1.3.1 Differentiation of mesenchymal stromal cells (MSC)
A third link between osteoblasts and adipocytes exists: they both differentiate from a common mesenchymal progenitor, the mesenchymal stromal or stem cell (MSC). In fact, this MSC is the common mesenchymal progenitor for all other mesenchymal cell lineages such as
chondrocytes, fibroblasts and myoblasts (Caplan, 2007). Mesenchymal cell fate commitment and differentiation towards the various lineages are driven by key transcription factors that confer identity to the cell. Fig. 1.2 summarizes the major transcription factors regulating MSC differentiation.
Mesenchymal stem/stromal cell
osteoblast chondrocyte fibroblast myoblast C/Ebpββββ
C/Ebpδδδδ
C/Ebpαααα Pparγγγγ2
β-catenin Runx2
Osterix
Sox9 L-Sox5, Sox6
MyoD Myogenin
Mrf4 Myf-5
brown adipocyte white
adipocyte
Sox9
Runx2/3
Prdm16
Figure 1.2: Transcription factors regulating mesenchymal cell fate decision. Mesenchymal stem/stromal cells have the capacity to differentiate into different lineages. The major transcription factors controlling lineage determination are indicated.
1.3.2 Chondrocyte differentiation
The key determinants to regulate chondrocyte differentiation are SOX9, L-SOX5, SOX6 and Runx2. In particular, SOX9 was shown to induce the condensation, proliferation and differentiation of mesenchymal progenitors to chondrocytes and to regulate the chondrogenic expression of collagen II and XI as well as aggrecan (Karsenty, 2008). Two other members of the SOX family, L-SOX5 and SOX6, are cooperating with SOX9, and have an essential function during differentiation and activation of collagen II expression (Lefebvre and Smits, 2005). Hypertrophic differentiation is inhibited by Sox genes, but activated by Runx2 and Runx3 (Hartmann, 2009).
Growth factors relevant for chondrogenesis are the members of the Transforming Growth Factor β super family (TGFβ-1, -2, -3 und BMPs), Fibroblast Growth Factors (FGFs) and
as well as chondrocyte proliferation, differentiation and apoptosis. A central mechanism of regulation is based on the Indian-hedgehog (IHH) / parathyroid-hormone-related-Protein (PTHrP) feedback loop. IHH is expressed by the prehypertrophic chondrocytes and is positively regulating the expression of PTHrP in the cells of the periarticular zone of the bone and the perichondrium, resulting in the formation of a gradient of PTHrP. PTHrP can therefore inhibit hypertrophic differentiation of chondrocytes in the zone of proliferation and the prehypertrophic region (Vortkamp et al., 1996).
1.3.3 Myoblast and brown adipocyte differentiation
Myoblast differentiation requires activation of the regulatory marker genes Myf5 and MyoD, belonging to the myogenic bHLH protein family, myogenin and Mrf4. MyoD and Myf5 that are induced by the Wnt and Shh pathway regulate the commitment of myoblasts. Mrf4 is supposed to positively regulate the early steps of differentiation as well as promoting terminal differentiation while myogenin is necessary for terminal myoblast differentiation (Berkes and Tapscott, 2005; Gilbert et al., 2006; Kassar-Duchossoy et al., 2004; Perry and Rudnick, 2000). Recently it was shown that brown adipocytes are more related to muscle cells than to white adipocytes as they originate from Myf5 expressing precursors (Seale et al., 2008). Cell fate decision between myoblasts and brown adipocytes is proposed to be regulated by the action of PRDM16 in complex with the transcription factor C/EBPβ (Kajimura et al., 2009).
1.3.4 Transcriptional control of adipogenesis
Adipogenesis is the process by which a mesenchymal progenitor cell differentiates via a preadipocyte to a mature adipocyte. The initial step of adipogenesis, called adipocyte determination is supposed to be regulated by BMP-signaling, leading to the commitment of the progenitor cell to the adipose lineage (MacDougald and Mandrup, 2002; Otto and Lane, 2005). The second step of adipogenesis, the terminal differentiation, occurs by activation of the basic leucine zipper protein family members C/EBPβ and C/EBPδ (CCAAT/enhancer binding protein β and δ). These two transcription factors initiate the second step of terminal differentiation by directly inducing the expression of Pparγ2 (peroxisome proliferator activated receptor gamma 2) (Farmer, 2006). PPARγ2, a member of the nuclear hormone receptor superfamily, was described to be the ‘master regulator’ of adipogenesis that is necessary as well as sufficient for adipocyte differentiation as shown by the overexpression of
Pparγ2 that can initiate adipogenic differentiation (Rosen and MacDougald, 2006). The ligand-activated transcription factor PPARγ2 subsequently dimerises with RXRα and induces, together with C/EBPβ and C/EBPδ, the expression of C/ebpα. Finally, C/EBPα and PPARγ2, that control the expression of late markers of adipocyte maturation, regulate expression of each other through a positive feedback loop (Farmer, 2006).
Figure 1.3: Adipocyte differentiation. Initiation of the terminal differentiation phase is regulated by the transcription factors C/ebpβ and C/ebpδ that activate the late markers of adipogenesis, Pparγ2 and C/ebpα.
(Green: pro-adipogenic factors, red: anti-adipogenic factors).
