D.O.S (DENEGACIÓN DE SERVICIO)

Although the cartilage is build up of only one cell type, the chondrocyte, different subtypes can be clearly distinguished histologically. In the resting zone, chondrocytes are small and roundish, mainly express collagen II and proliferate slowly, whereas in the proliferative zone, chondrocytes appear flattened and are highly proliferative (Fig 1.15, Fig 1.16). Differentiated pre-hypertrophic and hypertrophic chondrocytes increase their size, express collagen X and cease proliferation. An obvious question is how all these events, the induction of proliferation and differentiation, the synthesis of ECM and the cell shapes changes are regulated. Recent

work has identified several regulatory mechanisms: the Indian hedgehog (Ihh)-Parathyroid hormone-related peptide (PTHrP) crosstalk, growth factor and transcriptions factor signalling pathways. Interestingly, integrins were also shown to play an important role during most of these processes.

Fig 1.16. Organization of epiphyseal cartilage. Hematoxylin/Eosin staining of a cartilage section at E17.5. Cells in the resting zone are roundish, while cells in the proliferative zone appear flattened and form columnar structures. Pre-hypertrophic and hypertrophic chondrocyte are much larger. The cartilage is surrounded by a mesenchymal cell layer called perichondrium.

1.5.2.1. Ihh-PTHrP crosstalk

Targeted inactivation of PTHrP in mice leads to premature chondrocyte maturation and excessive bone formation at birth (Karaplis et al. 1994). Conversely, transgenic mice overexpressing PTHrP (using a Col2-promoter) fail to form bone in all skeletal elements which are formed by endochondral ossification (Weir et al. 1996). PTHrP is mainly secreted by cells at the periarticular cartilage, while the receptor for PTHrP (PPR) is expressed at lower levels in proliferating chondrocytes and is highly expressed in pre-hypertrophic chondrocytes. Therefore, it has been proposed that PTHrP diffuses through the bone to bind its receptor, which then antagonizes chondrocyte maturation. A somewhat similar but even more complex phenotype is caused by deletion of Ihh, which is at least at later time points of

null mice initially show a normal chondrocyte condensation, mice at the newborn stage display a prominent dwarfism characterized by increased calcification of the long bones and shortening of almost all skeletal elements. Due to a strongly reduced rib cage size, Ihh knockout mice can not breathe and die shortly after birth. The reduced size of the long bones in Ihh knockout mice is caused by impaired proliferation of chondrocytes in the growth plate. Interestingly, the expression of PTHrP in periarticular chondrocytes was absent in these animals indicating that Ihh is essential for the maintenance of PTHrP expression thereby controlling the transition from proliferating to hypertrophic chondrocytes (St-Jacques et al. 1999). But how can Ihh, expressed on pre-hypertrophic chondrocytes affect the secretion of PTHrP in periarticular chondrocytes?

One possibility could be that Ihh triggers PTHrP expression in a direct manner early during endochondral bone formation, when the distance between Ihh and PTHrP expressing cells is still small. At later time points is seems more reasonable that the regulation of PTHrP secretion by Ihh occurs in an indirect manner. It has been suggested that this indirect regulation depends on bone morphogenic proteins (BMPs) and the transforming growth factor beta (TGF-β). More detailed information can be found in recent reviews about the Ihh-PTHrP feed-back loop (Lai and Mitchell 2005).

1.5.2.2. Regulation of endochondral bone formation by growth factor signalling and transcription factors

Endochondral bone formation critically depends on growth factor receptor signalling. Activating mutations in the fibroblast growth factor receptor-3 (FGFR-3) leads to achondroplasia, characterized by a virtual absence of non-hypertrophic chondrocytes. Conversely, targeted inactivation of FGFR-3 in mice leads to an increased size of the growth plate and a prolonged growth of the axial and appendicular skeleton (Deng et al. 1996). Therefore, FGFR-3 is believed to act as a negative regulator of endochondral bone formation. Several studies have indicated that the transcription factor STAT1 is a mediator of FGF signalling by regulating the expression of cell cycle inhibitors like p21 in the growth plate. Indeed, FGFR-3 knockout mice display more proliferating chondrocyte in this area of the cartilage.

Other transcription factors like Sox9, Fos and Cbfa1 have been shown to play essential roles during endochondral bone formation. Sox9 is expressed throughout cartilage during development. In ES cell-chimeric mice, Sox9 null ES cells cannot contribute to cartilaginous tissues indicating that Sox9 is an essential factor for the initial condensation of the cartilaginous template (Bi et al. 1999). Since Sox9 can directly interact with enhancers of

collagen II, it is easy to envisage that Sox9 is crucial for the onset of collagen II expression in chondrocytes.

The important role of osteoblasts during endochondral bone formation has become evident by the generation of Cbfa1 knockout mice. Targeted deletion of this transcription factor, which is essential for the differentiation of osteoblasts leads to complete loss of bone formation in mice (Komori et al. 1997).

Fig 1.17. Regulatory signals and circuits during endochondral bone formation. A. The PTHrP-Ihh crosstalk regulates chondrocyte proliferation and differentiation. Ihh is expressed on pre-hypertrophic chondrocytes and induces the secretion of PTHrP. Activation of the PTHrP receptor (PPR), located at the pre-hypertrophic and hypertrophic zone, inhibits differentiation of chondrocytes. B. In chondrocytes, activation of the FGFR-3 pathway leads to inhibition of cell proliferation by STAT-dependent expression of cell cycle inhibitors. Several transcription factors affect endochondral bone formation by the regulation of ECM synthesis (Sox9), cell proliferation (Fos) or osteoblast differentiation (Cbfa1).