Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes (chondrogenesis). This process is characterized by a strong interdependence of cell shape, cytoskeletal organization, and the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. Here we investigated the role of the RhoA/ROCK pathway, a well characterized regulator of cytoskeletal organization, in chondrogenesis. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Suppression of Sox9 expression by ROCK signaling was achieved through repression of Sox9 promoter activity. These molecular changes were accompanied by reorganization of the actin cytoskeleton, where RhoA/ROCK signaling suppressed cortical actin organization, a hallmark of differentiated chondrocytes. This led us to analyze the regulation of Sox9 expression by drugs affecting cytoskeletal dynamics. Both inhibition of actin polymerization by cytochalasin D and stabilization of existing actin filaments by jasplakinolide resulted in increased Sox9 mRNA levels, whereas inhibition of microtubule polymerization by colchicine completely blocked Sox9 expression. In conclusion, our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.

No abstract

Chondrocyte differentiation is a multi-step process characterized by successive changes in cell morphology and gene expression. In addition to tight regulation by numerous soluble factors, these processes are controlled by adhesive events. During the early phase of the chondrocyte life cycle, cell-cell adhesion through molecules such as N-cadherin and neural cell adhesion molecule (N-CAM) is required for differentiation of mesenchymal precursor cells to chondrocytes. At later stages, for example in growth plate chondrocytes, adhesion signaling from extracellular matrix (ECM) proteins through integrins and other ECM receptors such as the discoidin domain receptor (DDR) 2 (a collagen receptor) and Annexin V is necessary for normal chondrocyte proliferation and hypertrophy. Cell-matrix interactions are also important for chondrogenesis, for example through the activity of CD44, a receptor for Hyaluronan and collagens. The roles of several signaling molecules involved in adhesive signaling, such as integrin-linked kinase (ILK) and Rho GTPases, during chondrocyte differentiation are beginning to be understood, and the actin cytoskeleton has been identified as a common target of these adhesive pathways. Complete elucidation of the pathways connecting adhesion receptors to downstream effectors and the mechanisms integrating adhesion signaling with growth factor-and hormone-induced pathways is required for a better understanding of physiological and pathological skeletal development.

The development of the cartilage template that precedes endochondral bone formation requires the condensation of mesenchymal cells and their subsequent differentiation to the chondrocytic lineage. We have previously shown that inhibition of the RhoA/ ROCK signaling pathway or actin dynamics enhances Sox9 mRNA expression, increases glycosaminoglycan production, and transforms cell shape to a spherical, chondrocyte-like morphology. However, we demonstrate here that in three-dimensional micromass cultures of mesenchymal cells, increased expression of Sox9 in response to these manipulations is not sufficient to induce the expression of established Sox9 target genes. This is illustrated by a decrease in the transcript levels of collagen II and aggrecan as well as reduced activity of a Sox9-responsive reporter gene in response to ROCK inhibition and cytochalasin D. We also demonstrate a decrease in mRNA levels of the transcriptional co-activators L-Sox5 and Sox6 upon ROCK inhibition and cytochalasin D. The decrease in Sox9 activity is likely partially due to reduced L-Sox5 and Sox6 levels but also to a delay in Sox9 phosphorylation following ROCK inhibition. In contrast, inhibition of the RhoA/ROCK pathway and cytochalasin D treatment in monolayer culture results in the enhancement of a number of markers of chondrogenesis such as Sox9 activity and collagen II and aggrecan transcripts levels. These data demonstrate that the effects of RhoA/ROCK signaling and actin polymerization inhibitors on chondrogenic gene expression are dependent on the cellular context.Endochondral ossification is a developmental process that forms the majority of the mammalian skeleton, as reviewed in Ref. 1. Beginning with condensations of mesenchymal cells and subsequent differentiation to the chondrocytic lineage, precisely shaped cartilage templates are formed (2). These templates are composed of proliferating and differentiating chondrocytes as well as vast amounts of extracellular matrix produced by chondrocytes (3). The commitment of cells to the chondrocytic lineage is marked by change in cell shape (from a spread, fibroblastic-like morphology to a spherical morphology), expression of the essential transcription factor Sox9 (4) and the matrix markers collagen II (5) and aggrecan (6), and increased glycosaminoglycan production (1).Although the importance of cell shape is recognized (7), molecular mechanisms controlling these processes had not been studied extensively.The RhoA/ROCK signaling pathway is an excellent candidate as its role as a master regulator of the actin cytoskeleton has been well documented (8 -10). In addition, RhoA signaling controls cell cycle progression (11), differentiation (12), and apoptosis in other cell types (13). We recently demonstrated that inhibition of the RhoA/ROCK pathway by the pharmacological inhibitor Y27632 induced spherical cell morphology, increased the transcription of the chondrogenic transcription factor Sox9, and enhanced glycosaminoglycan accumulation (14). We also demonstrated that RhoA overe...

Crosstalk, also known as ghosting or leakage, is a primary factor in determining the image quality of stereoscopic three dimensional (3D) displays. In a stereoscopic display, a separate perspective view is presented to each of the observer's two eyes in order to experience a 3D image with depth sensation. When crosstalk is present in a stereoscopic display, each eye will see a combination of the image intended for that eye, and some of the image intended for the other eye-making the image look doubled or ghosted. High levels of crosstalk can make stereoscopic images hard to fuse and lack fidelity, so it is important to achieve low levels of crosstalk in the development of high-quality stereoscopic displays. Descriptive and mathematical definitions of these terms are formalized and summarized. The mechanisms by which crosstalk occurs in different stereoscopic display technologies are also reviewed, including micropol 3D liquid crystal displays (LCDs), autostereoscopic (lenticular and parallax barrier), polarized projection, anaglyph, and time-sequential 3D on LCDs, plasma display panels and cathode ray tubes. Crosstalk reduction and crosstalk cancellation are also discussed along with methods of measuring and simulating crosstalk.

