No abstract

Recent experiments have established that Sox9 is required for chondrocyte differentiation. Here, we show that fibroblast growth factors (FGFs) markedly enhance Sox9 expression in mouse primary chondrocytes as well as in C3H10T1͞2 cells that express low levels of Sox9. FGFs also strongly increase the activity of a Sox9-dependent chondrocyte-specific enhancer in the gene for collagen type II. Transient transfection experiments using constructs encoding FGF receptors strongly suggested that all FGF receptors, FGFR1-R4, can transduce signals that lead to the increase in Sox9 expression. The increase in Sox9 levels induced by FGF2 was inhibited by a specific mitogen-activated protein kinase kinase (MAPKK)͞mitogen-activated protein kinase͞ERK kinase (MEK) inhibitor U0126 in primary chondrocytes. In addition, coexpression of a dual-specificity phosphatase, CL100͞MKP-1, that is able to dephosphorylate and inactivate mitogen-activated protein kinases (MAPKs) inhibited the FGF2-induced increase in activity of the Sox9-dependent enhancer. Furthermore, coexpression of a constitutively active mutant of MEK1 increased the activity of the Sox9-dependent enhancer in primary chondrocytes and C3H10T1͞2 cells, mimicking the effects of FGFs. These results indicate that expression of the gene for the master chondrogenic factor Sox9 is stimulated by FGFs in chondrocytes as well as in undifferentiated mesenchymal cells and strongly suggest that this regulation is mediated by the MAPK pathway. Because Sox9 is essential for chondrocyte differentiation, we propose that FGFs and the MAPK pathway play an important role in chondrogenesis.

The inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-␣ (TNF-␣) strongly inhibit the expression of genes for cartilage extracellular matrix proteins. We have recently obtained genetic evidence indicating that the high mobility group domain containing transcription factor Sox9 is required for cartilage formation and for expression of chondrocyte-specific genes including the gene for type II collagen (Col2a1). We show here that IL-1 and TNF-␣ cause a marked and rapid decrease in the levels of Sox9 mRNA and/or protein in chondrocytes. A role for the transcription factor NFB in Sox9 down-regulation was suggested by the ability of pyrrolidine dithiocarbamate, an inhibitor of the NFB pathway, to block the effects of IL-1 and TNF-␣. This role was further supported by the ability of a dominant-negative mutant of IB␣ to block the IL-1 and TNF-␣ inhibition of Sox9-dependent Col2a1 enhancer elements. Furthermore, forced expression of the NFB subunits p65 or p50 also inhibited Sox9-dependent Col2a1 enhancer. Because Sox9 is essential for chondrogenesis, the marked down-regulation of the Sox9 gene by IL-1 and TNF-␣ in chondrocytes is sufficient to account for the inhibition of the chondrocyte phenotype by these cytokines. The down-regulation of Sox9 may have a crucial role in inhibiting expression of the cartilage phenotype in inflammatory joint diseases.Cartilage is a highly specialized connective tissue with distinct biochemical and biomechanical properties. Its extracellular matrix is composed of a series of proteins such as collagen types II, IX, and XI; link protein; and aggrecan. The coordinated regulation of the genes for these proteins is likely to be essential for normal skeletal development and maintenance of cartilage in postnatal life, as mutations in these molecules lead to chondrodysplasias and degenerative joint diseases (1-5).Sox9 is a transcription factor with a high mobility group DNA-binding domain that is expressed in all prechondrocytic and chondrocytic cells during embryonic development in a pattern that closely parallels that of the gene for type II collagen (Col2a1) (6, 7). In humans, heterozygous mutations in and around the SOX9 gene cause campomelic dysplasia, a disease that is characterized by anomalies in a number of skeletal structures and is also often associated with XY sex reversal (8 -11). The disease is thought to be due to SOX9 haploinsufficiency, i.e. 50% of SOX9 being insufficient to fulfill the physiological function of SOX9. Recent work from our laboratory based on mouse embryo chimeras derived from Sox9 homozygous mutant embryonic stem cells obtained by gene targeting has demonstrated that Sox9 is a master regulatory factor for chondrocyte differentiation. Indeed, in these mouse embryo chimeras, Sox9 Ϫ/Ϫ mutant cells were blocked in their differentiation to become chondrocytes and persisted as mesenchymal cells; these cells were unable to express the genes for chondrocyte-specific markers such as collagen types II, IX, and XI and aggrecan (Col2a1, Col11a2, Col9a2, a...

