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.
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.
Osaki
Makoto
M
Beth Israel Deaconess Medical Center, Rheumatology Division, and New England Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Boston, MA 02115, USA.
Tan
Lujian
L
Choy
Bob K
BK
Yoshida
Yasuhiro
Y
Cheah
Kathryn S E
KS
Auron
Philip E
PE
Goldring
Mary B
MB
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*
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