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Genes to Cells (2008) 13, 839-850. doi:10.1111/j.1365-2443.2008.01209.x
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PI3K/Akt signaling as a key regulatory pathway for chondrocyte terminal differentiation

 Keisuke Kita1,2, Tohru Kimura1,*, Norimasa Nakamura2, Hideki Yoshikawa2 and Toru Nakano1,3,*

1 Department of Pathology, and
2 Department of Orthopedics, Graduate School of Medicine, and
3 Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Chondrogenesis is a well-coordinated multi-step differentiation process in which resting chondrocytes produce terminally differentiated hypertrophic chondrocytes through a proliferative stage. Here we show that phosphoinositide-3 kinase (PI3K) and its major downstream molecule, Akt, a serine–threonine kinase, play pivotal roles in this process. Akt signaling was activated in resting and proliferative chondrocytes but was reduced during terminal differentiation. We adopted two chondrocyte differentiation systems to investigate the roles of PI3K/Akt signaling in chondrogenesis. First, we employed an embryonic forelimb organ culture of transgenic mice expressing an Akt-Mer (a ligand-binding domain of a mutated estrogen receptor) fusion protein whose kinase activity was conditionally activated by treatment with 4-hydroxytamoxifen (4OHT). Activation of Akt signaling in embryonic chondrogenesis enhanced chondrocyte proliferation and inhibited hypertrophic differentiation, presumably due to the suppressed expression of Runx2, a transcription factor critical for chondrocyte terminal differentiation. Conversely, inhibition of PI3K by its inhibitor accelerated terminal hypertrophic differentiation, resulting in a shorter bone. Essentially the same results were obtained in a second line of experiments using human synovial stromal cells (hSSCs), which are mesenchymal progenitor cells isolated from adult joints. These findings demonstrate that PI3K/Akt signaling is a key regulator in terminal chondrocyte differentiation in both embryonic and adult chondrogenesis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
In vertebrates, there are two patterns of bone development: endochondral and intramembranous bone formation (Kronenberg 2003; Goldring et al. 2006). The former is initiated when mesenchymal cells condense and then differentiate into chondrocytes. The production and secretion of aggrecan, a matrix that is rich in proteoglycan and type II collagen, from chondrocytes is necessary to form cartilage. As cartilage enlarges, resting chondrocytes at both ends of bone rudiments differentiate into proliferative chondrocytes (Fig. 1A). After cell division ceases in proliferative chondrocytes, they enter terminal differentiation. The resulting hypertrophic chondrocytes mineralize their surrounding matrix, secrete type X collagen, and attract chondroclasts and osteoclasts through conducting vessels. Finally, the hypertrophic chondrocytes undergo apoptotic cell death, and the remaining matrix provides a scaffold for osteoblasts to form cancellous bone.


Figure 1
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  		Figure 1  Down-regulation of Akt signaling in the terminal differentiation of chondrocytes. (A) Scheme of chondrogenesis in mouse embryonic forelimbs. Resting chondrocytes are differentiated from mesenchymal precursor cells during endochondral bone development. Chondrocytes undergo multi-step differentiation processes to produce the terminally differentiated hypertrophic chondrocytes via proliferative and pre-hypertrophic phases. (B) Expression of EGFP in Akt-Mer transgenic mice. Akt-Mer transgenic mice at E14.5 showed strong EGFP fluorescence in their whole bodies and in cartilage chondrocytes. (C) Akt signaling activation in chondrogenesis. The forelimbs of wild-type and Akt-Mer transgenic mice were cultured in 4OHT and immunodetected with anti-phospho (Ser473)-Akt antibody (red). In wild-type mice, strong phospho-Akt signals were detected in resting and proliferative chondrocytes, but the signals gradually decreased in pre-hypertrophic and hypertrophic chondrocytes (upper panels). In contrast, significantly stronger signals were observed in all of the differentiation stages of chondrocytes of Akt-Mer transgenic mice (lower panels). Nuclei were counterstained with DAPI (blue).

