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First published online 10 July 2007
doi: 10.1242/jcs.005090

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PIASx is a key regulator of osterix transcriptional activity and matrix mineralization in osteoblasts

¹ Division of Cell Biology and Molecular Pharmacology, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata-city, Niigata 951-8514, Japan
² Division of Orthopedic Surgery, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata-city, Niigata 951-8514, Japan
³ Department of Molecular Pharmacology and 21st Century Center of Excellence (COE) Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Medical Research Institute, Tokyo Medical and Dental University, Kanda-Surugadai 2-3-10, Chiyoda-ku, Tokyo 101-0062, Japan.

View larger version (50K): [in this window] [in a new window]   Fig. 1. Expression of PIASx in osteoblastic cells. PIASx mRNA level in calvarial sutures (A) cultured for 6 hours in the absence (0 g) and presence (0.2 g) of TS, and in MC3T3-E1 cells (B) grown under conditions inducing differentiation toward a mature osteoblastic phenotype for 24 hours or 14 days in the absence (control) and presence (Flex) of cyclical strain by means of the FlexerCell system. (C) Expression patterns of genes encoding osteoblast differentiation markers and PIAS family proteins during osteogenic differentiation of MC3T3-E1 cells (same conditions as in B) without mechanical stimuli. Gene expression was determined by semi-quantitative RT-PCR analysis. The PCR products were analyzed by electrophoretic separation followed by Southern hybridization. These experiments were performed independently in triplicate, and representative results are shown. (D) Expression pattern of the PIASx gene during osteogenic differentiation of MC3T3-E1 cells determined by quantitative real-time RT-PCR analysis.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Knockdown of endogenous PIASx suppressed differentiation and matrix mineralization of MC3T3-E1 cells. (A) Effect of PIASx siRNA on the mRNA levels of osteoblast differentiation markers. Three days after confluence, osteogenic differentiation of MC3T3-E1 cells was initiated by changing the medium (day 0). At day 1 (24 hours later) cells were transfected with siRNA and RNA samples were collected at the indicated time periods. Expression was determined by semi-quantitative RT-PCR analysis. (B) Effect of PIASx siRNA on protein level. MC3T3-E1 cells were transfected with siRNA and lysates were collected after 72 hours for immunoprecipitation and western blotting as described in the Materials and Methods. (C) Effect of PIASx siRNA on the mRNA levels of osteoblast differentiation markers in calvarial osteoblasts. Three days after confluence, osteogenic differentiation of primary osteoblasts was initiated by changing the medium (day 0). At the same time, cells were transfected with siRNA and RNA samples were collected at the indicated time periods. Expression was determined by quantitative real-time RT-PCR analysis. (D) Blockage of endogenous PIASx expression by siRNA led to the inhibition of ALP activity in MC3T3-E1 cells. siRNA was transfected 24 hours after the induction of osteogenesis. 7 days later ALP staining was performed. (E) Knocking down of endogenous PIASx suppressed matrix mineralization in MC3T3-E1 cells. Cells were cultured for 27 days in DM and then subjected to Alizarin Red S (AR-S) staining. (F) To quantify the degree of mineralization, each stained culture was subjected to extraction, and samples of the AR-S extract were used for the assay. (G) A similar experiment to that described in E was performed using primary osteoblasts. Cells were cultured for 14 days in DM and then subjected to AR-S staining. (H) The degree of mineralization in primary culture was quantified.

View larger version (52K): [in this window] [in a new window]   Fig. 3. PIASx overexpression increased ALP activity and ALP, Osx and OCN mRNA levels. (A) MC3T3-E1 cells were transfected at 90% confluence with the indicated expression plasmids and subjected to ALP staining at days 6 and 9 in DM. (B) Cells were transfected at day 6 in DM with the indicated expression plasmids and subjected to ALP staining at day 10. (C) Cells were transfected with the expression plasmids at day 6 in DM and RNA samples were collected at day 9 for RT-PCR analysis. (D) mRNA expression level in C was also performed by quantitative real-time RT-PCR.

View larger version (29K): [in this window] [in a new window]   Fig. 4. PIASx enhanced Osx promoter-driven transcription and Osx transcriptional activity cooperatively with NFAT. (A) Osx upregulated its own promoter and PIASx further enhanced its activity. MC3T3-E1 cells were co-transfected with the expression plasmids pEx3.1Osx, pcDNA3.1-PIASx and pcDNA3.1-PIASxC362S, the reporter plasmid pGL4.18-Osx-Luc and the pRL-TK plasmid at day 1 in DM. 48 hours post-transfection the reporter assay was performed. (B) Endogenous Osx and PIASx are important for Osx promoter activation. Cells were transfected with siRNA at day 1 in DM and 72 hours later transfected with Luc expression and reporter plasmids. 48 hours after Luc transfection, the reporter assay was performed. (C) Effect of PIASx and NFAT on osterix transcriptional activity in the GAL4 transactivation system. MC3T3-E1 cells were transfected with the indicated plasmids at day 1 in DM. (D) Cells were transfected with siRNA at day 1 in the DM and 72 hours later Luc plasmids were transfected. 48 hours post-transfection of Luc plasmids, the reporter assay was performed. (E) Effect of PIASx on the transcriptional activity of Runx2. MC3T3-E1 cells were transfected with the indicated plasmids at day 1 in DM. Cells were transfected with siRNA at day 1 in DM and this was followed 24 hours later by transfection with Luc plasmids (F). 48 hours post-transfection of Luc plasmids the reporter assay was performed. (G) MC3T3-E1 cells were transfected with siRNA at day 1 in DM and this was followed 72 hours later by transfection with Luc plasmids. 48 hours post-transfection of Luc plasmids the reporter assay was performed. Data represent the mean (±s.d.) of three independent experiments, each of which was performed in triplicate.