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

• Published in: Biochemical journal Vol. 369; no. Pt 1; pp. 103 - 115
• Main Authors: Osaki, Makoto, Tan, Lujian, Choy, Bob K, Yoshida, Yasuhiro, Cheah, Kathryn S E, Auron, Philip E, Goldring, Mary B
• Format: Journal Article
• Language: English
• Published: England 01.01.2003
• Subjects: Base Sequence; Cell Line, Transformed; Chondrocytes - metabolism; Collagen Type II - genetics; DNA - metabolism; Humans; Interferon-gamma - physiology; Interferon-Stimulated Gene Factor 3; Janus Kinase 1; Janus Kinase 2; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins; RNA, Messenger - genetics; Sequence Homology, Nucleic Acid; TATA Box; Transcription Factors - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20020928

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.