 
Terminal chondrocyte differentiation is regulated by a transcription factor, Runx2. The expression of Runx2 dramatically increases in prehypertrophic chondrocytes and is maintained during hypertrophy (Enomoto et al. 2000). Hypertrophic chondrocyte genes such as Col10a1 and MMP-13 are under the control of Runx2 (Jimenez et al. 1999; Zheng et al. 2003). Runx2-deficient mice exhibit slower chondrocyte differentiation and lack of osteoblast differentiation (Komori et al. 1997; Otto et al. 1997; Inada et al. 1999; Kim et al. 1999). Furthermore, the enforced expression of Runx2 induces precocious chondrocyte maturation, whereas the dominant negative form of Runx2 inhibits chondrocyte terminal differentiation (Enomoto et al. 2000; Ueta et al. 2001). In light of these observations, it has been proposed that Runx2 is a critical transcriptional regulator for terminal chondrocyte differentiation. Meanwhile, the parathyroid hormone-related protein (PTHrP)/Indian hedgehog (Ihh) feedback loop has also been reported to control differentiation from proliferative to hypertrophic chondrocytes (Vortkamp et al. 1996). However, other than the roles of Runx2 and the PTHrP/Ihh feedback loop, the molecular basis of the control of chondrocyte terminal differentiation remains elusive.

Mesenchymal progenitor cells are cells that differentiate into various tissues, such as bone, cartilage, and adipose tissues (Pittenger et al. 1999). Although mesenchymal progenitor cells were first identified in bone marrow, they have also been identified in adipose tissue (Zuk et al. 2001), muscle (Jankowski et al. 2002), tendon (Salingcarnboriboon et al. 2003), and synovium (De Bari et al. 2001). Synovium-derived stromal cells (SSCs) have a higher chondrogenic potency than mesenchymal progenitor cells derived from bone marrow and adipose tissue (Sakaguchi et al. 2005). In fact, human SSCs (hSSCs) migrate into articular cartilage lesions, where they differentiate into chondrocyte progenitor cells (Hunziker & Rosenberg 1996). Considering that a sufficient amount of hSSCs can easily be obtained from knee joints without donor-site morbidity (Tateishi et al. 2007), hSSCs could be a promising source of cell-based cartilage repair.

Phosphoinositide-3 kinase (PI3K)/Akt signaling may regulate cell proliferation, growth, migration, death, adhesion, and tumorigenesis in various cell lineages (Cantley 2002). Akt, a serine–threonine kinase, is activated by phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3), which is generated from PtdIns(4,5)P2 by PI3K (Brazil et al. 2004). In this study, we examined the function of PI3K/Akt signaling in chondrogenesis (during endochondral bone formation) and chondrogenic differentiation in hSSCs. Activation of Akt inhibited the terminal differentiation of proliferative chondrocytes into hypertrophic chondrocytes in two independent experimental systems of chondrogenesis. In contrast, the transition from proliferation to hypertrophy was promoted by the inhibition of PI3K/Akt signaling. Thus, our results offer novel insights into the function of PI3K/Akt signaling in chondrocyte differentiation.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Down-regulation of Akt activation in terminally differentiated chondrocytes

We first examined the effects of Akt signaling activation on chondrocyte differentiation. We used transgenic mice expressing an Akt-Mer fusion protein (Murayama et al. 2007), a synthetic protein composed of a myristoylated constitutively active form of Akt and the ligand-binding domain of a mutant estrogen receptor (Mer) (Kohn et al. 1998). The kinase activity of Akt-Mer can be conditionally controlled because it is inactivated in the absence of 4-hydroxytamoxifen (4OHT) and activated in the presence of 4OHT (Watanabe et al. 2006). As Mer is insensitive to endogenous estrogen (Littlewood et al. 1995), Akt-Mer constitutes a valuable system to control this signal at the organ and tissue levels.

