title: Bcl-2-associated athanogene-1 (BAG-1): a transcriptional regulator mediating chondrocyte survival and differentiation during endochondral ossification.
abstract: BAG-1, an anti-apoptotic protein, was identified by its ability to bind to BCL-2, HSP70-family molecular chaperones and nuclear hormone receptor family members. Two BAG-1 isoforms, BAG-1L (50 kDa) and BAG-1S (32 kDa) were identified in mouse cells and BAG-1 expression was reported in murine growth plate and articular chondrocytes. The present study aimed to elucidate the role of BAG-1 in the regulation of molecular mechanisms governing chondrocyte differentiation and turnover during endochondral ossification. In long bones of skeletally immature mice, we observed expression of BAG-1 in the perichondrium, osteoblasts, osteocytes in the bone shaft, bone marrow, growth plate and articular chondrocytes. Monolayer cultures of murine chondrocytic ATDC5 cells, which exhibited robust expression of both BAG-1 isoforms and the Bag-1 transcript, were utilized as an in vitro model to delineate the roles of BAG-1. Overexpression of BAG-1L in ATDC5 cells resulted in downregulation of Col2a1 expression, a gene characteristically downregulated at the onset of hypertrophy, and an increase in transcription of Runx-2 and Alkaline phosphatase, genes normally expressed at the onset of chondrocyte hypertrophy and cartilage mineralization in the process of endochondral ossification. We also demonstrated the anti-apoptotic role of BAG-1 in chondrocytes as overexpression of BAG-1 protected ATDC5 cells, which were subjected to heat-shock at 48 degrees C for 30 min, against heat-shock-induced apoptosis. Overexpression of the SOX-9 protein in ATDC5 cells resulted in increased Bag-1 gene expression. To further investigate the regulation of Bag-1 gene expression by SOX-9, CHO cells were co-transfected with the human Bag-1 gene promoter-Luciferase reporter construct and the human pSox-9 expression vector. Activity of the Bag-1 promoter was significantly enhanced by the SOX-9 protein. In conclusion, a novel finding of this study is the role of BAG-1 as a transcriptional regulator of genes involved in chondrocyte hypertrophy and cartilage mineralization during the process of endochondral ossification. Additionally, we have demonstrated for the first time the regulation of Bag-1 gene expression by SOX-9 and the anti-apoptotic role of BAG-1 in chondrocytic cells. Modulation of Bag-1 expression can therefore mediate chondrocyte differentiation and turnover, and offer further insight into the molecular regulation of endochondral ossification.
authors.affiliation: Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK.
authors.forename: Rahul S
authors.initials: RS
authors.lastname: Tare
authors.forename: Paul A
authors.initials: PA
authors.lastname: Townsend
authors.forename: Graham K
authors.initials: GK
authors.lastname: Packham
authors.forename: Stefanie
authors.initials: S
authors.lastname: Inglis
authors.forename: Richard O C
authors.initials: RO
authors.lastname: Oreffo
mesh_terms: D000375:Aging / Q000502:physiology; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D010012:Osteogenesis; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: C091732:BCL2-associated athanogene 1 protein; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; D014157:Transcription Factors