title: Microarray analysis of thapsigargin-induced stress to the endoplasmic reticulum of mouse osteoblasts.
abstract: Activating transcription factor 4 (ATF4) protein has a dual role in osteoblasts. It functions as a responder to stress to the endoplasmic reticulum (ER) as well as a transcription factor for bone formation. Little is known about molecular pathways that can potentially lead to stress-induced apoptosis or homeostasis of extracellular matrix (ECM) molecules. Based on microarray-derived mRNA expression data for mouse osteoblasts (MC3T3 E1 cells, clone 4), we analyzed the ER-stress responses in the presence of 10 nM Thapsigargin using two computational approaches: "Gene Set Enrichment Analysis (GSEA)" and "Ingenuity Pathways Analysis (IPA)." GSEA presented a strong linkage to an expression pattern observed in the responses to hypoxia, and IPA identified two molecular pathways: ATF4-unlinked connective tissue development and ATF4-linked organ morphology. Real-time polymerase chain reaction (PCR) and Western blot analyses validated eIF2alpha-driven translational regulation as well as ATF4-linked transcriptional activation of transcription factors and growth factors including FOS, FGF-9, and BMP-2. Consistent with the role of p38 MAPK in hypoxia, phosphorylation of p38 MAPK was activated in nonapoptotic osteoblasts under surviving ER stress. Furthermore, the level of phosphorylated PERK was elevated. These results support cross-talk between p38 MAPK and ER kinase, presenting a similarity to the responses to hypoxia as well as a pathway toward connective tissue development and organ morphology.
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University, Indianapolis, IN 46202, USA.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Hamamura
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Liu
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yokota
mesh_terms: D016475:3T3 Cells; D051701:Activating Transcription Factor 4 / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D002999:Clone Cells; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D020869:Gene Expression Profiling; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D019284:Thapsigargin / Q000009:adverse effects*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D019892:eIF-2 Kinase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D051701:Activating Transcription Factor 4; D019284:Thapsigargin; D019892:eIF-2 Kinase