Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor.

Abstract

Abstract. The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 ixg/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (M r ~'-~80 kD; K d = 3.9 nM; 3.2 x 105 sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action. IT has been suggested that when limb mesenchymal cells move away from the influence of the apical ectodermal ridge, their cAMP content increases, triggering chondrogenic differentiation (38). The response of limb mesenchymal cells to dibutyryl cAMP is a responsive property of the cells, which changes temporally and spatially during limb differentiation (57). Elevation of cAMP content of the subridge mesenchymal cells precludes the necessity of cells passing through a condensation phase before overt cartilage formation (38). Thus, the increase in intracellular cAMP is hypothesized to be a key cellular response to cellular condensation that is a prerequisite for limb chondrogenesis (46,49). We previously reported that parathyroid hormone (PTH) 1 stimulates sulfate incorporation into cartilagecharacteristic proteoglycans followed by elevation of intracellular cAMP in primary growth plate chondrocytes (23,59,60). This stimulation of sulfate incorporation was mainly caused by elongation of glycosaminoglycan chains rather than increased synthesis of proteoglycan core protein (23). Chondrocytes respond to PTH through the adenylate cyclase-coupled surface recepto...

CCAAT/Enhancer-binding Protein β Regulates the Repression of Type II Collagen Expression during the Differentiation from Proliferative to Hypertrophic Chondrocytes

“…ATDC5 cells show chondrogenic and hypertrophic differentiation in nodules, and undifferentiated ATDC5 cells remain fibroblastic morphology around nodules. Subsequently, the cells form numerous nodules, and hypertrophic differentiation of ATDC5 cells progresses (31). Thus, mixing the cells at various differentiation stages may cause higher expression of Col1a1 in ATDC5 cells than in primary chondrocytes.…”

Zinc finger protein Zac1 is expressed in chondrogenic sites of the mouse

“…These cells can undergo in vitro chondrogenesis starting from cellular condensation and leading to differentiation into mature and hypertrophic chondrocytes (Atsumi et al, 1990;Shukunami et al, 1996). The ATDC5 cells were grown in monolayers in the presence or absence of insulin for 3, 7, 14, 21, and 28 days.…”

In vitro differentiation of chondrogenic ATDC5 cells is enhanced by culturing on synthetic hydrogels with various charge densities

“…Toward the end of examining the effect of hydrogels on chondrogenic differentiation, we have used murine chondrogenic ATDC5 cells in this study. ATDC5 cells that represent a chondroprogenitor clone can be induced through an insulin-dependent pathway into chondrogenic differentiation that closely reduplicates the cartilage development in vivo [18]. The purpose of this study was to clarify the differentiation behavior of ATDC5 cells when cultured on the PAMPS, PDMAAm, and P(AMPS-co-DMAAm) gels, not only in standard insulin-supplemented medium but also in insulin-free medium.…”