Activation of the PTH/PTHrP Receptor Increases Cyclin-D1 mRNA Levels

Objectives: Parathyroid Hormone (PTH) regulates cartilage development. This G-protein coupled receptor (GPCR) activates critical downstream signaling in chondrogenesis, however details of the regulation are still poorly understood. Previously, we have reported that PTH decreases apoptosis and increases longitudinal growth of primary cartilage. Here we asked: Does activation of the PTH/PTHrP receptor modulate mRNA levels of Cyclin D1, a positive regulator of proliferation in cartilage? Methods: Day fourteen embryonic chick sterna were harvested without perichondrial membranes and placed in organ culture for 8 days. The sterna were grown in Ham's F12 media with dexamethasome, insulin, T3, and ascorbic acid with or without PTH (1-34) . RNA was isolated and quantified using RT-PCR with a Stratagene MX4000 with SYBR green fluorescent labeling. Primers for Cyclin D1 and 18s, an internal control, were designed, tested and standard curves established. Results: R-square values of .965 and .981 were obtained on the MX-400 for the standard curves of 18s and Cyclin D1. There were equal amounts of 18s internal RNA in all samples. However, Cyclin D1 mRNA levels significantly increased (P < .0122) in cartilage samples treated with PTH (n=14) over controls (n=14). The mean dR (fluorescent relative amount) levels of Cyclin D1 mRNA in the PTH group were 0.468, compared to the dR of the control group, 0.369. Conclusions: Activation of the PTH/PTHrP receptor may be a positive regulator of chondrocytic mitogenesis. This is concurrent with previous observations in our lab that PTH increases Ki-67, a proliferation marker, in the cephalic region during chondrogenesis. Downstream signaling of this common receptor may increase sternal cartilaginous length as a result of Cyclin D1 induced proliferation. NIH/DE 446951