The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-γ-mediated inhibition in human chondrocytes: Requirement for Stat1α, Jak1 and Jak2

Abstract

Interferon-γ (IFN-γ) 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-γ 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-γ 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-γ 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 γ-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1α-binding activity in nuclear extracts. These responses to IFN-γ were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1α Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-γ had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (γ2A)- and Stat1α (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-γ was rescued by overexpression of Stat1α, but not by either Stat1α Y701F or Stat1β. Functional analysis using deletion constructs showed that the IFN-γ 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-γ persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN-γ requires Jak1, Jak2 and Stat1α and suggest that this response involves indirect interaction of activated Stat1α with the general transcriptional machinery that drives constitutive COL2A1 expression.