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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.

^* Author for correspondence (e-mail: kawashim{at}dent.niigata-u.ac.jp)

Accepted 18 May 2007

	Summary

Top Summary Introduction Results Discussion Materials and Methods References

 
We recently reported that tensile stress induces osteoblast differentiation and osteogenesis in the mouse calvarial suture in vitro. Using this experimental system, we identified PIASx, a splice isoform of Pias2, as one of the genes most highly upregulated by tensile stress. Further study using cell culture revealed that this upregulation was transient and was accompanied by upregulation of other differentiation markers, including osterix, whereas expression of Runx2 was unaffected. Runx2 and osterix are the two master proteins controlling osteoblast differentiation, with Runx2 being upstream of osterix. Targeted knockdown of PIASx by small interfering RNA (siRNA) markedly suppressed osteoblastic differentiation and matrix mineralization, whereas transient overexpression of PIASx caused the exact opposite effects. Regardless of PIASx expression level, Runx2 expression remained constant. Reporter assays demonstrated that osterix enhanced its own promoter activity, which was further stimulated by PIASx but not by its sumoylation-defective mutant. NFATc1 and NFATc3 additionally increased osterix transcriptional activity when co-transfected with PIASx. Because osterix has no consensus motif for sumoylation, other proteins are probably involved in the PIASx-mediated activation and NFAT proteins may be among such targets. This study provides the first line of evidence that PIASx is indispensable for osteoblast differentiation and matrix mineralization, and that this signaling molecule is located between Runx2 and osterix.

Key words: PIASx, Osterix, Mineralization, Osteoblast

	Introduction

Top Summary Introduction Results Discussion Materials and Methods References

 
We have previously reported that application of tensile stress (TS) to mouse calvarial sutures in culture induces osteoblast differentiation, which later leads to osteogenesis (Ikegame et al., 2001). Further investigation of the molecules involved in the early stage of mechanical stress-induced osteoblast differentiation revealed that the gene encoding one of the protein inhibitors of activated signal transducer and activator of transcription (STAT; PIAS) is markedly upregulated. Although it is known that some of the PIAS family members suppress the transforming growth factor- (TGF-) (Imoto et al., 2003) and bone morphogenetic protein (BMP) (Imoto et al., 2004) signaling pathways, it remains unknown whether any member of the PIAS family has any specific action on osteoblasts.

PIAS proteins were initially identified as specific cofactors inhibiting DNA binding and transcriptional activation by the STAT family of transcription factors. These proteins typically contain an N-terminal SAP domain, a central RING-like zinc-finger domain and a C-terminal acidic domain (Shuai and Liu, 2005; Rosas-Acosta et al., 2005). Five mammalian PIAS proteins have been identified: PIAS1, PIAS3, PIASx (also known as ARIP3, androgen receptor interacting protein), PIASx (also known as Miz1, Msx-interacting-zinc finger), and PIASy. PIASx and PIASx are the products of alternatively spliced mRNAs from the same gene. These two proteins differ only in the C-terminal region, in which PIASx contains an extension of the 31-amino acid serine-rich domain (Schmidt and Muller, 2003). PIAS proteins mediate their functions in either a sumoylation-dependent (as E3 ligase) or independent manner. In some cases both modes of action are found to be working (Sharrocks, 2006). PIAS-mediated sumoylation of target proteins has been established to play important roles in a wide range of cellular processes, including transcriptional control and protein-protein interactions (Hay, 2005; Johnson, 2004; Seeler and Dejean, 2003). Many interacting partners of PIAS proteins such as AR, p53, septins and the DJ-1 protein (Gostissa et al., 1999; Johnson and Blobel, 1999; Poukka et al., 2000; Rodriguez et al., 1999; Takahashi et al., 2001) are modified by SUMO-1. PIAS1 and PIAS3 bind to STAT1 and STAT3, respectively, and inhibit their action (Liu et al., 1998; Chung et al., 1997). PIAS1 is shown to localize the Msx1 homeodomain transcription factor to the nuclear periphery (Lee et al., 2006). PIASy targets LEF1 to nuclear bodies and represses its transcriptional activation (Sachdev et al., 2001). Pias1^–/– mice show an increased resistance to pathogenic infection (Liu et al., 2004). ARIP3 and other members of this family function as coregulators for the androgen receptor and other steroid receptors (Moilanen et al., 1999; Kotaja et al., 2000; Tan et al., 2000). PIASx/Miz1 directly interacts with the homeobox protein Msx2 and enhances its DNA-binding affinity (Wu et al., 1997). PIASx-deficient mice have mild defects in testicular development (Santti et al., 2005). PIASx is a transcriptional cofactor for TFII-I (Tussie-Luna et al., 2002b). It also physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation (Tussie-Luna et al., 2002a).