Because the Akt-Mer cDNA is ligated to a cDNA encoding an internal ribosomal entry site-enhanced green fluorescent protein (IRES-EGFP), the expression of Akt-Mer can be monitored using EGFP fluorescence. Akt-Mer transgenic mice at embryonic day 14.5 (E14.5) showed strong EGFP fluorescence in cartilage rudiments (Fig. 1B). Immunohistochemistry using an anti-phosphorylated Akt antibody revealed strong signals in the resting and proliferative chondrocytes of wild-type cartilage rudiments (Fig. 1C). As differentiation proceeded, the signals gradually decreased, and only weak signals could be detected in the hypertrophic zone. In contrast, significantly stronger signals were observed in all of the chondrocyte differentiation stages in Akt-Mer transgenic mice treated with 4OHT (Fig. 1C).

Inhibition of chondrocyte terminal differentiation by Akt

To investigate the role of PI3K/Akt signaling in chondrocyte differentiation, we used a mouse embryonic forelimb explant culture system. This system allowed us to cultivate cartilage rudiments free of the effects of systemic hormonal or neuronal stimulation that could influence cartilage metabolism during the endochondral ossification process, and to analyze the effects of any cytokines or chemicals. Radii were isolated from each E14.5 Akt-Mer transgenic mouse embryo and a wild-type littermate control and cultured for 5 days in 4OHT. There were no detectable differences between the groups of mice at the beginning of the culture (Fig. 2A). During the culture, however, bone rudiment elongation was significantly enhanced in Akt-Mer transgenic mice (Fig. 2A,B). At day 5 of the culture, two opaque zones that corresponded macroscopically to hypertrophic zones became apparent on both sides of the ossificated zones in wild-type rudiments (arrowheads in Fig. 2A). In contrast, no opaque zones were observed in the transgenic rudiments, showing that hypertrophic differentiation was impaired in 4OHT-treated transgenic chondrocytes.


Figure 2
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  		Figure 2  Promotion of bone-rudiment elongation and inhibition of terminal differentiation of embryonic chondrocytes by Akt signaling. (A) Macroscopic photos of cultured bone rudiments. Bone rudiments of E14.5 wild-type and Akt-Mer transgenic mice were cultured in 5 µM 4OHT. No significant differences were observed between the radii of the two groups of mice at day 0. At day 3 of the culture, bone rudiment elongation began to be more pronounced in Akt-Mer transgenic mice. At day 5, two opaque zones corresponding to hypertrophic zones became visible in wild-type mice but not in Akt-Mer transgenic mice (arrowheads in the lower panels). The ossification zones are marked with brackets. (B) Lengths of bone rudiments in culture. Bones were significantly longer in the 4OHT-treated Akt-Mer transgenic mice at days 3 and 5 of the culture (*P < 0.01 by Student's t-test). Data are shown as mean ± SD of six samples. (C) Histology of radius sections stained with Alcian blue. In Akt-Mer transgenic mice, the proliferation zone was elongated but the numbers of pre-hypertrophic and hypertrophic chondrocytes were dramatically lower. The nuclei were counterstained with Kernechtrot. Bar = 0.2 mm. (D) Expression of the Runx2 mRNA. Runx2 expression was examined by in-situ hybridization using an antisense Runx2 probe. Runx2-positive cells were detected in pre-hypertrophic and hypertrophic zones of wild-type mice, but not in Akt-Mer transgenic mice. Bars = 0.4 mm. (E) Immunostaining with an antibody against Type X collagen. Type X collagen was deposited in the hypertrophic zone of wild-type mice but not in the bone rudiments of Akt-Mer transgenic mice. Bars = 0.2 mm.

 
Morphological analysis of sections stained with Alcian blue revealed that the proliferative zone was elongated; only a few hypertrophic chondrocytes were detectable in 4OHT-treated transgenic bone rudiments (Fig. 2C). These phenotypes were not observed in untreated transgenic rudiments (data not shown). Furthermore, Akt activation suppressed the expression of hypertrophic chondrocyte markers such as Runx2 and type X collagen (Fig. 2D,E). These results indicate that Akt activation induced the expansion of proliferative chondrocytes and inhibited terminal differentiation into hypertrophic chondrocytes.

