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
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
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DOI：
10.1042/bj20020928
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发表时间：
2003-01-01
影响因子：
4.1
通讯作者：
Goldring, MB
中科院分区：
文献类型：
--
作者：
Osaki, M;Tan, LJ;Goldring, MB
干扰素-γ（IFN-γ）抑制软骨特异性细胞外基质蛋白II型胶原的合成，并在转录水平抑制II型胶原基因（COL 2A 1）的表达。为了进一步研究这种机制，在永生化的人软骨细胞系C-28/12中研究了COL 2A 1调节序列对IFN-γ的反应以及Janus激酶/信号转导子和转录激活子（JAK/STAT）途径组分的作用。IFN-γ抑制COL 2A 1和聚集蛋白聚糖的mRNA水平，但不抑制Sox 9、L-Sox 5和Sox 6的mRNA水平，所有这些细胞都表达为分化表型的标志物。IFN-γ抑制荧光素酶报告基因结构的表达，该结构含有跨越-6368至+125 bp的COL 2A 1启动子序列，在不存在和存在内含子增强子的情况下，并刺激γ-干扰素激活位点（GAS）荧光素酶报告基因载体的活性，与诱导细胞核提取物中的Stat 1 α结合活性相关。这些对IFN-γ的反应可通过JAK抑制剂JAK结合蛋白（JAB）的过表达而被阻断，或通过在JAK磷酸化位点Tyr-701处含有突变的显性阴性Stat 1 alpha Y 701 F而被逆转。IFN-γ对Jak 1（U4 A）-、Jak 2（gamma 2A）-和Stat 1 alpha（U3 A）-缺陷细胞系中的COL 2A 1启动子表达没有影响。在U3 A细胞系中，对IFN-γ的反应被Stat 1 α的过表达所拯救，但不是由Stat 1 α Y 701 F或Stat 1 β。使用缺失构建体的功能分析表明，IFN-γ应答保留在COL 2A 1核心启动子区域，跨越-45至+11 bp，含有TATA盒和富含GC的序列，但没有Stat 1结合元件。IFN-γ对COL 2A 1启动子活性的抑制在-45/+11 bp区域内存在多个缺失的情况下持续存在。我们的研究结果表明，抑制COL 2A 1基因转录IFN-γ需要Jak 1，Jak 2和Stat 1alpha，并建议这种反应涉及间接相互作用的激活Stat 1alpha与一般的转录机制，驱动组成型COL 2A 1表达。
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-28/12. 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 Stat1alpha-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 Stat1alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma2A)- and Stat1alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1alpha, but not by either Stat1alpha Y701F or Stat1beta. 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 Stat1alpha and suggest that this response involves indirect interaction of activated Stat1alpha with the general transcriptional machinery that drives constitutive COL2A1 expression.