Screening of chondrogenic factors with a real-time fluorescence-monitoring cell line ATDC5-C2ER: identification of sorting nexin 19 as a novel factor.
OBJECTIVE
To establish a cell culture system for noninvasive and real-time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors.


METHODS
The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP-2), or transforming growth factor beta (TGFbeta) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene.


RESULTS
We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5-C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP-2, or TGFbeta, showing good correspondence to the chondrogenic markers. Screening using the ATDC5-C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain-of-function and loss-of-function analyses revealed a potent chondrogenic activity of SNX19.


CONCLUSIONS
We established the ATDC5-C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation.
Kan
Akinori
A
Faculty of Medicine, University of Tokyo, Tokyo, Japan.
Ikeda
Toshiyuki
T
Saito
Taku
T
Yano
Fumiko
F
Fukai
Atushi
A
Hojo
Hironori
H
Ogasawara
Toru
T
Ogata
Naoshi
N
Nakamura
Kozo
K
Chung
Ung-Il
UI
Kawaguchi
Hiroshi
H
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D054337:Cell Dedifferentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D020219:Chondrogenesis / Q000502:physiology*; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D018450:Disease Progression; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D055747:SOX Transcription Factors / Q000494:pharmacology; D058305:Sorting Nexins; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D055396:Bone Morphogenetic Protein 2; C499177:COL2A1 protein, human; D002352:Carrier Proteins; D024043:Collagen Type II; D055747:SOX Transcription Factors; C524095:Snx19 protein, mouse; D058305:Sorting Nexins; D016212:Transforming Growth Factor beta; D033921:Vesicular Transport Proteins