Promotion of chondrocyte terminal differentiation by PI3K signaling inhibition

Akt signaling was activated in proliferative chondrocytes but down-modulated during the course of terminal differentiation (Fig. 1C). To determine the physiological relevance of endogenous PI3K/Akt signaling in chondrogenesis, we cultured wild-type forelimbs with a PI3K inhibitor, LY294002, or the vehicle DMSO. Bone rudiment elongation was significantly suppressed by treatment with LY294002 in a dose-dependent manner (Fig. 3A). Macroscopic analysis showed that the opaque zones corresponding to hypertrophic zones were more prominent in LY294002-treated bones than in DMSO-treated bones (Fig. 3B). Immunohistochemical analysis revealed that the deposition of type X collagen increased in rudiments treated with LY294002 (Fig. 3C). These results indicate that terminal chondrocyte differentiation was accelerated by the inhibition of PI3K signaling. These results, together with those of the Akt-Mer transgenic mice studies, suggest that PI3K/Akt signaling regulates the transition from proliferative to hypertrophic chondrocytes.


Figure 3
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  		Figure 3  Promotion of chondrocyte differentiation by a PI3K inhibitor. (A) Effects of a PI3K inhibitor on bone-rudiment elongation. Bone rudiments of wild-type mice at E14.5 were cultured for 5 days in various concentrations of LY294002. Bone-rudiment elongation was suppressed by LY294002 treatment in a dose-dependent manner (*P < 0.05, **P < 0.01 by Student's t-test). Means ± SD of six samples are as follows; 0.82 ± 0.04 (0 µM), 0.77 ± 0.05 (0.5 µM), 0.71 ± 0.03 (10 µM), 0.62 ± 0.11 (50 µM), 0.53 ± 0.02 (100 µM) for day 3; 0.96 ± 0.07 (0 µM), 0.78 ± 0.04 (0.5 µM), 0.69 ± 0.02 (10 µM), 0.63 ± 0.05 (50 µM), 0.43 ± 0.07 (100 µM) for day 5. (B) Macroscopic photographs of bone rudiments cultured in PI3K inhibitor. Bone rudiments were cultured in 1 µM LY294002 or DMSO. The opaque zones corresponding to hypertrophic zones were more prominent in LY294002-treated wild-type bones (arrowheads). The ossification zones are marked with brackets. (C) Immunostaining with an antibody against Type X collagen. Type X collagen deposition increased in the hypertrophic zones of bone rudiments treated with LY294002. Bars = 0.2 mm.

 
Inhibition of chondrocyte terminal differentiation of human SSCs by activation of Akt signaling

In contrast to embryonic chondrogenesis during endochondral bone formation, chondrogenesis in adult tissues may originate in mesenchymal progenitor cells. To assess the function of PI3K/Akt in adult chondrogenesis, hSSCs were infected with lentivirus expressing Akt-Mer and differentiated into chondrocytes through a cartilaginous pellet culture procedure (Johnstone et al. 1998). The Akt-Mer-transducted hSSCs and control hSSCs were aggregated by centrifugation and cultured for 21 days in TGF-β1. The pellets were significantly larger in 4OHT-treated Akt-Mer-transduced hSSCs than the others (Fig. 4A,B). The Akt-Mer-transduced pellets were larger than those from control hSSCs even in the absence of 4OHT, presumably due to the weak and leaky activation of Akt-Mer protein even in the absence of 4OHT, as evidenced by anti-phospho-Akt staining (Fig. 4A).


