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...

PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT-PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activity at both stages. Butaprost, an EP2-selective agonist, showed effects similar to those of 11-deoxy-PGE1 only at confluency. Another and more differentiated osteoblastic marker, osteocalcin production, was detectable and was stimulated by 11-deoxy-PGE1 only 5 days after confluency. The exposure of these cells to EP1 agonist changed the cell shape to a more fibroblastic appearance. These results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast. Furthermore, the decreased response to EP2-specific agonist 5 days after confluency suggests that the expression of PGE receptor subtype is dependent on the stage of osteoblastic differentiation. This is the first report to determine PGE receptor subtypes in the bone.

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We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.The mammalian ras protooncogenes, Ha-, Ki-, and N-ras, are expressed in a variety of tissues (1). For example, Ha-ras is highly expressed in skin and skeletal muscle; Ki-ras in gut and thymus; and N-ras in testis and thymus. Ras proteins bind guanine nucleotides and possess intrinsic GTPase activity, serving as transducers of diverse physiological signals including those controlling cellular proliferation and differentiation (2). Some of the biological activities of Ras proteins are known to be modulated by other proteins, including Krev-1 (3) and Ha-rev107 (4). However, physiological functions of Ras proteins and these regulatory proteins in mammalian cells remain largely unknown.Chondrogenesis is a key event in skeletal development in vertebrates. We previously reported that chondrogenesis could be induced in chondroprogenitor-like EC cells, ATDC5, at a high incidence when cultured in the presence of insulin and that ATDC5 cells keep track of the overt chondrogenesis in vitro (5-9). Clonal mouse embryonic fibroblast cells, C3H10T1/2, retain the properties of pluripotent mesodermal progenitors and have been shown to differentiate into adipocytes, myoblasts, osteoblasts, as well as chondrocytes (10) under distinct cultural conditions, including the presence of 5-azacytidine or high dose bone morphogenetic protein-2 (10, 11). In this study, comparison by differential display of mRNAs expressed in undifferentiated ATDC5 cells with those in undifferentiated C3H10T1/2 cells led us to isolate a novel cDNA clone encoding a Ha-rev107-related protein predominantly expressed in skeletal muscle, heart, hippocampus, and bone marrow as well as ATDC5 cells. EXPERIMENTAL PROCEDURESCells and Culture Conditions-ATDC5 cells were plated in six-multiwell plates at an initial cell density of 6 ϫ 10 4 cells/well and cu...

In children with end-stage liver disease, little is known regarding the long-term effects of liver transplantation on bone. In this 2-year prospective study, we evaluated the effects of liver transplantation on bone mineral density (BMD) and on other parameters of bone metabolism in 30 consecutive children with biliary atresia who underwent liver transplantation after failed Kasai operation. Tacroli B one metabolism in the perioperative period of liver transplantation changes dynamically with the function of the liver and the systemic status of the recipient. 1 Objective information regarding the impact of liver transplantation on this aspect is lacking, especially for transplants in children. Biliary atresia (BA) is one of the most typical cholestatic diseases that cause end-stage liver disease in children. In the course of BA, multifactorial pathologic phenomena disturb normal bone metabolism to various degrees, causing atraumatic fractures and severe growth retardation. 2 Bone mineral density (BMD) is a reliable objective parameter of bone metabolism. The aims of this study were, first, to assess, with BMD, the growth retardation and defective skeletal mineralization before liver transplantation of pediatric patients with end-stage liver disease caused by BA, and, second, to evaluate the effects of liver transplantation for these patients and the mechanism of improvement of the bone metabolism after liver transplantation. Patients and MethodsThirty-six consecutive patients with BA who underwent liver transplantation in our institution were included in this study. Informed consent was obtained from the families of all the patients. To adequately evaluate the effect of the liver transplantation, 6 patients (16%) who developed life-threatening posttransplant complications, (peritonitis [4 cases], sepsis [1] or chronic rejection [1]) were excluded from this study. Four children with peritonitis had surgical repair and did not receive standard immunosuppressants because of their severe septic condition. They were not given the steroids, and two of them died. The other 2 patients could not continue to follow up because of poor general condition. The patients with sepsis and chronic rejection died 1 month and 3 months after transplantation, respectively. The remaining 30 patients (15 boys and 15 girls), age ranging from 7 months to 181 months (median, 34 months), were enrolled. All patients had previously undergone a Kasai operation at around 2 months of age. The indications for liver transplantation in these patients were gastrointestinal bleeding (9 cases), recurrent cholangitis (7), growth retardation (5), intractable ascites (3), worsening of jaundice (3), hepatopulmonary syndrome (2), and hypersplenism (1). All patients underwent living-related liver transplantation as described elsewhere. 3 The postoperative immunosuppressant regimen was the combination of tacrolimus and steroids. The patients were weaned off steroids with stable liver function at around 6 months after the transplantation. 4 All children had r...