Development of PTH-responsive adenylate cyclase activity during chondrogenesis in cultured mesenchyme from chick limb buds

Abstract

The present study investigated the development of parathyroid hormone (PTH)-responsive adenylate cyclase (AC) activity in chondrogenic cells differentiating from chick limb mesenchyme in culture. Mesenchyme from stage 25 chick embryos was removed from the distal tip (0.3 mm) of limb buds and cultured for a 6 day period in high density micromass cultures. Under these conditions, initial appearance of cartilage matrix and chondroblasts occurred on day 3 of culture and rapidly progressed over the next 3 days to produce, by day 6, a highly confluent and homogeneous layer of cartilage matrix and chondrocytes. Cells initially dissociated from limb mesenchyme on day 0 were essentially unresponsive to PTH, but development of AC-coupled, PTH receptors occurred rapidly during the initial 24 hours of culture. Based on data from dose-response experiments, prechondrogenic cells on day 1 of culture had synthesized their full complement of these receptors relative to fully differentiated chondrocytes in cultures at day 6. Inhibition of chondrocyte differentiation by retinoic acid did not significantly affect the initial development of AC-coupled, PTH receptors but it almost completely prevented synthesis of cartilage matrix. The results indicate that development of AC-coupled PTH receptors during chondrogenesis precedes, by at least 48 hours, overt differentiation of chondrocytes and the accumulation of cartilage-specific extracellular matrix and appears to represent one of the earliest reported events in chondrocyte differentiation. The lack of effect of retinoids on development of these receptors indicates that the inhibitory effects of retinoids on differentiating cartilage are at least somewhat specific for genes regulating synthesis of extracellular matrix molecules.

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

“…Elevation of binding capacity was correlated with progression of chondrogenic differentiation of cells. Capehart (9) and Zull (65) described in their earlier reports that the appearance of PTH-sensitive adenylate cyclase was one of the earliest events in chick limb differentiation. It was unclear at the time, however, whether acquisition of PTH responsiveness occurred in cells of the chondrogenic or osteoblastic lineage.…”

Differential effects of parathyroid hormone fragments on collagen gene expression in chondrocytes.

“…The other fragments were regarded as inactive metabolites whose functional importance was confined to processing and intracellular transport events during hormone secretion by the cells of the parathyroid gland (PTH 53-84, references 35,46). However, there is now increasing evidence for a broader spectrum of target tissues, including cartilage (26,33,34), and of hormone action in growth (30,48) and dif-ferentiation processes (8,13) which are mediated by additional functional domains on the mid-regional (23) and COOH-terminal portion (39,40,44) of PTH. For example, PTH (53-84) increases alkaline phosphatase activity in osteoblastic cell lines (39) and more recent studies showed that PTH and PTH (53-84) dose dependently stimulate the differentiation of osteoclast precursors into osteoclast-like cells (25).…”

“…Several studies (8,13,30) have indicated that the response of chondrocytes to PTH depends on the source and developmental stage of the cartilage. In embryonic transient cartilage, such as epiphyseal cartilage of long bone rudiments, chondrocytes rapidly proliferate and undergo a series of differentiation steps.…”

“…Tel. : 49 9131 856 990 Fax: 49 9131 856 341. ferentiation processes (8,13) which are mediated by additional functional domains on the mid-regional (23) and COOH-terminal portion (39,40,44) of PTH. For example, PTH (53-84) increases alkaline phosphatase activity in osteoblastic cell lines (39) and more recent studies showed that PTH and PTH dose dependently stimulate the differentiation of osteoclast precursors into osteoclast-like cells (25).…”