Figure 4
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  		Figure 4  Effects of Akt activation on chondrogenic differentiation in hSSCs. (A) Chondrogenic differentiation of Akt-Mer-transducted hSSCs. hSSCs were infected with Akt-Mer-expressing lentivirus or control lentivirus. Chondrogenic differentiation was subsequently induced using a pellet procedure as described in the text. The cultures were incubated for 21 days in the presence or absence of 4OHT. Pellet sections were stained with anti-phospho-Akt antibody (lower panels). Strong phospho-Akt signals were observed in Akt-Mer-transduced hSSCs cultured in 4OHT. Weak and leaky phospho-Akt signals were also detected in Akt-Mer-transduced pellets cultured without 4OHT. (B) Pellet size. The 4OHT-treated Akt-Mer-transduced pellets were significantly larger than the controls (*P < 0.01 by Student's t-test). Even without 4OHT treatment, the Akt-Mer-transduced pellets were significantly larger than the control virus-transduced pellets (**P < 0.05 by Student's t-test). The data shown are means ± SD of nine samples. (C) Sections stained with Alcian blue. The stained areas were enlarged in 4OHT-treated Akt-Mer-transduced pellets (upper panels; bar, 500 µm), and their cells were larger and had a round chondrocytic morphology and cartilaginous lacuna-like cavities. The cells of the other pellets had a fibroblastic phenotype (middle panels; bar, 50 µm; lower panels; bar, 7.5 µm). Nuclei were counterstained with Kernechtrot. (D) GAG levels in pellets. The 4OHT-treated Akt-Mer-transduced pellets produced higher amounts of GAG than control pellets (**P < 0.05 by Student's t-test). n.s., not significantly different. The data shown are means ± SD of three samples. (E) Semi-quantitative RT-PCR analysis. Akt activation did not affect the expression of the early chondrogenic markers Sox9, Col2a1, or Aggrecan, but suppressed the expression of the terminal chondrogenic markers Runx2 and Col10a1.

 
The area stained with Alcian blue was significantly enlarged by Akt activation (upper panels of Fig. 4C). The 4OHT-treated Akt-Mer-transducted cells had round chondrocytic morphology, whereas the others were reminiscent of a fibroblastic phenotype (middle and bottom panels of Fig. 4C). Cartilaginous lacuna-like cavities were also induced by Akt activation (middle and bottom panels of Fig. 4C), and glycosaminoglycan (GAG) levels were higher in Akt-activated pellets (Fig. 4D). Semi-quantitative RT-PCR analysis revealed no obvious differences in the mRNA levels of the early chondrogenic markers Sox9, Col2a1, and Aggrecan in Akt-activated cells and the other cells (Fig. 4E). However, the expression of the terminal chondrogenic marker genes Runx2 and Col10a1 in Akt-activated pellets was lower and undetectable, respectively (Fig. 4E). These results show that the hyperactivation of Akt signaling inhibited terminal differentiation in chondrogenesis from multi-potent hSSCs, which is essentially the same result as that obtained in the previous analysis of Akt-Mer transgenic mice.

Next, we added the PI3K inhibitor LY294002 to a pellet culture of parental hSSCs. The pellets were significantly smaller (Fig. 5A,B) and the area stained in Alcian blue was lower in pellets treated with LY294002 (Fig. 5C). Decreased pellet size and inhibited chondrocytic differentiation were similarly observed in cultures treated with the Akt inhibitor NL-71–101 (Fig. S1 in Supplementary Material). RT-PCR analysis showed that the inhibition of PI3K decreased the expression of the early chondrocyte markers Sox9, Col2a1, and Aggrecan but increased the expression of the terminal differentiation markers Runx2 and Col10a1 (Fig. 5D). Thus, endogenous activation of PI3K/Akt negatively regulates the transition from the proliferative to the hypertrophic stage, not only in embryonic chondrogenesis but also in adult chondrogenesis in hSSCs.


