cause a change in phosphor-ERK1/2 (P-ERK1/2), total-ERK1/2 (T-ERK1/2), phosphor-p38 (P-p38) and total-p38 (T-p38) protein levels in MSC culture at day 2. (F) Densitometry quantification of activated P-ERK1/2 and P-p38, expressed as a ratio to total ERK and total p38 by P/T ratio. Data are means ± s.d. of three independent experiments. (*, p < 0.05 compared with shRNA-lentivirus control MSCs).</p

Inhibition of Notch signaling by deletion of RBPJK enhances inducted MSCs osteogenic differentiation.

<p>(A) An increase in osteogenic nodule formation was observed in shRBPJK lentivirus infected MSCs at both day 3 and day 14, with maximal staining of Alizarin red at day 14. Scale bars, 100 μm. (B) Quantification of Alizarin Red Staining indicates a significant increase (p < 0.05) in mineral content between lentivirus control (Co) and shRBPJK lentivirus infected MSCs. (C, D, E, F) Runx2, Osterix (OSX), Osteopontin (OPN), and type I collagen (COL1a1) gene expression were significantly increased in RBPJK deficient MSCs at day 14. Data are means ± s.d. of three independent experiments performed in duplicate and the shRNA-lentivirus control (Co) gene expression level was set at 1. (*, p < 0.05 compared with control at day 3)</p

Deletion of RBPJK enhances cell spontaneous differentiation in regular MSC culture.

<p>Mesenchymal stem cells were infected with Control shRNA(Co) or RBPJK shRNA (shRBPJK) lentivirus before being harvested for alkaline phosphatase (ALP) staining, western blot, RT-PCR analysis and BrdU ELISA. (A) Western Blot showed a significant decrease of RBPJK protein expression at day 3 and 14 following shRBPJK lentiviral infection. (B) Fold change in protein level of RBPJK in Western blots was determined by measuring band intensity with ImageJ. (C) Deletion of RBPJK resulted in increased ALP staining at days 3 and 14, compared to controls (Co). Scale bars, 100μm. (D) ALP gene expression was increased at days 3 and 14, with maximal increase at day 14 in RT-PCR analysis. (E) BrdU ELISA to monitor both MSCs proliferation at days 3 and 14 after lentiviral infection. Data are means ± s.d. of three independent experiments performed in duplicate and the control gene expression level at day 3 was set at 1. (*, <i>p</i> < 0.05 compared with control at same time point).</p