Expression of the prostaglandin E₂ (PGE₂) receptor subtype EP₄ and its regulation by PGE₂ in osteoblastic cell lines and adult rat bone tissue¹

Abstract

Prostaglandins E (especially PGE₂) stimulate bone formation and increase bone mass in several species including man. The mechanism for this effect, the target cells, and the receptors involved are not known. Specific cell-surface receptors for PGE₂ (EP_1–4) have been cloned and characterized. EP₄ was reported to be the major receptor in embryonic and neonatal bone tissue in mice, especially in preosteoblasts; however, no data are available regarding its expression in adult bone. This study examines the expression of EP₄ in bone tissue of young adult rats, in which PGE₂ is markedly anabolic, and in various osteoblastic cell lines. Using northern blot analysis, we found that osteoblastic cell lines RCT-1, RCT-3, TRAB-11, and RP-1, primary osteoblastic cells harvested from fetal rat calvaria, as well as tibiae and calvariae of 5-week-old rats express 3.8 kb EP₄ messenger RNA (mRNA). Treatment of periosteal cells (RP-1) in vitro with 10⁻⁶ mol/L PGE₂ increased the levels of both EP₄ mRNA and EP₄ protein, peaking at 1–2 h. Similarly, systemic administration of an anabolic dose of PGE₂ (3–6 mg/kg) to young adult rats upregulated the expression of EP₄ in the tibia and calvaria, also peaking at 1–2 h. Using in situ hybridization, we found increased expression of EP₄ in bone marrow cells of the tibial metaphysis in response to systemic PGE₂ treatment. The preosteoblastic nature of these EP₄-expressing cells was suggested by the fact that dexamethasone-treated bone marrow stromal cells in culture express EP₄ mRNA, which is upregulated by PGE₂. Northern blot analysis failed to detect both basal and PGE₂-induced EP₂ mRNA in the bone samples or cell lines tested. Taken together, these data implicate EP₄ as the major cyclic AMP-related PGE₂ receptor subtype expressed in bone tissue and osteoblastic cells and indicate that this receptor is upregulated by its ligand, PGE₂.

Introduction

Prostaglandins (PGEs, especially PGE₂) have multiple effects on bone, including stimulating both resorption and formation.5, 31 Systemic administration of PGE₂ or E₁ to infants33, 41 and animals13, 14, 15, 16, 24, 39, 42 is clearly anabolic, stimulating bone formation and increasing bone mass. The ability of local administration of PGE₂ or E₁ to stimulate new bone formation23, 48 suggests that PGE₂ induces osteogenesis by acting directly on bone tissue. Histological analysis suggests that PGE₂ increases the number of osteoblasts present on the bone surface. However, the target cells responsible for PGE₂ anabolic action on bone have not been conclusively identified, although bone marrow and/or periosteal osteoprogenitor cells have been suggested.21, 45, 46 In addition, it has not been established whether PGE stimulation of osteoblastic activity in vivo involves effects on cell proliferation, differentiation, and/or survival.

PGEs act on a variety of cells via cell-surface receptors divided into four subtypes, EP_1–4, which differ in their signal transduction pathways and their relative sensitivity to selective agonists and antagonists.1, 8, 25 The recent isolation and characterization of cDNAs for human, mouse, and rat EP receptors2, 4, 10, 18, 28, 32, 43 demonstrated that they all belong to theG-protein-coupled receptor family and activate different secondary messenger systems such as adenylate cyclase or phospholipase C (PLC). Of these four receptors, EP₄ and EP₂ activate adenylate cyclase, EP₁ activates PLC, and EP₃ either lowers intracellular cyclic AMP levels or activates PLC, depending on the alternatively spliced isoform.

In MC3T3-E1 osteoblastic cells in vitro, PGE₂ stimulates both phosphatidylinositol and cAMP-initiated signal transduction pathways.40 Accordingly, both EP₁ and EP₄, found in these cells,37 have been proposed to play a role in the biological action of PGE₂ in bone tissue. Initial characterization by in situ hybridization of in vivo expression of EP₁, EP₃, and EP₄, at first designated “EP₂,” showed that, in embryonic and neonatal mice, EP₄ is the major form found in bone tissue, especially in preosteoblasts, whereas EP₃ can be found in perichondrium.12, 17 However, a recent report showed that expression of EP₂ (current designation) is detected in fetal rat calvariae and long bone by reverse transcription-polymerase chain reaction (RT-PCR) and in fetal rat calvariae by in situ hybridization,26 suggesting a role for EP₂ in addition to EP₄ in PGE₂ effects on bone. Although EP₄ and EP₂ are expressed in many tissues of adult animals, no data are available regarding the expression of these receptors in adult bone tissue. This study therefore examined the expression of EP₂ and EP₄ and the effect of PGE₂ on their expression in adult rat bone and in several osteoblastic cells, including a cell line (RP-1) derived from adult rat periosteum and bone marrow stromal cells. We found that EP₄ is the major cAMP-related receptor expressed in adult rat bone tissue and bone-derived cell lines and that its expression is upregulated by PGE₂.