Analysis of N‐cadherin function in limb mesenchymal chondrogenesis in vitro

DeLise, Anthony M.; Tuan, Rocky S.

doi:10.1002/dvdy.10151

Cited by 163 publications

(120 citation statements)

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“…The formation of cellular condensations precedes the commitment of mesenchymal cells to the chondrogenic lineage (6,7). Inhibition of Rac1 signaling resulted in decreased mesenchymal condensation, as confirmed by decreased PNA staining and reduced expression of N-cadherin.…”

Section: Discussionmentioning

confidence: 99%

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

Woods¹,

Wang

Dupuis

et al. 2007

Journal of Biological Chemistry

109

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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...

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Section: Discussionmentioning

confidence: 99%

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

Woods¹,

Wang

Dupuis

et al. 2007

Journal of Biological Chemistry

109

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“…Studies have shown that the expression of the deletion mutant form of N-cadherin, which lacks either the extracellular homotypic interaction domains or the intracellular β-catenin binding site, results in decreased cellular condensation and impaired chondrogenesis (21,22). These findings suggest that both extracellular homotypic interaction and intracellular interaction with the catenin complex are essential for proper N-cadherin signaling (23).…”

mentioning

confidence: 99%

Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis

Bian

Guvendiren

Mauck

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

357

300

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Methacrylated hyaluronic acid (HA) hydrogels provide a backbone polymer with which mesenchymal stem cells (MSCs) can interact through several cell surface receptors that are expressed by MSCs, including CD44 and CD168. Previous studies showed that this 3D hydrogel environment supports the chondrogenesis of MSCs, and here we demonstrate through functional blockade that these specific cell-material interactions play a role in this process. Beyond matrix interactions, cadherin molecules, a family of transmembrane glycoproteins, play a critical role in tissue development during embryogenesis, and N-cadherin is a key factor in mediating cellcell interactions during mesenchymal condensation and chondrogenesis. In this study, we functionalized HA hydrogels with Ncadherin mimetic peptides and evaluated their role in regulating chondrogenesis and cartilage matrix deposition by encapsulated MSCs. Our results show that conjugation of cadherin peptides onto HA hydrogels promotes both early chondrogenesis of MSCs and cartilage-specific matrix production with culture, compared with unmodified controls or those with inclusion of a scrambled peptide domain. This enhanced chondrogenesis was abolished via treatment with N-cadherin-specific antibodies, confirming the contribution of these N-cadherin peptides to chondrogenesis. Subcutaneous implantation of MSC-seeded constructs also showed superior neocartilage formation in implants functionalized with N-cadherin mimetic peptides compared with controls. This study demonstrates the inherent biologic activity of HA-based hydrogels, as well as the promise of biofunctionalizing HA hydrogels to emulate the complexity of the natural cell microenvironment during embryogenesis, particularly in stem cell-based cartilage regeneration.M esenchymal stem cells (MSCs) have emerged as a clinically relevant cell source for regenerative medicine due to their potential to differentiate into several mesenchymal lineages including cartilage, bone, and fat (1, 2). The multipotent differentiation of MSCs is tightly regulated by both soluble and physical cues present in the pericellular microenvironment, including cell-cell and cell-matrix interactions, cues that can be engineered into a variety of natural and synthetic biomaterial scaffolds (3). These materials may be either permissive to chondrogenesis (inert materials including agarose and PEG) or inductive to chondrogenesis by mimicking components of the natural pericellular microenvironment (4, 5). For example, photopolymerizable hydrogels composed of methacrylated (Me) hyaluronic acid (HA) may provide biological cues such as CD44 and CD168 interactions based on the role of HA in cellular signaling (6-8) (Fig. 1). Coincident with the onset of condensation and the first appearance of cartilage in the embryo is the appearance of specific binding sites for HA on bud limb mesenchymal cells (9). Large HA molecules are involved in the aggregation of these cells during condensation via multivalent cross-bridging (10), and HA has already been shown to enhan...

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“…Mesenchymal condensation is a prerequisite for chondrogenesis and is facilitated by cell adhesion molecules. Upregulation of cell adhesion proteins such as N-cadherin is a hallmark of condensing cells, whereas loss of cell adhesion molecules is able to abolish condensation (Delise and Tuan 2002;Bobick et al 2009). The molecular mechanisms governing condensation are not fully understood, though several genes have been implicated in this process such as bone morphogenic proteins (BMPs) and SRY (sex determining region Y)-box 9 (Sox9) (Fromental-Ramain et al 1996;Wada et al 1998;Lu et al 2008).…”

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confidence: 99%

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

Wang

et al. 2016

Genetics

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Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-b1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-b/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation. KEYWORDS Cdc42; condensation; osteoblast; chondrocyte S EVERAL successive steps, including mesenchymal condensation of undifferentiated cells, chondrocyte differentiation, proliferation of chondrocytes, and differentiation into hypertrophic chondrocytes followed by mineralization and ossification, exist in the process of endochondral ossification (Long and Ornitz 2013). Mesenchymal condensation is a prerequisite for chondrogenesis and is facilitated by cell adhesion molecules. Upregulation of cell adhesion proteins such as N-cadherin is a hallmark of condensing cells, whereas loss of cell adhesion molecules is able to abolish condensation (Delise and Tuan 2002;Bobick et al. 2009). The molecular mechanisms governing condensation are not fully understood, though several genes have been implicated in this process such as bone morphogenic proteins (BMPs) and SRY (sex determining region Y)-box 9 (Sox9) (Fromental-Ramain et al. 1996;Wada et al. 1998;Lu et al. 2008). Conditional inactivationof Sox9 in limb mesenchymal progenitors leads to the absence of condensations, while simultaneous deletion of Bmp2 and Bmp4 in limb mesenchymes leads to the loss of zeugopod elements and to defective joint articulations (Reversade et al. 2005). During endochondral ossification, condensed cells undergo the differentiation into chondrocytes that secrete an abundance of cartilage matrices including types II, IX, and XI collagen. Sox9 is also the earliest known transcription...

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Analysis of N‐cadherin function in limb mesenchymal chondrogenesis in vitro

Cited by 163 publications

References 56 publications

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

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