We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. These observations indicate that the MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. These observations suggest a model in which Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1.

We generated Prx1CreER-GFP transgenic mice that express tamoxifen-inducible Cre recombinase and GFP under the control of a 2.4 kb Prx1 promoter. The transgene is expressed in osteochondro progenitor cells in the developing limb buds and in a subpopulation of periosteal cells that is closely associated with the cortical bone. GFP-expressing cells isolated from the diaphyses of long bones by cell sorting express multiple markers of periosteal cells, including Prx-1, Fgf18, Tenascin-W, Periostin, and Thrombospondin 2. In addition, these cells undergo chondrogenic and osteogenic differentiation in culture upon induction. Cell fate analysis using the Rosa26 LacZ reporter indicated that transgene-expressing cells give rise to some of the chondrocytes and osteoblasts in the fracture callus. Collectively, these observations strongly suggest that the transgene-expressing cells are osteochondro progenitor cells in the periosteum. The established Prx1CreER-GFP mice would offer novel approaches for analyzing the functions of periosteal cells in vitro and in vivo.

Osteoblasts and chondrocytes arise from common osteo-chondroprogenitor cells. We show here that inactivation of ERK1 and ERK2 in osteo-chondroprogenitor cells causes a block in osteoblast differentiation and leads to ectopic chondrogenic differentiation in the bone-forming region in the perichondrium. Furthermore, increased mitogen-activated protein kinase signaling in mesenchymal cells enhances osteoblast differentiation and inhibits chondrocyte differentiation. These observations indicate that extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in the lineage specification of mesenchymal cells. The inactivation of ERK1 and ERK2 resulted in reduced beta-catenin expression, suggesting a role for canonical Wnt signaling in ERK1 and ERK2 regulation of skeletal lineage specification. Furthermore, inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation. Thus, our results indicate that ERK1 and ERK2 not only play essential roles in the lineage specification of osteo-chondroprogenitor cells but also support osteoclast formation in vivo.The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK MAPK) pathway is activated by various stimuli, including a number of growth factors and cytokines. The activation of the Raf members of MAPK kinase kinase leads to the activation of the MAPK kinase, MEK1, and MEK2. MEK1 and MEK2 then phosphorylate and activate MAPK, ERK1, and ERK2. ERK1 and ERK2 phosphorylate various cytoplasmic and nuclear target proteins, ranging from cytoplasmic adaptor proteins and transcription factors to kinases, including ribosomal S6 kinase (RSK) (4,17,18,23,36,46). In this pathway, multiple mutations that cause syndromes with various skeletal manifestations have recently been identified. Missense-activating mutations in KRAS, BRAF, MEK1, and MEK2 have been identified in Costello, Noonan, LEOPARD, and Cardio-facio-cutaneous syndromes, while loss-of-function mutations in RSK2, a kinase downstream of ERK1 and ERK2, cause Coffin-Lowry syndrome (2, 42). These observations highlight the importance of the ERK MAPK pathway in human skeletal development.Both chondrocytes and osteoblasts arise from common osteo-chondro progenitor cells. Bone growth is achieved through two major ossification processes, endochondral ossification and intramembranous ossification, in which chondrocytes and osteoblasts are involved (5,13,30,31,38). In normal endochondral ossification, the skeletal element is formed as a cartilaginous template that is subsequently replaced by bone. Condensed mesenchymal cells differentiate into chondrocytes. Chondrocytes first proliferate in columnar stacks to form the growth plate and then exit the cell cycle and differentiate into hypertrophic chondrocytes. Hypertrophic chondrocytes are removed by apoptotic cell death, and the cartilaginous matrix is resorbed by chondroclasts/osteoclasts and replaced by trabecular bone. Chondroclast/osteoclast formation is supported by receptor activator of nucl...

