Stat1-independent regulation of gene expression in response to IFN-gamma

Abstract

Although Stat1 is essential for cells to respond fully to IFN-gamma, there is substantial evidence that, in the absence of Stat1, IFN-gamma can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-gamma in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-gamma in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-gamma, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-gamma in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-gamma receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-gamma, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling.

Figure 1

Regulation of immediate-early gene expression in response to IFN-γ in Stat1-null and wild-type fibroblasts. Total RNA, prepared from serum-starved cells, untreated or treated with mouse IFN-γ (1,000 units/ml) or 10% serum for the times indicated, was subjected to RNase protection analysis.

Figure 2

IFN-γ-dependent regulation of chemokine gene expression in serum-starved Stat1-null and wild-type fibroblasts. Total RNA was prepared from MEFs, untreated or treated with mouse IFN-γ (1,000 units/ml). RNase protection analysis was used.

Figure 3

The transcription factors EGR and C/EBP-β are induced by IFN-γ independently of Stat1. (A) Western analysis of tyrosine-phosphorylated Stat1 and EGR-1. (B) Electrophoretic mobility-shift assays with an EGR probe. Whole-cell extracts were prepared from serum-starved Stat1-null or wild-type fibroblasts treated with IFN-γ. (C) Western analysis of C/EBP-β in Stat1-null and wild-type fibroblasts.

Figure 4

PDGF-dependent induction of gene expression in Stat1-null and wild-type fibroblasts. (A) Stat1-null or wild-type MEFs were untreated or treated for 30 min with IFN-γ or PDGF alone, or with PDGF plus IFN-γ. c-Jun mRNA levels were determined by Northern analysis. (B) Stat1-null or wild-type MEFs were untreated or treated with PDGF for 2 h. MCP-1 mRNA levels were determined by Northern analysis.

Figure 5

Mutation of the Stat1 docking site of IFNGR1 allows IFN-γ to induce the expression of c-myc and c-jun. (A) Cell cycle analysis. Cells grown to 20% confluence in 5% serum were serum-starved for 48 h and then not treated (control) or treated with 10% serum, alone or in combination with human IFN-γ. The cells were stained with propidium iodide 24 h later, and the DNA content was analyzed by flow cytometry. The percentages of cells in the G₁, S, and G₂ phases of the cell cycle are indicated. (B) Induction of immediate-early genes. Subconfluent, serum-starved hGR or Y440F cells were untreated or treated with 1,000 units/ml of human IFN-γ for 30 min, 1 h, or 4 h. c-Myc, c-jun, and GAPDH mRNA levels were determined by Northern analysis.