Prostaglandin E2 modulates murine chondrocyte maturation in vitro

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology, 2008.Clinically, the use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with impaired bone healing. NSAIDs inhibit arachidonic acid metabolism. Interruption of arachidonic acid metabolism during fracture healing results in cartilage-related defects of the fracture callus. Prostaglandin E2 (PGE2) is the major product of arachidonic acid metabolism in cartilage. Together, these observations suggest depression of PGE2 levels (and possibly, PGE2-mediated signaling events) in cartilage may be the mechanism by which NSAIDs inhibit bone healing. This prompted us to examine the role of PGE2 in chondrogenesis and chondrocyte hypertrophy—two processes involved in callus formation and removal during fracture repair. The hypothesis tested was comprised of three separate, but interconnected components: (i) expression of the EP receptors is maturationally-regulated, (ii) PGE2’s promotion of chondrogenesis and depression of chondrocyte hypertrophy are dependent upon activation of EP receptor signaling, and (iii) PGE2 inhibits chondrocyte hypertrophy by decreasing BMP signaling. Primary murine cell culture systems possessing the ability to undergo chondrogenesis and chondrocyte hypertrophy in vitro were used to study the effect of PGE2 on these processes. A murine limb bud-derived mesenchymal cell model known to undergo chondrogenesis in vitro was chosen to examine the role of PGE2 during chondrogenesis. As the cells became more chondrogenic, the expression levels of the PGE2 receptors EP1, EP2, and EP4 increased. Signaling by these receptors has previously been shown to induce chondrogenesis in other cell systems. Timing of increased expression of these receptors in our model coincided with the first effects of PGE2 on the expression of genes induced during chondrogenesis and cartilage matrix accumulation. These results suggest the initial limited effects exerted by PGE2 during murine chondrogenesis may be a consequence of low expression levels of its receptors. A primary murine sternal chondrocyte model previously shown to undergo chondrocyte hypertrophy in vitro was used in the second set of experiments. PGE2’s effects on chondrocyte maturational markers were gene-specific, with the biggest effect seen in type X collagen (col10A1) gene expression. The mechanism by which PGE2 inhibited type X collagen (col10A1) gene expression was examined further. Cultures were treated with or without BMP-2, a master regulator of chondrocyte maturation, in the presence or absence of PGE2. PGE2 treatment diminished the ability of BMP-2 to (i) phosphorylate its downstream effectors, Smads 1, 5, and 8, and (ii) stimulate col10A1 gene expression. Of the EP receptors, EP4 exhibited the highest expression when the effect of PGE2 on BMP-2-mediated Col10A1 expression was maximal. This receptor can activate adenylyl cyclase activity. Artificial enhancement of adenylyl cyclase activity by dibutrylyl cyclic AMP treatment impeded BMP-2’s ability to activate a BMP-Smad-specific Col10A1 promoter. Activation of Smad signaling has been shown to enhance Col10A1 promoter activity and gene transcription. Collectively, these findings imply activation of adenylyl cyclase activity by PGE2 may depress BMP-Smad signaling events; resulting in decreased col10A1 gene transcription. The results demonstrate activation of adenylyl cyclase signaling, as well as addition of PGE2, stimulates chondrogenesis, and the expression of the EP receptors changes as chondrogenesis progresses. The timing of the first PGE2 effects coincides with the up-regulation of the EP2 and EP4 receptors. Combined, these data imply the reason for the delayed response of the cells to PGE2 may be attributed to initial low levels of EP2 and EP4, the receptors PGE2 utilizes to activate adenylyl cyclase activity. The findings from the chondrocyte hypertrophy studies also strongly implicate the EP4 receptor and adenylyl cyclase activity in PGE2-induced effects on Col10A1 expression. They also suggest crosstalk between the PGE2 and BMP signaling pathways may work to regulate the timing of the transition from proliferation to hypertrophy in chondrocytes