Osteoblasts, which play central roles in bone formation, originate from undifferentiated mesenchymal cells (Pittenger et al., 1999). The osteogenic master regulators Runx2/Cbfa1 (Ducy et al., 1997; Komori et al., 1997) and osterix (Osx) (Nakashima et al., 2002) are indispensable for osteoblast commitment and maturation. Runx2 directly regulates osteoblast-specific genes, including the osteocalcin gene, through its binding to the specific DNA element OSE2 (Ducy et al., 1997). Targeted disruption of Runx2 results in the complete lack of bone formation by osteoblasts (Komori et al., 1997), and inherited mutations of the Runx2 gene in humans cause cleidocranial dysplasia, which is characterized by severely impaired osteogenesis (Mundlos et al., 1997). Osterix-deficient mice show an abnormal skeletogenesis with a complete lack of bone formation in spite of unaffected Runx2 expression (Nakashima et al., 2002). These findings indicate that osteoblast differentiation is mainly regulated by Runx2 and osterix. It is extremely important to identify any possible modifiers of Runx2 and/or osterix, because the rate of osteoblast differentiation must be tightly regulated in bone homeostasis. Several transcription factors including C/EBP, C/EBP, ETS1, Menin, Smad1 and Smad5, interact with Runx2 and enhance its transcriptional activity. Grg5, Rb, TAZ and p204 interact with Runx2 and function as transcriptional co-activators (Komori, 2005; Liu et al., 2005). Other transcription factors and co-regulators including C/EBP, Dlx3, Msx2, PPAR, Twist, Stat1, Smad3, Yes and TLE, reduce the transcriptional activity of Runx2 (Kang et al., 2005; Komori, 2005). However, little is known about factors that interact with osterix. Recently, it was shown that NFATc1 interacts with osterix and enhances Col1a1 promoter activity (Koga et al., 2005) whereas Runx2 specifically upregulates osterix promoter activity (Nishio et al., 2006).

In this study we demonstrate that a transient increase of PIASx is crucial for osteoblast differentiation and mineralization to take place. This effect of PIASx is probably mediated through activation of osterix transcription based on the following reasons: (1) the expression level of the osterix gene closely follows that of PIASx, and (2) knockdown and overexpression of PIASx markedly reduce and enhance, respectively, osterix gene expression as well as mineralization in MC3T3-E1 cells. We also show that PIASx stimulates osterix transcriptional activity in a sumoylation-dependent manner, but does not affect Runx2 transcriptional activity. Furthermore, we provide evidence for the autoregulation of the osterix promoter. This is the first demonstration that PIASx plays a crucial role in osteoblast differentiation and mineralization.

	Results

Top Summary Introduction Results Discussion Materials and Methods References

 
Mechanical stress upregulated PIASx expression in calvariae and MC3T3-E1 cells
We previously determined that TS applied to an organ culture of mouse calvarial sutures induced osteoblast differentiation, which eventually led to osteogenesis. In that experimental system, gene-chip microarray analysis revealed a 5.2-fold upregulation of the PIASx gene in cells exposed to TS. The expression level of PIASx mRNA in suture tissue from organ cultures with or without stress was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization. PIASx expression was markedly upregulated as early as 6 hours following the initiation of loading (Fig. 1A).

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.

PIASx mRNA expression is transiently upregulated during the matrix maturation stage of osteoblast differentiation
To gain some insight into a possible role of PIASx in osteoblast differentiation and osteogenesis, we examined PIAS family gene expression together with other marker genes in an in vitro mineralization assay. MC3T3-E1 cells were grown in -MEM [with 10% fetal bovine serum (FBS)] until they reached confluence. Three days after confluence, the mineralization assay was begun (this is designated as day 0) by changing the medium to that supplemented with -glycerophosphate, ascorbic acid and dexamethasone as described in the Materials and Methods. The cells were harvested at days 0, 3, 9, 15, 21 and 27 and total RNA was isolated for RT-PCR analysis. The expression patterns of osteoblast differentiation markers and PIAS family members are shown in Fig. 1C. PIASx mRNA increased as osteoblastic differentiation progressed up to day 9 when expression levels reached a maximum (during the matrix maturation stage of osteoblast differentiation), and then gradually declined until day 27 when mineralization was peaking (Fig. 1C, Fig. 1D). Expression of other PIAS family members remained unchanged throughout the differentiation process (Fig. 1C). The expression levels of alkaline phosphatase (ALP) and the osterix gene closely paralleled with that of PIASx (see also Fig. 2C), followed by that of osteocalcin (OCN). Runx2 gene expression did not change appreciably. These data suggest that PIASx may play an important role in osteoblast differentiation and mineralization. To test this possibility, we next examined the effect of knockdown of this gene expression.

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.

