title: The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-gamma-mediated inhibition in human chondrocytes: requirement for Stat1 alpha, Jak1 and Jak2.
abstract: Interferon-gamma (IFN-gamma) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene ( COL2A1 ) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-gamma and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-gamma inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-gamma suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning -6368 to +125 bp in the absence and presence of the intronic enhancer and stimulated activity of the gamma-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1 alpha-binding activity in nuclear extracts. These responses to IFN-gamma were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1 alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma 2A)- and Stat1 alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1 alpha, but not by either Stat1 alpha Y701F or Stat1 beta. Functional analysis using deletion constructs showed that the IFN-gamma response was retained in the COL2A1 core promoter region spanning -45 to +11 bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-gamma persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN-gamma requires Jak1, Jak2 and Stat1 alpha and suggest that this response involves indirect interaction of activated Stat1 alpha with the general transcriptional machinery that drives constitutive COL2A1 expression.
authors.affiliation: Beth Israel Deaconess Medical Center, Rheumatology Division, and New England Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Boston, MA 02115, USA.
authors.forename: Makoto
authors.initials: M
authors.lastname: Osaki
authors.forename: Lujian
authors.initials: L
authors.lastname: Tan
authors.forename: Bob K
authors.initials: BK
authors.lastname: Choy
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yoshida
authors.forename: Kathryn S E
authors.initials: KS
authors.lastname: Cheah
authors.forename: Philip E
authors.initials: PE
authors.lastname: Auron
authors.forename: Mary B
authors.initials: MB
authors.lastname: Goldring
mesh_terms: D001483:Base Sequence; D002461:Cell Line, Transformed; D019902:Chondrocytes / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3; D007371:Interferon-gamma / Q000502:physiology*; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D024043:Collagen Type II; D050832:Interferon-Stimulated Gene Factor 3; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; C079868:gamma interferon activation factor; D007371:Interferon-gamma; D004247:DNA; D011505:Protein-Tyrosine Kinases; C507904:JAK1 protein, human; C507924:JAK2 protein, human; D053613:Janus Kinase 1; D053614:Janus Kinase 2