Figure 5
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  		Figure 5  Promotion of terminal differentiation of chondrocytes in hSSCs using a PI3K inhibitor. (A) Pellets induced by PI3K inhibition. Chondrogenic differentiation was induced in hSSCs using a pellet procedure. At day 7 of the culture, 1 µM LY294002 or DMSO was added. The pellet cultures were then incubated for additional 14 days. (B) Pellet size. LY294002-treated pellets were significantly smaller than DMSO-treated controls (*P < 0.01 by Student's t-test). The data shown are means ± SD of three samples. (C) Sections stained with Alcian blue. Stained areas were smaller in LY294002-treated pellets (upper panels, bar, 500 µm; lower panels, bar, 200 µm). Nuclei were counterstained with Kernechtrot. (D) Semi-quantitative RT-PCR analysis. Akt activation decreased the expression of the early chondrogenic markers Sox9, Col2a1, and Aggrecan, but enhanced the expression of the terminal chondrogenic markers Runx2 and Col10a1.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We have identified a novel function of PI3K/Akt signaling in chondrogenesis using two experimental systems: an ex vivo mouse embryonic forelimb culture and a system for the in vitro differentiation of human mesenchymal progenitor cells, hSSCs. In both analyses, Akt signaling activation induced the expansion of proliferative chondrocytes and inhibited terminal differentiation into hypertrophic chondrocytes. In contrast, inhibition of PI3K/Akt signaling accelerated terminal differentiation. These findings clearly demonstrate that terminal differentiation is negatively regulated by PI3K/Akt signaling in embryonic and adult chondrogenesis.

Several studies have reported that PI3K/Akt signaling exhibits pleiotropic functions in chondrogenesis. Analyses using prechondrogenic ATDC5 cells have shown that PI3K/Akt signaling promotes the differentiation of chondrocyte precursors into early-stage chondrocytes. A constitutively active form of Akt accelerates the insulin-dependent chondrogenic differentiation of ATDC5 cells (Hidaka et al. 2001). Conversely, PI3K inhibitor attenuates the expression of the early chondrocyte differentiation marker Col2a1 and the production of proteoglycan in ATDC5 cells (Fujita et al. 2004b). Meanwhile, PI3K/Akt signaling is required for the proliferation of chondrocyte cell lines and the production of sulfated GAG in human primary articular chondrocytes (Oh & Chun 2003; Starkman et al. 2005; Priore et al. 2006; Qureshi et al. 2007). Our results are also in accordance with the notion that proliferation and cartilage matrix synthesis are augmented by PI3K/Akt signaling during the proliferative differentiation stage. Thus, PI3K/Akt promotes chondrogenic commitment and preserves the chondrogenic phenotype and function in the early differentiation stages.

PI3K/Akt signaling may also promote terminal chondrocyte differentiation in cooperation with Runx2 (Fujita et al. 2004a). Although the overexpression of Runx2 induces Col10a1 expression in ATDC5 cells, this Col10a1 induction is completely abrogated by the expression of a dominant-negative Akt. Furthermore, the levels of Akt and the PI3K subunits p85 and p110β are upregulated by Runx2 overexpression, whereas PI3K/Akt signaling enhances the DNA-binding ability of Runx2. These observations collectively suggest that PI3K/Akt signaling and Runx2 constitute a positive feedback loop to accomplish terminal differentiation in Runx2-expressing hypertrophic chondrocytes. However, the role of PI3K/Akt signaling in the transition from the proliferative stage to terminal differentiation remained unclear, because hypertrophic differentiation was artificially induced by overexpressing Runx2 in the study of Fujita et al. (Fujita et al. 2004a).

To circumvent this problem, we used a forelimb organ culture in which chondrogenesis transitions to hypertrophy as it does in vivo (Serra et al. 1999). Combining this organ culture and a conditional Akt activation system (Murayama et al. 2007) provided compelling evidence that PI3K/Akt signaling inhibits the transition from proliferative to hypertrophic differentiation during endochondral ossification. In addition, we showed that PI3K/Akt signaling activation causes essentially the same phenomenon in chondrogenic differentiation in hSSCs. These results, together with those of Fujita (Fujita et al. 2004a), suggest that PI3K/Akt signaling exerts completely opposite influences on two aspects of terminal differentiation. Namely, it inhibits the transition from the proliferative stage to terminal differentiation, but once chondrocytes enter into the hypertrophic differentiation stage, it drives terminal differentiation to completion.