Targeted knockdown of PIASx by siRNA inhibits osteoblast differentiation
Three days after confluence, MC3T3-E1 cells were fed with differentiation medium (DM) (day 0). At day 1 (24 hours later) they were transfected with siRNA and expression of marker genes was evaluated at the times indicated. As shown in Fig. 2A, the addition of siRNA reduced PIASx expression by more than 80% after 24 hours and the maximum knockdown effect was observed 3 days after treatment. The effect then gradually decreased but persisted until the end of the culture period. The scrambled siRNA, a nonsilence control, had no effect, demonstrating the PIASx siRNA sequence specificity. The level of expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal control was not changed in either PIASx siRNA or the scrambled siRNA transfected cells, confirming the specificity and indicating the lack of toxicity. PIASx gene expression in the control cells again peaked at day 9 of culture, in good agreement with the data shown in Fig. 1A. It should be noted that the endogenous PIASx protein level was also markedly reduced after siRNA treatment (Fig. 2B).

The mRNA levels of the osteoblast differentiation markers ALP, Osx and OCN were all downregulated in PIASx siRNA-treated cells but the Runx2 mRNA level was unchanged. These data clearly indicate that PIASx suppresses osteoblastic differentiation of MC3T3-E1 cells. Similar results were obtained using primary cultured osteoblasts, as shown in Fig. 2C.

At day 7, ALP staining was performed. PIASx knockdown inhibited ALP (an early stage osteoblast differentiation marker enzyme) activity significantly, as measured histochemically (Fig. 2D).

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.

Together, these data clearly demonstrated that reduced PIASx expression is the cause of inhibition of osteoblast differentiation and matrix mineralization.

Transient overexpression of PIASx increased ALP activity and upregulated the expression of ALP, Osx and OCN genes
We next examined whether overexpression of PIASx affects osteoblast differentiation. At 90% confluence, MC3T3-E1 cells were transiently transfected with the PIASx expression plasmid. Three days after confluence, the culture was fed with DM (day 0). ALP staining was then performed at day 6 and day 9. As shown in Fig. 3A, PIASx overexpression barely affected ALP activity at day 6 compared with empty vector-treated control, whereas at day 9 it was slightly increased. When cells were transfected at day 6 and the staining was performed at day 10, the ALP activity increased significantly (Fig. 3B). This result suggests that PIASx exerts its effect in a differentiation stage-dependent manner. We also examined the effect of PIASx overexpression on osteoblast differentiation markers. MC3T3-E1 cells were transfected at day 6 with PIASx expression plasmid, and RNA samples were collected at day 9 for RT-PCR analysis. As shown in Fig. 3C, PIASx overexpression upregulated the expression levels of ALP, Osx and OCN, but did not affect that of Runx2, again consistent with the data shown in Fig. 1C and Fig. 2A. These semi-quantitative RT-PCR data were confirmed by real-time PCR as shown in Fig. 3D. Taken together, these results indicate that PIASx controls the osteoblast differentiation process.

Autoregulation of osterix synthesis: a crucial step in osteoblast differentiation
To gain an insight into the mechanism of how PIASx controls osterix gene expression, we cloned a 1269-bp fragment of the osterix promoter to upstream of the luciferase reporter. We found that forced expression of osterix protein upregulated osterix promoter activity (Fig. 4A), indicating that the osterix gene is autoregulated by positive feedback on its own promoter. Co-expression of PIASx and osterix further increased osterix promoter activity. Co-transfection of a sumoylation-defective mutant of PIASx (C362S) and osterix failed to produce this additive effect (Fig. 4A). Moreover, knocking down of endogenous osterix and PIASx expression by siRNA in MC3T3-E1 cells reduced the basal promoter activity, suggesting that both endogenous osterix and PIASx activated the osterix promoter (Fig. 4B). These results indicate that the osterix promoter is autoregulated by osterix itself and suggest that PIASx activates osterix transcriptional activity.

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.

PIASx enhances the transcriptional activity of osterix and NFAT but not Runx2

PIASx knockdown and overexpression experiments showed that Runx2 gene expression is unaffected by PIASx (Figs 2, 3). To provide further information, we co-transfected PIASx and Runx2 expression plasmids with a reporter plasmid p6OSE2-Luc (containing six copies of the Runx2-binding site). We found that PIASx or its sumoylation-defective mutant (C362S) had no effect on Runx2 transcriptional activity (Fig. 4E). Knockdown of endogenous PIASx by siRNA also had no effect on transcriptional activity as compared with control siRNA treatment (Fig. 4F). As some of the STAT family members play a role in osteoblast differentiation (Itoh et al., 2006; Kim et al., 2003; Xiao et al., 2004), we tried to seek whether PIASx has any effect on the transcriptional activity of STAT. As shown in Fig. 4G, knockdown of endogenous PIASx by siRNA in MC3T3-E1 cells resulted in activation of the interferon response element (ISRE) and STAT promoters. The gamma-interferon activation site (GAS) promoter was unaffected (Fig. 4G). ISRE-Luc possesses the binding site for the STAT1 and STAT2 heterodimers and STAT-Luc has the binding site for all STAT homodimers and heterodimers.

As a whole, our results indicate that PIASx enhances osterix transcriptional activity in a sumoylation-dependent manner. Although we do not know which SUMO substrates may be involved in this case, NFATc1, NFATc3 and/or STAT1 may be among such targets.