Activating mutations in FGFR3 cause achondroplasia and thanatophoric dysplasia, the most common human skeletal dysplasias. In these disorders, spinal canal and foramen magnum stenosis can cause serious neurologic complications. Here, we provide evidence that FGFR3 and MAPK signaling in chondrocytes promote synchondrosis closure and fusion of ossification centers. We observed premature synchondrosis closure in the spine and cranial base in human cases of homozygous achondroplasia and thanatophoric dysplasia as well as in mouse models of achondroplasia. In both species, premature synchondrosis closure was associated with increased bone formation. Chondrocyte-specific activation of Fgfr3 in mice induced premature synchondrosis closure and enhanced osteoblast differentiation around synchondroses. FGF signaling in chondrocytes increases Bmp ligand mRNA expression and decreases Bmp antagonist mRNA expression in a MAPK-dependent manner, suggesting a role for Bmp signaling in the increased bone formation. The enhanced bone formation would accelerate the fusion of ossification centers and limit the endochondral bone growth. Spinal canal and foramen magnum stenosis in heterozygous achondroplasia patients, therefore, may occur through premature synchondrosis closure. If this is the case, then any growth-promoting treatment for these complications of achondroplasia must precede the timing of the synchondrosis closure.

Accumulating in vitro evidence suggests that the p38 mitogenactivated protein kinase (MAPK) pathway is involved in endochondral ossification. To investigate the role of this pathway in endochondral ossification, we generated transgenic mice with expression in chondrocytes of a constitutively active mutant of MKK6, a MAPK kinase that specifically activates p38. These mice had a dwarf phenotype characterized by reduced chondrocyte proliferation, inhibition of hypertrophic chondrocyte differentiation, and a delay in the formation of primary and secondary ossification centers. Histological analysis with in situ hybridization showed reduced expression of Indian hedgehog, PTH͞PTH-related peptide receptor (PTH, parathyroid hormone), cyclin D1, and increased expression of p21 in chondrocytes. In addition, both in vivo and in transfected cells, p38 signaling increased the transcriptional activity of Sox9, a transcription factor essential for chondrocyte differentiation. In agreement with this observation, transgenic mice that express a constitutively active mutant of MKK6 in chondrocytes showed phenotypes similar to those of mice that overexpress SOX9 in chondrocytes. These observations are consistent with the notion that increased activity of Sox9 accounts at least in part for the phenotype caused by constitutive activation of MKK6 in chondrocytes. Therefore, our study provides in vivo evidence for the role of p38 in endochondral ossification and suggests that Sox9 is a likely downstream target of the p38 MAPK pathway.E ndochondral ossification, a process involving a cartilage intermediate, is responsible for the formation of most vertebrate skeletal elements. After the condensation of mesenchymal chondroprogenitor cells (1), cells differentiate into chondrocytes, which express cartilaginous matrix molecules and form cartilage that prefigures future skeletal elements. Endochondral bone growth takes place at the growth plate, where chondrocytes undergo unidirectional proliferation and then become hypertrophic chondrocytes. Hypertrophic chondrocytes eventually undergo apoptosis and are replaced by bone cells (2). This complex process of endochondral ossification is under the concerted regulation of various cytokines and growth factors, including fibroblast growth factors (FGFs), parathyroid hormone (PTH)-related peptide, Indian hedgehog (Ihh), and bone morphogenetic proteins (3-7).Several transcription factors, including Sox9, Sox5, Sox6, Osterix, and Runx2, have critical roles in endochondral ossification (8-12). In particular, we previously showed that Sox9 has an essential role at sequential steps in the chondrocyte differentiation pathway (8, 9). Indeed, Sox9 is needed for the condensation of chondrogenic mesenchymal cells; it is also required for the overt differentiation of these cells into chondrocytes, in part because Sox9 is needed for the expression of Sox5 and Sox6, which are also needed at this step. A further role for Sox9 is inhibition of the proliferation of chondrocytes and of the transition of these cells...