Hypoxia occurs in the interior of cartilaginous tissues during endochondral bone formation (Schipani et al. 2001). Culturing embryonic forelimbs and mesenchymal C3H10T1/2 cells under low oxygen levels leads to phenotypes that are similar to those induced by Akt activation (Hirao et al. 2006). Hypoxic treatment promotes chondrocytic commitment, increases cartilaginous matrix synthesis, and suppresses terminal chondrocyte differentiation. Thus, the transition to terminal differentiation is regulated by at least two extracellular environmental factors: growth factors and oxygen supply.

The hypoxia-induced inhibition of terminal differentiation is presumably caused by reduced Runx2 expression, which could result from the suppression of Smad signaling and the activation of histone deacetylase 4 (HDAC4) (Hirao et al. 2006). We demonstrated that Runx2 expression is decreased by Akt activation but increased by PI3K inhibition, which raises the possibility that Runx2 is repressed at the transcriptional level by PI3K/Akt signaling. Because hypoxia-inducible factor 1 (HIF-1) is a transcription factor that is activated by both hypoxia and PI3K/Akt signaling (Bardos & Ashcroft 2004), it is possible that HIF-1 is involved in the regulation of Runx2 in chondrogenesis. Alternatively, other downstream Akt transcription factors such as FOXO might play a role. Future studies should resolve the regulation of Runx2 expression by PI3K/Akt signaling and hypoxia.

Cellular differentiation in various stem-cell systems is controlled by PI3K/Akt signaling. For instance, the pluripotent differentiation capacities of embryonic stem (ES) cells are prohibited by PI3K/Akt signaling activation (Paling et al. 2004; Ivanova et al. 2006; Watanabe et al. 2006). Enhancing PI3K/Akt activation causes the dedifferentiation of primordial germ cells into pluripotent embryonic germ (EG) cells in vitro and into testicular teratoma in vivo (Kimura et al. 2003, 2008). In addition, PI3K/Akt activation induces the expansion of stem/progenitor cell populations in epithelial stem cell systems (He et al. 2007; Murayama et al. 2007). Because these phenomena induced by PI3K/Akt signaling activation are accompanied by enhanced proliferation, it is likely that PI3K/Akt signaling inhibits cellular differentiation via mitotic activation in these stem-cell systems. Our results suggest that this is also the case with chondrogenesis, because PI3K/Akt signaling activation expanded proliferative chondrocytes but inhibited the differentiation into mitotically quiescent terminally differentiated cells.

In conclusion, we provide evidence that each step of chondrogenesis is regulated both positively and negatively by PI3K/Akt signaling, depending on the differentiation stage of the chondrocytes. Even if PI3K/Akt signaling is overactivated, this mechanism could prevent the exhaustion of immature precursors and the overproduction of terminally differentiated cells. Conversely, when this signaling is down-modulated, the mechanism could ensure that precursors enter into terminal differentiation. This kind of Yin Yang regulation may serve as a safeguard mechanism to the vulnerabilities inherent to unidirectional differentiation systems.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Organ culture of embryonic limb explants

Male Akt-Mer transgenic mice were crossed with female BDF1 mice (Charles River, Osaka, Japan) (Murayama et al. 2007). The forelimbs of E14.5 embryos of Akt-Mer transgenic mice and wild-type littermates were stripped of their skin and muscle and the radii removed. The radii were cultured for 5 days at 37 °C in a humidified 5% CO2 incubator in 24-well plates in alpha-MEM (Gibco BRL, Rockville, MD) supplemented with 0.05 ng/mL ascorbic acid, 0.3 mg/mL L-glutamine, 1% penicillin/streptomycin, 1 mM β-glycerophosphate, and 0.2% bovine serum albumin (Serra et al. 1999). Cultures of Akt-Mer transgenic and wild-type rudiments were supplemented with 5 µM 4-hyroxytamoxifen (4OHT; Sigma, St Louis, MO) or 0.5–100 µM LY294002 (Calbiochem, San Diego, CA). Photographs of cultured bones were taken with a Leica MZ 16 F (Leica Microsystems, Wetzlar, Germany), and bone length was measured using Leica IM500 software.