	Discussion

Top Summary Introduction Results Discussion Materials and Methods References

 
It is well established that mechanical stress induces osteoblast differentiation and osteogenesis both in vivo and in vitro (Meyer et al., 2006; Burr et al., 2002). The mechanism by which mechanical stress induces these effects, however, remains largely unknown. In the present study we have identified PIASx as one of the genes most highly upregulated by mechanical stress in both ex vivo (calvarial organ culture) and in vitro (MC3T3-E1 in flexer cell strain) experimental systems. To examine a possible role of this gene in mechanical stress-induced osteoblast differentiation and mineralization, we also studied the time course of expression compared with other differentiation markers in MC3T3-E1 cells without mechanical stress. We found that PIASx was transiently upregulated, peaking at the ninth day of culture in DM. This was paralleled by upregulation of ALP and osterix, followed by upregulation of osteocalcin and then mineralization. The expression levels of other PIAS family members were unchanged. These data suggest that transient upregulation of PIASx is necessary for osteoblast differentiation and mineralization irrespective of the presence of mechanical stress stimuli.

Knockdown of endogenous PIASx suppressed both ALP activity and mineralized bone nodule formation, two classical cytochemical markers of osteoblast differentiation. The expression of the osteoblast differentiation markers ALP and OCN and the transcription factor osterix was also downregulated following inhibition of endogenous PIASx. These data indicate that other PIAS family members fail to compensate for the PIASx knockdown effect and that among PIAS family genes only PIASx is essential for osteoblast differentiation and bone mineralization. Knockout mice of PIAS family genes show different phenotypes but there is no previous study on the skeletal phenotype. Further studies of de novo bone formation in PIASx-deficient mice should improve understanding of the importance of PIASx in osteogenesis.

Forced expression of PIASx enhanced ALP activity as well as ALP, Osx and OCN mRNA levels, supporting the notion that PIASx acts as an anabolic agent for bone formation. Early stage transfection did not increase ALP activity significantly; however, middle-stage transfection markedly enhanced ALP activity. This suggests that PIASx expression must be strictly controlled in a stage-dependent manner during osteoblast differentiation.

Both knockdown and overexpression experiments clearly show that PIASx is crucial for the expression of ALP, Osx and OCN genes. Runx2 and Osx are the two key transcriptional regulators of osteoblast differentiation, but the Runx2 mRNA level was unchanged in the presence or absence of PIASx.

To explore the mechanism by which PIASx affects osterix mRNA levels in our experiments, osterix transcriptional regulation was examined by luciferase assay. To evaluate PIASx action on the osterix promoter, we cloned a 1269-bp fragment upstream of the luciferase reporter. Surprisingly, we found that Osx itself positively upregulated this promoter activity. There is no previous report of this autoregulation. Such autoregulation may ensure the optimum concentrations of important master regulators of cell differentiation to maintain the available number of osteoblastic cells in a committed differentiation state. Positive autoregulation has been described for several transcription factors, such as NFATc1, NFATc4, MyoD, Pit-1, GATA-1 and GATA-3 (Asagiri et al., 2005; Arron et al., 2006; Lun et al., 1997; Rhodes et al., 1993; Tsai et al., 1991; Ouyang et al., 2000). The Runx2 gene is known to be autoregulated by negative feedback on its own promoter (Drissi et al., 2000), which may stringently control Runx2 expression and function during bone formation. Our result uncovered an important autoactivation pathway of osterix that is crucial for osteoblast differentiation. This autoregulation may provide an additional mechanism for timely control of osterix activity to mediate osteoblast differentiation and for proper execution of the osteogenic program. Further study revealed that co-transfection of PIASx with osterix increased osterix transcriptional activity and a sumoylation-defective mutant of PIASx failed to show this activity. Again, knockdown of endogenous osterix or PIASx repressed the basal promoter activity. These results demonstrate the two different modes of osterix promoter regulation, one is autoregulation and the other is PIASx-mediated. Because there is no consensus motif for sumoylation in the osterix protein, other proteins interacting with osterix are likely to be the substrate for PIASx.

