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Novel Early Target Genes of Parathyroid Hormone-Related Peptide in Chondrocytes

Departments of Pediatrics (J.H., E.P., R.M., J.M.W., M.K.) and Endocrinology and Metabolic Diseases (C.W.G.M.L., M.K.), Leiden University Medical Center, 2333 ZA Leiden, The Netherlands

Address all correspondence and requests for reprints to: Dr. Marcel Karperien, Department of Endocrinology, Leiden University Medical Center, C4R89, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail: karperien{at}lumc.nl.

We have performed microarray analysis to identify PTHrP target genes in chondrocytes. ATDC5 cells were cultured as micromasses to induce chondrocyte differentiation. On d 8 of culture, the cells had a prehypertrophic appearance. This time point was chosen for isolation of RNA at 0, 1, 2, and 4 h after a challenge with 10^–7 M PTHrP. Samples were subjected to a cDNA microarray using competition hybridization. A list of 12 genes (P < 10^–3), the expression regulation of which by PTHrP was confirmed by quantitative PCR analysis, was generated. This included seven up-regulated and five down-regulated genes. Three genes were known to be involved in PTHrP regulation, and six were previously found in growth plate chondrocytes. Most of the genes (10 of 12) were implicated in signal transduction and regulation. PTHrP also induced expression of the up-regulated genes in KS483 osteoblasts, suggesting involvement in a more generalized response to PTHrP. The vast majority of the up-regulated genes (six of seven) contained cAMP response element-binding protein- and/or activating protein-1 transcription factor-binding sites in their promoter regions. Remarkably, a number of PTHrP-regulated genes contained signal transducer and activator of transcription factor (Stat)-binding sites in their promoters. In transient transfection assays, we show that PTHrP is able to positively regulate the activity of Stat3-specific and negatively regulate the activity of Stat5-specific promoter-reporter constructs in ATDC5 and UMR106 cells. In combination with the expression regulation of genes involved in Janus kinase/Stat signaling, this data suggest a previously unrecognized interaction between PTHrP and Janus kinase/Stat signaling.