Novel Late Response Genes of PTHrP in Chondrocytes

Abstract

To gain more insight into the downstream effectors of parathyroid hormone (PTH) related peptide (PTHrP) signaling in chondrocytes, we performed microarray analysis to identify late PTHrP response genes using the chondrogenic ATDC5 cell line and studied their response in the osteoblastic KS483 cell line and explanted metatarsals. At day 8 of micromass culture, ATDC5 cells have pre-hypertrophic-like characteristics and at this time point the cells were stimulated with PTHrP for 24 and 72 h and RNA was isolated. PTHrP treatment inhibited outgrowth of cartilage matrix and decreased the expression of Col10a1 mRNA, which is in line with the inhibitory effects of PTHrP on chondrocyte differentiation. Using cDNA microarray analysis, a list of 9 genes (p< 10^–3) was generated, including 3 upregulated (IGFBP4, Csrp2, and Ecm1) and 6 downregulated (Col9a1, Col2a1, Agc, Hmgn2, Calm1, and Mxd4) response genes. Four out of 9 genes are novel PTHrP response genes and 2 out of 9 have not yet been identified in cartilage. Four out of 9 genes are components of the extra-cellular matrix and the remaining genes are involved in signal transduction and transcription regulation. The response to PTHrP was validated by quantitative PCR, using the same RNA samples as labeled in the microarray experiments and RNA samples isolated from a new experiment. In addition, we examined whether these genes also reacted to PTHrP in other PTHrP responsive models, like KS483 osteoblasts and explanted metatarsals. The expression of late PTHrP response genes varied between ATDC5 chondrocytes, KS483 osteoblasts and metatarsals, suggesting that the expression of late response genes is dependent on the cellular context of the PTHrP responsive cells.