A reporter assay using the GAL4 transactivation system confirmed that PIASx enhanced the transcriptional activity of osterix and a sumoylation-defective mutation deprived this effect. This result provides evidence that sumoylation is indispensable for PIASx-mediated regulation of osterix transcriptional activity. Recently, Koga et al. reported that NFAT transcription factors play an important role in the transcriptional program of osteoblasts (Koga et al., 2005). NFAT and Osx form a complex that binds to DNA, and this interaction is important for the transcriptional activity of osterix. In our microarray data we also found that NFATc3 was markedly upregulated during TS-induced osteoblast differentiation and osteogenesis in organ culture of mouse calvarial suture. On the basis of this information we consider that NFAT proteins may be involved in PIASx-induced osterix activation. When transfected with osterix, neither NFATc1 nor NFATc3 affected the promoter activity. However, when co-transfected with PIASx and Osx, NFATc1 or NFATc3 further enhanced PIASx/osterix transcriptional activity, consistent with the data of Koga et al. (Koga et al., 2005). NFATc3 had a similar but a lesser effect as compared with NFATc1. The reason behind this may be its low expression in osteoblasts (Koga et al., 2005). Inhibition of endogenous NFATc3 in MC3T3-E1 cells by siRNA caused a partial reduction in matrix mineralization (data not shown). Together, these results suggest that PIASx may act on the NFAT-Osx complex. As osterix has no consensus motif for sumoylation, PIASx may sumoylate NFAT, thereby causing a conformational shift that favors NFAT binding with osterix and enhancing promoter activity. Terui et al. showed that NFATc2 was sumoylated at two different sites (amino acid positions 684 and 897) and other members (NFATc3, NFATc4) also possess the sumoylation motif (Terui et al., 2004). They also mentioned two important functions of this sumoylation-nuclear anchorage and transcriptional activation. PIASx may influence nuclear translocation of NFAT by sumoylation. In our experimental system, NFATc1/NFATc3 alone enhanced osterix transcriptional activity in an HEK293T cell line as previously reported (data not shown). By contrast, NFATc1/NFATc3 alone had no effect on osterix transcriptional activity in an MC3T3-E1 cell line (Fig. 4C). This difference may reflect cell type-specific phenomena. In HEK293T cells NFAT naturally translocates to the nucleus, whereas in MC3T3-E1 cells the NFAT proteins may be retained in the cytoplasm in the absence of nuclear translocation signals such as PIASx-mediated activation.

It remains to be determined whether endogenous proteins other than NFATs are also sumoylated by PIASx, which then cooperate with the NFAT-Osx complex to activate the osterix promoter or independently play roles at other stages throughout the osteoblast differentiation process.

Next we determined the effect of PIASx on Runx2 transcriptional activity. PIASx or its sumoylation-defective mutant had no effect on Runx2 transcriptional activity. Knockdown of endogenous PIASx also did not affect transcriptional activity. These results suggest that the effect of PIASx on osteoblast differentiation is downstream of Runx2 and upstream of osterix.

PIASx also upregulated expression of the middle-stage differentiation marker ALP. Hatta et al. reported that there is no involvement of osterix in the regulation of ALP promoter activity (Hatta et al., 2006). Consequently, PIASx may regulate ALP activity either directly or indirectly through a mechanism independent of osterix activation.

Other signaling molecules that may affect osterix transcriptional activity are STAT protein family members. Reports on the effects of STAT proteins in bone formation are limited and controversial. The STAT3 signal is essential for osteoblast differentiation (Itoh et al., 2006). By contrast, STAT1 functions as a negative regulator of bone formation (Kim et al., 2003; Xiao et al., 2004), and PIAS proteins inhibit the transcriptional activity of STATs in a sumoylation-dependent and/or independent manner.

Among STAT family members only STAT1 is modified by SUMO E3 ligase, whereas others do not have the sumoylation consensus site (Song et al., 2006). Although PIAS1, PIAS3 and PIASx all sumoylate STAT1 (Rogers et al., 2003; Ungureanu et al., 2003), it remains to be determined whether PIASx also sumoylates STAT1. Xiao et al. recently showed that the osterix mRNA level was increased by 40% in osteoblasts from STAT^–/– mice, whereas the Runx2 mRNA level was unchanged (Xiao et al., 2004). Because these mice have increased bone volume, it is postulated that STAT1 may be a negative regulator of osterix expression. Thus, PIASx may increase the osterix mRNA level through sumoylation of STAT1, thereby relieving the negative regulation. This is consistent with our data that PIASx knockdown enhanced the ISRE-Luc and STAT-Luc reporter activity (Fig. 4G). As these promoters posses the binding site for STAT1 dimer, STAT1 can be a substrate for PIASx-mediated sumoylation. This mechanism may also contribute to PIASx-mediated osteoblast differentiation. Further studies are required to clarify this.

Osteoporosis remains a major public health problem. Most treatments available so far target osteoclasts and inhibit bone resorption, so there is no way to restore the decreased bone volume. Our data herein demonstrate that the E3 ligase PIASx plays specific roles in regulating osterix transcriptional activity as well as osteoblast differentiation. Thus, PIASx could be a potential molecular target for anabolic agents that would accelerate osteoblast differentiation and promote bone formation. It has been reported that osterix mediates antitumor activity in murine osteosarcoma (Cao et al., 2005). As osterix activity is positively regulated by PIASx, PIASx may be a novel therapeutic target for osteosarcoma as well.

We have demonstrated for the first time that the SUMO E3 ligase PIASx plays a key role in upregulating osterix transcriptional activity, thereby stimulating osteoblast differentiation and bone formation. Our study will shed light on the largely unknown physiological roles of PIAS proteins in regulating cellular signaling pathways other than through SUMO-E3-ligase activity in bone biology but also in other fields of biology in health and diseases.

