The effects of prostaglandin E2 on gene expression of IDG-SW3-derived osteocytes in 2D and 3D culture

Abstract

Prostaglandin E₂ (PGE₂) is a key signaling molecule produced by osteocytes in response to mechanical loading, but its effect on osteocytes is less understood. This work examined the effect of PGE₂ on IDG-SW3-derived osteocytes in standard 2D culture (collagen-coated tissue culture polystyrene) and in a 3D degradable poly(ethylene glycol) hydrogel. IDG-SW3 cells were differentiated for 35 days into osteocytes in 2D and 3D cultures. 3D culture led to a more mature osteocyte phenotype with 100-fold higher Sost expression. IDG-SW3-derived osteocytes were treated with PGE₂ and assessed for expression of genes involved in PGE₂, anabolic, and catabolic signaling. In 2D, PGE₂ had a rapid (1 h) and sustained (24 h) effect on many PGE₂ signaling genes, a rapid stimulatory effect on Il6, and a sustained inhibitory effect on Tnfrsf11b and Bglap. Comparing culture environment without PGE₂, osteocytes had higher expression of all four EP receptors and Sost but lower expression of Tnfrsf11b, Bglap, and Gja1 in 3D. Osteocytes were more responsive to PGE₂ in 3D. With increasing PGE₂, 3D led to increased Gja1 and decreased Sost expressions and a higher Tnfrsf11b/Tnfsf11 ratio, indicating an anabolic response. Further analysis in 3D revealed that EP4, the receptor implicated in PGE₂ signaling in bone, was not responsible for the PGE₂-induced gene expression changes in osteocytes. In summary, osteocytes are highly responsive to PGE₂ when cultured in an in vitro 3D hydrogel model suggesting that autocrine and paracrine PGE₂ signaling in osteocytes may play a role in bone homeostasis.

Keywords: 3D culture; Gene expression; Hydrogel; Osteocyte; Prostaglandin E(2).