In vitro chondrogenesis of hSSCs and transduction with lentivirus vectors

hSSCs were isolated from the synovium of the human adult knee joint during arthroscopic surgery in accordance with a protocol approved by the Osaka University Institutional Ethics Committee (Tateishi et al. 2007). They were then cultured in high-glucose Dulbecco's modified Eagle's medium (HG-DMEM; Gibco BRL) containing 10% feral bovine serum and 1% penicillin/streptomycin. The cDNA encoding Akt-Mer was inserted into lentivirus vector pWPI. Subconfluent 293T cells were co-transfected with pWPI-Akt-Mer, pCMV-{Delta}R8.91, and pMD2G-VSVG using FuGENE 6 transfection reagent (Roche Diagnostics, Basel, Switzerland). After 24 h, the medium was changed, and recombinant lentivirus was harvested 36 h later (Wiznerowicz & Trono 2003). The hSSCs of passage three were infected with the lentivirus, and the cells of passage five were used for chondrogenic differentiation. A total of 2 x 105 cells were placed in a 15-mL polypropylene tube and centrifuged at 500 g for 5 min. The pellets were cultured for 14 or 21 days in chondrogenic medium of HG-DMEM supplemented with 10 ng/mL TGFβ1 (R&D Systems, Minneapolis, MN), 50 µg/mL ascorbic acid, 100 µg/mL sodium pyruvate, 100 nM dexamethasone, and 50 mg/mL each of insulin, transferrin, and selenious acid (ITS Premix; BD Biosciences, San Jose, CA). The agents 4OHT, LY294002, and NL71-101 (Calbiochem) were used at 1 µM. The cross-sectional area of the pellets was measured using Leica IM500 software.

Histological analysis

Cultured limb explants and pellets were fixed overnight in 4% paraformaldehyde at 4 °C, embedded in methyl methacrylate, and cut into 5-µm sections. The sections were stained for 2 h in 1% Alcian blue (Alcian Blue 8GX; Sigma) and subsequently counterstained with Kernechtrot.

Quantification of GAG levels

The pellets were digested for 4 h at 65 °C with a Papain solution (Sigma) and assayed for GAG as a measurement of proteoglycan content. GAG levels were measured using the 1,9-demethylmethylene blue binding assay on the basis of a chondroitin sulfate standard curve (Nacalai Tesque, Kyoto, Japan) (Karran et al. 1995; Tateishi et al. 2007).

Antibodies

Antibodies against phospho (Ser473)-Akt, and type X collagen were purchased from Cell Signaling Technology (Danvers, MA), and LSL (Cosmo Bio, Tokyo, Japan), respectively.

In situ hybridization

Digoxigenin-11-UTP-labeled single-stranded RNA probes were prepared using a DIG RNA Labeling Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. Hybridization was performed as described previously (Hirakawa et al. 1994; Kawahata et al. 2003).

Semi-quantitative reverse transcription PCR analysis

Total RNA was extracted using Sepasol(R)-RNA 1 Super (Nacalai Tesque), and 1 µg of total RNA was used for cDNA synthesis. First-strand cDNA was synthesized using the ThermoScript RT-PCR System (Gibco BRL). PCR reactions were optimized to allow semi-quantitative comparisons within the log phase of amplification. The primer sequences and amplification cycles are listed in Supplementary Table S1 (Girotto et al. 2003; Ikeda et al. 2005). The PCR products were analyzed by agarose gel electrophoresis and visualized using ethidium-bromide staining.


   Acknowledgements
 
This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the 21st Century COE "CICET."


   Footnotes
 
Communicated by: Tetsuya Taga

* Correspondence: tkimura{at}patho.med.osaka-u.ac.jp; tnakano{at}patho.med.osaka-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 11 May 2008





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