	Materials and Methods

Top Summary Introduction Results Discussion Materials and Methods References

 
Application of mechanical stress
For organ culture calvariae were aseptically removed from 2-3-day-old neonatal mice. TS was applied to the midsagittal cranial sutures of the calvariae by means of helical springs, as described previously (Hickory and Nanda, 1987; Ikegame et al., 2001), in serum-free BGJb medium (Invitrogen, Carlsbad, CA) containing 0.1% bovine serum albumin (BSA; Invitrogen). The cranial sutures were then freed from the calvarial bones with blades under a dissection microscope, from which total RNA was extracted for RT-PCR.

A Flexercell FX-4000 strain unit was used to generate cyclic tensile strain in the MC3T3-E1 cells. Cells were seeded onto BioFlex type I collagen-coated six-well plates and incubated at 37°C. Differentiation was induced 3 days after confluence (day 0) and from that time the cells were subjected to mechanical strain of 8-12% elongation at three cycles/minute, each cycle consisting of 10 seconds of strain and 10 seconds of relaxation, according to the manufacturer's instructions. The strain unit consists of a computer-controlled vacuum unit and base plates to hold the culture dishes. The computer system controls the frequency of deformation and the negative pressure applied to the culture plates. Control cells (0% elongation) were cultured on similar plates and kept in the same incubator without mechanical strain. Cells from stretched and non-stretched cultures were harvested and mRNA was extracted to quantify expression by RT-PCR.

Cell culture
A mouse osteoblastic cell line, MC3T3-E1 (ATCC, Manassa, VA), was cultured in -modified minimum essential medium (-MEM; Gibco BRL, Rockville, MD) with 10% heat-inactivated FBS (JRH, Lenexa, KS). To isolate primary osteoblasts, calvaria from 2-3-day-old neonatal mice were dissected free of adherent connective tissue and submerged in phosphate-buffered saline (PBS) containing 100 U/ml penicillin G and 100 µg/ml streptomycin. Then the calvariae were washed in PBS and digested with 0.1% collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ) and 0.06% trypsin (Gibco BRL, Life Technologies) in MEM (Gibco) for 1 hour with gentle shaking at 37°C. The cells were collected by centrifugation at 2000 g for 5 minutes and seeded into 35 mm culture dishes at a density of 10⁶ cells/dish in -MEM (Gibco) containing 10% FBS. Cells were maintained at 37°C in a 95% air/5% CO₂ atmosphere. To induce osteogenesis, cells were fed with DM that consisted of -MEM with 10% FBS, 10 mM -glycerophosphate, 50 µg/ml L-ascorbic acid and 100 nM dexamethasone. Medium was renewed every third day.

Semi-quantitative and quantitative real-time RT-PCR and Southern hybridization
Total RNA was extracted according to the acid guanidium isothiocyanate-phenol-chloroform method. Then, first-strand cDNA was synthesized using random primers (nine-mers) (Takara Shuzo, Osaka, Japan) and Superscript III reverse transcriptase (Invitrogen). For PCR, samples of synthesized cDNA were added to PCR mixtures containing forward and reverse primers (0.2 mM each), dNTP mixture (0.2 mM each) (Bioneer, Daejeon, Korea) and Taq polymerase (0.05 U/µl) (Bioneer). Cycling conditions and primer sequences were described previously (Saito et al., 2002; Yoshizawa et al., 2004), and are as follows. For the mouse ALP gene the conditions were 94°C (30 seconds), 55°C (35 seconds) and 72°C (60 seconds) for 24 cycles, and the primers were 5'-GACTGGTACTCGGATAACGAGATGC-3' and 5'-TGCGGTTCCAGACATAGTGG-3'. For the mouse osteopontin gene the conditions were 94°C (30 seconds), 62°C (45 seconds) and 72°C (30 seconds) for 24 cycles, and the primers were 5'-CATTGCCTCCTCCCTCCCGGTG-3' and 5'-GCTATCACCTCGGCCGTTGGGG-3'. For the mouse PIASx, PIASx and Pias3 genes, the conditions were: 94°C (30 seconds), 55°C (45 seconds) and 72°C (30 seconds) for 30 cycles, and the primers were 5'-GAAGATCTATGGCGGATTTCGAGGAGTTG-3' and 5'-GGAATTCTCACTGTTGCACAGTATCAGAAG-3' for the PIASx gene, 5'-ATGTCATCAGATTTGCCAGG-3' and 5'-GTTGCAAAAATCAGCTTCCA-3' for the PIASx gene and 5'-AAGGAGAAACTGACCGCTGA-3' and 5'-CTCTGATGCCTCCTTCTTGG-3' for the Pias3 gene. For the mouse Pias1 and PIASy genes the conditions were 94°C (30 seconds), 55°C (45 seconds) and 72°C (30 seconds) for 27 cycles, and the primers were 5'-TGGGTTTGTCCTGTCTGTGA-3' and 5'-CGAGGCTTGATGAGGAAGAC-3' for the Pias1 gene and 5'-AGACCCTTAAGCCGGAGGTA-3' and 5'-GTGGCCGAGGACAGATACAT-3' for the PIASy gene. PCR products were fractionated on 1% agarose gels, transferred to positively charged nylon membranes and cross-linked with UV light. Membranes were hybridized with digoxigenin (Roche Diagnostics, Indianapolis, IN)-labeled DNA probes and detected with CDP-Star substrate (New England BioLabs, Ipswich, MA). In most cases, the results were further confirmed by quantitative real-time PCR. Real-time RT-PCR was performed using 2x SYBR Premix Ex Taq (Takara) and Thermal Cycler Dice^TM Real Time System TP 800 (Takara). The specificity of detected signals was confirmed by a dissociation curve consisting of a single peak. The relative amount of each mRNA was normalized by GAPDH expression as an internal control. The primer sequences used were as follows: for GAPDH, forward 5'-TGTGTCCGTCGTGGATCTGA-3', reverse 5'-TTGCTGTTGAAGTCGCAGGAG-3'; for osteocalcin, forward 5'-TGCTTGTGACGAGCTATCAG-3', reverse 5'-GAGGACAGGGAGGATCAAGT-3'; for Runx2, forward 5'-AAATGCCTCCGCTGTTATGAA-3', reverse 5'-GCTCCGGCCCACAAATCT-3'; for osterix, forward 5'-CCCTTCTCAAGCACCAATGG-3', reverse 5'-AGGGTGGGTAGTCATTTGCATAG-3'; for ALP, forward 5'-TGACCTTCTCTCCTCCATCC-3', reverse 5'-CTTCCTGGGAGTCTCATCCT-3'; for PIASx, forward 5'-CCTTATTCCAGTTGATCCCCAGT-3', reverse 5'-TATGACCCCTGTCTCACTCCT-3'.