Coordinated proliferation and differentiation of growth plate chondrocytes is required for normal growth and development of the endochondral skeleton, but little is known about the intracellular signal transduction pathways regulating these processes. We have investigated the roles of the GTPase RhoA and its effector kinases ROCK1/2 in hypertrophic chondrocyte differentiation. RhoA, ROCK1, and ROCK2 are expressed throughout chondrogenic differentiation. RhoA overexpression in chondrogenic ATDC5 cells results in increased proliferation and a marked delay of hypertrophic differentiation, as shown by decreased induction of alkaline phosphatase activity, mineralization, and expression of the hypertrophic markers collagen X, bone sialoprotein, and matrix metalloproteinase 13. These effects are accompanied by activation of cyclin D1 transcription and repression of the collagen X promoter by RhoA. In contrast, inhibition of Rho/ROCK signaling by the pharmacological inhibitor Y27632 inhibits chondrocyte proliferation and accelerates hypertrophic differentiation. Dominant-negative RhoA also inhibits induction of the cyclin D1 promoter by parathyroid hormone-related peptide. Finally, Y27632 treatment partially rescues the effects of RhoA overexpression. In summary, we identify the RhoA/ROCK signaling pathway as a novel and important regulator of chondrocyte proliferation and differentiation.The development and growth of endochondral bones (such as ribs, vertebrae, and the long bones of vertebrate limbs) are regulated through the highly controlled rates of proliferation and hypertrophic differentiation of growth plate chondrocytes (1-3). In the growth plate, chondrocytes first undergo a series of cell divisions along the longitudinal axis of the growing bone, thereby forming characteristic columns of clonal cells. Chondrocytes then withdraw from the cell cycle and begin to increase their cell volume until reaching the fully differentiated state of hypertrophic chondrocytes. Transition from a proliferating to a hypertrophic phenotype involves numerous changes in gene expression, for example the induction of the collagen X (4, 5), matrix metalloproteinase 13 (6, 7), and bone sialoprotein (BSP) 1 (8 -10) genes. The latter two genes are also expressed by osteoblasts, suggesting similar biological properties of hypertrophic chondrocytes and osteoblasts. The fate of hypertrophic chondrocytes is still debated, but it appears that the majority of these cells undergo apoptosis and are replaced by bone tissue (11). Longitudinal growth of endochondral bones therefore requires both proliferation and differentiation-associated hypertrophy of growth plate chondrocytes.Disruption of chondrocyte proliferation and/or differentiation by gene mutations commonly results in chondrodysplasias that are characterized by skeletal deformities and reduced growth (12)(13)(14). Mutations in genes encoding extracellular matrix molecules, growth factors, receptors, and transcription factors have been identified as causes of several chondrodysplasias. Fo...

Small GTPases of the Rho family have been implicated in the regulation of many intracellular processes. However, their tissue-specific roles in mammalian growth and development in vivo remain largely unknown. Here we describe the effects of cartilage-specific inactivation of the Rac1 gene in mice. Mice carrying this mutation show increased lethality, skeletal deformities, severe kyphosis and dwarfism. Rac1-deficient growth plates are disorganized and hypocellular, with chondrocytes of abnormal shape and size. Rac1-deficient chondrocytes also display reduced adhesion and spreading on collagen II and fibronectin as well as altered organization of the actin cytoskeleton, suggesting that Rac1 is required for normal cell-extracellular matrix interactions in cartilage. This phenotype is accompanied by reduced proliferation, increased apoptosis and deregulated expression of the cell cycle genes cyclin D1 and p57 in vivo. Moreover, phosphorylation of p38 MAP kinases is greatly reduced and expression of a key regulator of cartilage development, Indian hedgehog, is increased in mutant mice. In summary, these data identify a novel, essential and tissue-specific role of Rac1 in skeletal development and demonstrate that Rac1 deficiency affects numerous regulatory pathways in cartilage.

The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.Chondrocytes are the cellular component of cartilage, responsible for generating and maintaining its extracellular environment (1). Cartilage has multiple functions such as providing a cushion on the articular surfaces of joints (2), providing a template for the formation of endochondral bone (3), and contributing to fracture repair (4). The formation of cartilage templates in endochondral ossification begins with condensation of mesenchymal cells, increased expression of the cell adhesion molecules N-cadherin and N-CAM, and therefore increased cell-cell interactions (5-7). As cells become chondrogenic, the expression of these adhesion molecules is decreased (8). Cells within these condensations commit to the chondrogenic lineage, acquire a spherical cell morphology and induce expression of the essential chondrogenic transcription factor Sox9 (9, 10). Sox5 and Sox6 cooperate with Sox9 to control chondrogenesis and are themselves under the transcriptional control of Sox9 (9,11,12). Together, these transcription factors activate transcription of the major chondrogenic matrix genes, collagen II and aggrecan (11,(13)(14)(15). Furthermore, increased glycosaminoglycan (GAG) 3 content is another marker of the chondrogenic extracellular matrix (1). The production of glycosaminoglycans are partially regulated by the enzymes chondroitin 4-sulfotransferase 11 (Chst 11) and chondroitin 6-sulfotransferase 3 (Chst 3), which have been determined to be important for proper cartilage and bone formation (16,17).Although many...