title: Transmission of ER stress response by ATF6 promotes endochondral bone growth.
abstract: BACKGROUND
  We reported earlier that X-box binding protein1 spliced (XBP1S), a key regulator of the unfolded protein response (UPR), as a bone morphogenetic protein 2 (BMP2)-inducible transcription factor, positively regulates endochondral bone formation by activating granulin-epithelin precursor (GEP) chondrogenic growth factor. Under the stress of misfolded or unfolded proteins in the endoplasmic reticulum (ER), the cells can be protected by the mammalian UPR. However, the influence of activating transcription factor 6 (ATF6), another transcriptional arm of UPR, in BMP2-induced chondrocyte differentiation has not yet been elucidated. In the current study, we investigate and explore the role of ATF6 in endochondral bone formation, focus on associated molecules of hypertrophic chondrocyte differentiation, as well as the molecular events underlying this process.
  
  
  METHODS
  High-cell-density micromass cultures were used to induce ATDC5 and C3H10T1/2 cell differentiation into chondrocytes. Quantitative real-time PCR, immunoblotting analysis, and immunohistochemistry were performed to examine (1) the expression of ATF6, ATF6α, collagen II, collagen X, and matrix metalloproteinase-13 (MMP13) and (2) whether ATF6 stimulates chondrogenesis and whether ATF6 enhances runt-related transcription factor 2 (Runx2)-mediated chondrocyte hypertrophy. Culture of fetal mouse bone explants was to detect whether ATF6 stimulates chondrocyte hypertrophy, mineralization, and endochondral bone growth. Coimmunoprecipitation was employed to determine whether ATF6 associates with Runx2 in chondrocyte differentiation.
  
  
  RESULTS
  ATF6 is differentially expressed in the course of BMP2-triggered chondrocyte differentiation. Overexpression of ATF6 accelerates chondrocyte differentiation, and the ex vivo studies reveal that ATF6 is a potent stimulator of chondrocyte hypertrophy, mineralization, and endochondral bone growth. Knockdown of ATF6 via a siRNA approach inhibits chondrogenesis. Furthermore, ATF6 associates with Runx2 and enhances Runx2-induced chondrocyte hypertrophy. And, the stimulation effect of ATF6 is reduced during inhibition of Runx2 via a siRNA approach, suggesting that the promoting effect is required for Runx2.
  
  
  CONCLUSIONS
  Our observations demonstrate that ATF6 positively regulates chondrocyte hypertrophy and endochondral bone formation through activating Runx2-mediated hypertrophic chondrocyte differentiation.
authors.affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Zhangyuan
authors.initials: Z
authors.lastname: Xiong
authors.affiliation: Laboratory of Stem Cells and Tissue Engineering, Chongqing Medical University, Chongqing, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Jiang
authors.affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Han
authors.affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing, 400016, China. guofengjin919@vip.sohu.com.
authors.forename: Feng-Jin
authors.initials: FJ
authors.lastname: Guo
mesh_terms: D051702:Activating Transcription Factor 6 / Q000096:biosynthesis*; D000818:Animals; D001846:Bone Development / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D020219:Chondrogenesis / Q000502:physiology*; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H
chemical_list: D051702:Activating Transcription Factor 6; C497112:Atf6 protein, mouse