Expression and reporter plasmids
For a mammalian expression plasmid encoding PIASx, the RT-PCR-amplified mouse PIASx open reading frame was subcloned into the pcDNA3.1/myc-HisA (Invitrogen). The expression plasmid pcDNA3-Runx2/Osf2 and the reporter plasmid p6OSE2-Luc were generated as described previously (Saito et al., 2002). For pcDNA3-Flag-NFATc3, NFATc3 was subcloned into pcDNA3-Flag (N-terminal Flag expression vector) generated with a PCR-based approach. The expression plasmid pSGOSX was made up of the near-full-length protein (residues 27-428) fused in-frame with the DNA-binding domain of the yeast transcription factor GAL4 in the pSG424 vector (Nakashima et al., 2002). pG5E1b-Luc contains five copies of the GAL4 binding sequence and the adenoviral E1b promoter extending upstream into the luciferase coding sequence. In the osterix expression plasmid pEx3.1Osx, osterix cDNA (amino acid 3-428) was cloned into the pTriEx1.1 expression vector.

The sumoylation-defective mutant of PIASx, C362S (mutation in zinc-finger or SP-RING motif at amino acid position 362, where a cysteine residue is replaced by serine) (Schmidt and Muller, 2002), was constructed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's recommendations.

The Osx promoter sequence spans nucleotides –1360 to –10 of the Osx gene locus relative to the position (+1) of the initiation of methionine for the Osx open reading frame (Lu et al., 2006). This sequence was amplified from mouse (C57BL/6) tail tissue genomic DNA by PCR using the following 5' and 3' primers, respectively: 5'-ACAGGTACCCACACATACACG-3' and 5'-CAAGCAGAGAGGACGCCATCCTCGAGCT-3'. The resulting PCR product was digested with KpnI and XhoI and inserted into the pGL4.18 luciferase reporter vector (Promega, Madison, WI).

pSTAT RE-TK hRluc, pISRE-TK hRluc and pGAS RE-TK hRluc constructs were kindly provided by RIKEN Bio-Resource Center (Tsukuba, Japan). The nucleotide sequences of the response element were as follows: 5'-GATCCAGTTCCCGTCAATCG-3' for STAT response element, 5'-GATCCAGAAACAAAAACAAG-3' for ISRE response element and 5'-GATCCTTCCGGGAATTCTGGGAAG-3' for GAS response element. The HSV-TK promoter from the pRL-TK vector expressing Renilla luciferase was removed by digestion from the BamHI site to the HindIII site. It was then cloned into the pGL4.18 vector (Promega), expressing firefly luciferase.

All constructs were confirmed by the ABI PRISM^TM 377 DNA Sequencing System (PE Company, Foster City, CA).

