Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation

Abstract

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...

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

“…Interestingly, mutant mice harboring the MTK1 kinase-inactive mutant allele, which is incompetent for p38 signaling, display abnormalities in axial skeletogenesis, although the role of MTK1 in terminal chondrocyte differentiation was not addressed (23). In addition, transgenic mice expressing a Col2a1 promoter/enhancerdriven, constitutively active MKK6 mutant have shorter limbs associated with delayed chondrocyte terminal differentiation and reduced Col10a1 expression (24), suggesting that excessive MKK6 signaling inhibits chondrocyte proliferation and subsequent differentiation. In contrast, enforced MKK6 activation of the p38 pathway in synovial fibroblasts induces terminal chondrocyte differentiation markers, including type X collagen, and mineralization (65).…”

“…Ablation of the MAPK kinase kinase (MAP3K), MEKK4/MTK1, causes skeletal patterning defects in the mouse embryo (23). Moreover, Col2a1 promoterdriven MKK6 transgene overexpression leads to decreased chondrocyte proliferation and delayed terminal differentiation to hypertrophy (24). Constitutive activation of the MAP2K, MEK1, responsible for ERK activation caused persistence of proliferating chondrocytes and delayed hypertrophic maturation (25), whereas in vitro studies using pharmacological inhibition showed that ERK activation is required for chondrocyte hypertrophy (26).…”

Focus on the p38 MAPK signaling pathway in bone development and maintenance

“…For instance, p38 has been found to regulate the transcriptional activity of SOX9, the master transcription factor controlling chondrocyte differentiation. 18 Moreover, ATF2 is expressed in resting and proliferating chondrocytes during endochondral bone development. Atf2-deficient mice display a uniform dwarfism due to reduced chondrocyte proliferation, disorganized chondrocyte columns and impaired endochondral ossification.…”

Sox9 modulates cell survival and adipogenic differentiation of multipotent adult rat mesenchymal stem cells

“…Among these extra functions are control of cell metabolism and cellular differentiation (Pestell et al, 1999). To control and balance chondrocyte proliferation during limb bud development, cyclin D1 expression is reduced in the resting and proliferating chondrocytes of mice embryos, a phase in chondrogenesis where Sox9 is highly induced and where it plays a decisive role (Zhang et al, 2006). Moreover, Akiyama et al showed that in the growth plate of E16.5 Sox9 knock-in mutant mice (mice in which all chondrocytes overexpressed Sox9) the expression of cyclin D1 mRNA and protein was severely reduced (Akiyama et al, 2004).…”