A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation
Elena Kozhemyakina1, Andrew B. Lassar1,* and Elazar Zelzer2,*
Decades of work have identified the signaling pathways that regulate the differentiation of chondrocytes during bone formation, from their initial induction from mesenchymal progenitor cells to their terminal maturation into hypertrophic chondrocytes. Here, we review how multiple signaling molecules, mechanical signals and morphological cell features are integrated to activate a set of key transcription factors that determine and regulate the genetic program that induces chondrogenesis and chondrocyte differentiation. Moreover, we describe recent findings regarding the roles of several signaling pathways in modulating the proliferation and maturation of chondrocytes in the growth plate, which is the ‘engine’ of bone elongation.
KEY WORDS: Chondrogenesis, Chondrocyte hypertrophy, Growth plate, Sox9, Ihh, PTHrP, Fgfr3
The commitment of mesenchymal cells to the chondrogenic lineage is a key event in the formation of bones. With the exception of the bones in the cranial vault, parts of the jaw and the medial part of the clavicle [which all form via intramembranous ossification (Hall and Miyake, 1992; Ornitz and Marie, 2002)], the vertebrate skeleton is formed by the process of endochondral bone formation.
During this process, each skeletal element is first established from mesesenchymal progenitors and is then patterned as cartilage, which is later replaced by bone (Fig. 1). Mesenchymal progenitors that originate from the cranial neural crest, somites and lateral plate mesoderm contribute to the craniofacial, axial and limb skeleton, respectively. Condensations of such mesenchymal cells express the transcription factor Sox9, which is a key regulator of chondrogenesis, and give rise to cartilage primordia consisting of round immature chondrocytes that continue to express Sox9 (Bi et al., 1999; Akiyama et al., 2002). Cells lying within the central regions of the cartilage primordia then undergo maturation (Fig. 1). During this process, chondrocytes withdraw from the cell cycle and increase ∼20-fold in volume (Cooper et al., 2013), giving rise to cells that are termed hypertrophic chondrocytes. As the cartilage continues to grow longitudinally, it continually deposits hypertrophic chondrocytes in its wake. These are subsequently replaced by bone in a region known as the primary ossification center. The production andmaturation of chondrocytes is eventually restricted to the end of the bone (the epiphysis) in a structure termed the growth plate. A secondary ossification center then arises within the epiphysis, separating the growth plate from the distal ends of the long bones.
During the process of endochondral bone formation, the cellular features and expression profiles of chondrocytes progressively change (Fig. 2). Within the growth plate, small round distally located chondrocytes initially give rise to flattened chondrocytes that are more centrally located in the cartilage primordia, and which proliferate and stack into longitudinal columns. These immature chondrocytes express the transcription factors Sox5,
Sox6 and Sox9, and the structural proteins collagen, type II, α1 (Col2a1) and aggrecan (Acan). The next stage of maturation into prehypertrophic chondrocytes is marked by the expression of both parathyroid hormone 1 receptor (Pth1r) and Indian hedgehog (Ihh). This is followed by maturation into early hypertrophic chondrocytes that express collagen, type X, α1 (Col10a1). Notably the induction of Pth1r, Ihh and, subsequently, Col10a1 correlates with the loss of Sox5, Sox6, Sox9, Col2a1 and Acan expression.
Finally, Col10a1-expressing cells lose expression of this collagen and progress to become late hypertrophic chondrocytes, which express vascular endothelial growth factor A (VEGFA), matrix metalloproteinase 13 (Mmp13) and secreted phosphoprotein 1 (also known as, osteopontin/bone sialoprotein 1; Spp1). VEGFA and Mmp13 expression herald the invasion of the growth plate by endothelial cells, osteoclasts and osteoblast precursors. Osteblast precursors that arise from both the perichondrium (Maes et al., 2010) and the hypertrophic chondrocytes (Yang et al., 2014) work together with osteoclasts to remodel the growth plate matrix to form trabecular bone.
Work over the past few decades, using both in vitro and in vivo systems, has identified a multitude of signaling and transcription factors, as well as changes in cell shape (see Box 1), that regulate these progressive changes in chondrocytes, from their initial induction to their terminal maturation. These findings have implications both for understanding the basic biology of cartilage and bone, and for understanding how disruption of this finely tuned process of chondrocyte maturation results in various skeletal pathologies. In this Review, we first describe the signaling pathways and transcription factors that regulate the specification of mesenchymal cells as chondroprogenitors. We then concentrate on the establishment of the growth plate and the factors that regulate the balance between chondrocyte proliferation and differentiation.
The initiation of chondrogenesis
The molecular events that regulate the differentiation of mesenchymal cells into chondrocytes are still largely unknown.
Although some of the signaling molecules that are necessary for the induction of this process have been identified, our understanding of the downstreammolecular pathways that promote chondrogenesis is still a work in progress. Below, we summarize current knowledge of the molecular players that participate in initiating chondrogenesis. 1Department of Biological Chemistry and Molecular Pharmacology, Harvard
Medical School, Building C-Room 305A, 240 Longwood Avenue, Boston, MA 02115, USA. 2Weizmann Institute of Science, Department of Molecular Genetics,