C/EBPα and PPARγ2 regulate the expression of various proteins establishing adipocyte function, for instance of the insulin-controlled glucose transporter Glut4, of fatty-acid-binding protein Ap2 (Fabp4), that is involved in the transport of free fatty acids, as well as of lipolytic and lipogenic genes (Lowe et al., 2011).
Numerous additional factors have been described to regulate adipocyte differentiation. For instance preadipocyte factor 1 (Pref1), a transmembrane protein that is cleaved to generate its active form (Kim et al., 2007). Pref-1 is expressed in pre-adipocytes and has to be repressed during adipocyte differentiation as it inhibits adipogenesis through regulating C/ebpβ and C/ebpδ promoter activities (Lowe et al., 2011). A known positive regulator of adipogenesis is the insulin-regulated sterol regulatory element binding protein SREBP-1c that has been described to increase PPARγ activity and to regulate the generation of Pparγ ligands in vitro as well as to increase the expression of genes regulating fatty acid synthesis (White and Stephens, 2009). Other important factors described to inhibit adipocyte differentiation are two
C/EBPααα α
transcription factors (GATA2 and GATA 3) and the Kruppel-like factor (KLF2) (Lowe et al., 2011). However, other members of the Klf family such as KLF5 and KLF15 have been described to be positive regulators of adipocyte differentiation (Lowe et al., 2011).
In vitro, differentiation to adipocytes can be induced by a cocktail of insulin, dexamethasone and 3-isobutyl-1-methylxanthine (IBMX). In several cell culture systems, the first step is a phase of mitotic clonal expansion, followed by the terminal differentiation phase. The components of the adipogenic cocktail activate specific transcription factors regulating the differentiation process. In particular, IBMX inhibits phosphodiesterases, leading to elevated cAMP levels. As a result, cAMP responsive element binding protein 1 (CREB1) is phosphorylated and is therefore able to induce the expression of C/ebpβ (Berry et al., 2010).
C/ebpβ expression is also induced by serum mitogens via KROX20 (Chen et al., 2005).
C/ebpδ expression is activated by the synthetic glucocorticoid dexamethasone via the inhibition of Pref1 expression. The third component of the adipogenic cocktail, insulin, targets Pparγ via Srebp1c (Farmer, 2006).
1.3.5 Osteoblast differentiation
The major factors regulating differentiation of mesenchymal stromal cells to pre-osteoblasts are β-catenin downstream of the wnt signaling pathway and Runx2 (Cbfa1) (Komori, 2006).
Runt related transcription factor 2, Runx2, was described to be necessary for osteoblastogenesis as mice with a homozygous deletion of Runx2 display a lack of osteoblasts. However, it was suggested that Runx2 also inhibits proliferation and terminal differentiation to osteoblasts, as overexpression of Runx2 in mice leads to a reduced number of mature osteoblasts (Nakashima and de Crombrugghe, 2003). For efficient transcriptional activity, formation of a heterodimeric transcription factor complex with Cbfβ is essential (Nakashima and de Crombrugghe, 2003; Yoshida et al., 2002). Other factors that were described to be involved in early regulation of osteoblast differentiation are ATF4, IHH, IGFs and BMP/TGF-β signalling (Karsenty and Wagner, 2002). Osterix (Osx/Sp7), one of the osteoblastic genes directly activated by Runx2, drives osteoblast maturation. As for Runx2, Osx deficiency in mice was shown to result in a complete lack of bone formation (Zhang, 2010). In vitro, osteoblast differentiation can be induced by stimulation with ascorbic acid and β-glycerophosphate.
1.3.6 Reciprocal regulation of osteoblastogenesis and adipogenesis
Several factors have been described as regulators of cell fate decisions between osteoblasts and adipocytes by promoting commitment or differentiation into one lineage at the expense of the other (Takada et al., 2007b). PPARγ, for instance, a transcription factor that plays a crucial role in adipocyte differentiation, decreases bone formation by repressing the activity of Runx2 and Osx (Muruganandan et al., 2009). One isoform of another key transcription factor regulating adipogenesis, namely of C/EBPβ, activates factors involved in osteoblastogenesis for instance by acting as coactivator of Runx2, thereby regulating bone formation (Hata et al., 2005; Henriquez et al., 2011). Runx2 is also activated by TAZ (Wwtr1), an osteoblastogenic factor of mesenchymal stem cell differentiation, which additionally represses the function of PPARγ (Marie, 2008).
A reciprocal regulation of osteoblastogenesis and adipogenesis has also been reported for the wnt signaling pathway. Activation of the wnt signaling was described to promote osteoblastogenesis, while repressing C/ebpα and Pparγ induction, thereby inhibiting adipogenesis (Muruganandan et al., 2009). Therefore wnt signalling is inhibited during adipogenesis (Bennett et al., 2002). For the canonical wnt signaling pathway, amongst others Wnt1, 3a and 10b were described to negatively regulate adipogenesis and positively osteoblastogenesis (Kang et al., 2007), in regard to the non-canonical wnt pathway, Wnt-5a was reported inhibit PPARγ activity (Takada et al., 2007a).
A positive role in switching osteoblast versus adipocyte differentiation has also been shown for various other factors, for instance Msx2 (Ichida et al., 2004; Qadir et al., 2011), steroids like glucocorticoids (Chen et al., 2007) or the Fos family members of the AP-1 transcription factor.