Abstract
Parathyroid hormone (PTH) binds to its receptor PTH1R (parathyroid hormone 1 receptor) in osteoblastic cells to regulate bone remodeling and calcium homeostasis. While prolonged exposure to PTH causes increased bone resorption, intermittent injections of PTH have an anabolic effect on bone. The molecular mechanisms regulating these processes are still largely unknown. Here, we present our results on gene expression profile changes in the PTH-treated osteoblastic cell line, UMR 106-01, using DNA microarray analysis. A total of 125 known genes and 30 unknown expressed sequence tags (ESTs) were found to have at least 2-fold expression changes after PTH treatment at 4, 12, and 24 h. 14 genes were previously known to be PTH-regulated but many were unknown to be regulated by PTH prior to our experiments. Real-time reverse transcriptase-PCR confirmed that 90 and 50% of the genes are regulated more than 2-fold by PTH in UMR 106-01 and rat primary osteoblastic cells, respectively. Most genes belong to the following protein families: hormones, growth factors, and receptors; signal transduction pathway proteins; transcription factors; proteases; metabolic enzymes; structural and matrix proteins; transporters; etc. These results provide a comprehensive and deeper knowledge about PTH regulation of osteoblastic gene expression. Next, we designed a computational method to extract information about transcription factors likely involved in regulating these genes. These factors include those previously known to be involved in PTH signaling (AP-1 and the cAMP response element-binding protein), those that were identified by microarray data (C/EBP), and some novel transcription factors (AP-2, AP-4, SP1, FoxD3, etc.). Our results suggest that a reliable bioinformatics approach can be easily applied for other systems.
Highlights
Bone is a mineralized tissue that confers multiple mechanical and metabolic functions to the body
Our results suggest that a reliable bioinformatics approach can be applied for other systems
Treatment of UMR 106-01 cells with 10Ϫ8 M Parathyroid hormone (PTH) inhibits proliferation of these cells by arresting them in G1 [9] and has a similar effect on gene expression patterns of UMR 106-01 cells as high dose PTH does on osteoblasts in the skeleton [10, 11]
Summary
Microarray Analysis—UMR 106-01 cells were seeded in 100-mm dishes at 1.2 ϫ 104 cells/cm in Eagle’s minimal essential medium (MEM) supplemented with 5% fetal bovine serum overnight. The sequences of the 125 PTH-regulated genes revealed by microarray experiments were retrieved from GenBankTM and matched using BLAST against the rat mRNA data base to ensure the longest rat mRNA entries were found. Each of these longest rat mRNAs was matched using BLAST against human mRNA. Genes having a 5,000-bp continuous region upstream of the first exon start site were kept for further transcription factor site analysis We term this PTH-regulated gene list as the sample list. Transcription Factor Binding Site Analysis and Statistical Analysis—For each gene in the sample list, 500-, 1000-, and 2000-bp upstream of the first exon start site were retrieved as possible promoter sequence. A matrix and its corresponding transcription factor are considered to have significantly higher occurrence in the promoter regions of the sample list if it meets the following conditions. 1) The transcription factor should belong to the vertebrate category. 2) The observed number should be higher than the expected number. 3) 2 should be higher than 8 (p Ͻ 0.005) in at least one matrix cutoff in 2 of 3 promoter distances
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