Small interfering RNA (siRNA) duplex preparation and transfection
The mouse PIASx/Miz1 and Sp7/osterix SMARTpool siRNA duplexes were chemically synthesized by Dharmacon RNA Technologies (Chicago, IL). The SMARTpool siRNA is a mixture of four different siRNA duplexes targeting mouse PIASx (GenBank^TM accession number NM_008602) and osterix (GenBank accession number AF 184902). The sequences of the SMARTpool siRNA are proprietary. All siRNA sequences were subjected to a BLAST search against mouse genome sequences in GenBank to ensure specificity. The siRNA duplexes were dissolved in 1x universal RNA oligo buffer (20 mM KCl, 6 mM HEPES-KOH, pH 7.5, 0.2 mM MgCl₂).

MC3T3-E1 cells and calvarial osteoblasts were plated on 35 mm culture dishes (2 ml medium/dish). Three days post-confluence, the cultures were fed with DM (day 0). siRNA transfection was performed at day 0 for calvarial osteoblasts and at day 1 for MC3T3-E1 cells using DharmaFECT1 transfection reagent (Dharmacon RNA Technologies) according to the manufacturer's instructions: 4 µl/dish DharmaFECT1 containing 100 nM siRNA. Scrambled nonsilence control siRNA/RISC-free control siRNA was added to each transfection reaction to keep the total amount of RNA constant. Cells were maintained for the times indicated in the figure legends. Specific RNA interference effects were confirmed by at least three independent experiments.

Transient co-transfection and luciferase reporter assay
Cells were transfected with various plasmid DNAs using Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's recommendations. Forty-eight hours after transfection, cells were harvested, lysed and assayed using firefly luciferase and Renilla luciferase substrates in the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity in the same cell extract to correct for variation in transfection efficiency. The total amount of transfected DNA was kept constant by the addition of empty vectors pSG424 or pcDNA3. After 48 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) on a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA).

Preparation of rabbit polyclonal antisera
Polyclonal antisera against PIASx was raised against a synthetic peptide, H₂N-CSVTVASDASKKKIDVID-COOH, corresponding to the residue sequence 454-471 in the PIASx protein by immunizing rabbits. The peptide was coupled to keyhole limpet hemocyanin (KLH) as a carrier. The antisera (Ab2) were affinity-purified against immunized peptide and employed for immunoprecipitation and western blot analysis.

Immunoprecipitation and western blotting
MC3T3-E1 cells at 70% confluence in a 100-mm cell culture plate were transfected with the indicated siRNA using DharmaFECT1 (Dharmacon) according to the manufacturer's instructions. Cells were washed with ice-cold PBS at 72 hours after transfection, collected, and lysed in 1 ml of TNE buffer [10 mM Tris-HCl (pH 7.8), 1% NP-40, 0.15 M NaCl, 1 mM EDTA] containing 1:1000 diluted protease inhibitor cocktail (Sigma) for 30 minutes and passed through a 27-gauge needle ten times. After centrifugation at 12,000 g for 30 minutes, supernatants were first cleared with protein G-Sepharose beads for 30 minutes. Equal amounts of the pre-cleared lysates were used for immunoprecipitation with 5 µg anti-PIASx antisera (Ab2) on a rocking platform/rotating wheel at 4°C overnight. Then protein G-Sepharose was added to the immunocomplexes for 30 minutes. The beads were washed five times with 0.5 ml of TNE buffer to remove nonspecific adsorbed proteins and immunoprecipitates were eluted by boiling in sodium dodecyl sulfate (SDS)-sample buffer. Proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto polyvinylidene difluoride (PVDF) membrane. Membrane was blocked with 5% non-fat dried milk in TBS supplemented with 0.1% Tween-20 and incubated with anti-PIASx antisera (Ab2) [1:100] for 1 hour at room temperature. Subsequently, membrane was incubated with horseradish-peroxidase-conjugated anti-rabbit IgG secondary antibody [1:25,000]. Antigen-antibody complexes were detected using a proprietary kit according to the manufacturer's instructions (Immobilon; Millipore Corporation, Billerica, MA, USA).

Mineralization assay
Three days after confluence, MC3T3-E1 cells and primary osteoblasts were fed with DM (day 0). Cultures were then continued until day 27 and 14, respectively, with medium changed every 3 days. At the end of each culture, cells were subjected to Alizarin Red S staining and quantification for calcium as described previously (Saito et al., 2002).

ALP staining
ALP activity was analyzed histochemically as described previously (Saito et al., 2002).

	Acknowledgments

 
The authors are grateful to Hiroshi Takayanagi of Tokyo Medical and Dental University and Melissa Brown of Northwestern University School of Medicine for their kind gift of NFATc1 expression vector plasmid. pSTAT RE-TK hRluc, pISRE-TK hRluc and pGAS RE-TK hRluc constructs deposited by Yokoyama K. were kindly provided by the DNA Bank, RIKEN Bio-Resource Center (Tsukuba, Japan) with the support of the National Bio-Resources Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (No. 16390531) (H.K.), a Grant-in-Aid for Young Scientists (A) from MEXT (No. 17689050) (T.Y.), a Grant-in-Aid for Young Scientists (B) from MEXT (No. 17791319) (O.I.), and by a grant from Novartis Pharma Japan.

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