STAT3 Protein-Protein Interaction Analysis Finds P300 as a Regulator of STAT3 and Histone 3 Lysine 27 Acetylation in Pericytes

Abstract

Background: Signal transducer and activator of transcription 3 (STAT3) is a member of the cytoplasmic inducible transcription factors and plays an important role in mediating signals from cytokines, chemokines, and growth factors. We and others have found that STAT3 directly regulates pro-fibrotic signaling in the kidney. The STAT3 protein-protein interaction plays an important role in activating its transcriptional activity. It is necessary to identify these interactions to investigate their function in kidney disease. Here, we investigated the protein-protein interaction among three species to find crucial interactions that can be targeted to alleviate kidney disease.

Method: In this study, we examined common protein-protein interactions leading to the activation or downregulation of STAT3 among three different species: humans (Homo sapiens), mice (Mus musculus), and rabbits (Oryctolagus cuniculus). Further, we chose to investigate the P300 and STAT3 interaction and performed studies of the activation of STAT3 using IL-6 and inhibition of the P300 by its specific inhibitor A-485 in pericytes. Next, we performed immunoprecipitation to confirm whether A-485 inhibits the binding of P300 to STAT3.

Results: Using the STRING application from ExPASy, we found that six proteins, including PIAS3, JAK1, JAK2, EGFR, SRC, and EP300, showed highly confident interactions with STAT3 in humans, mice, and rabbits. We also found that IL-6 treatment increased the acetylation of STAT3 and increased histone 3 lysine acetylation (H3K27ac). Furthermore, we found that the disruption of STAT3 and P300 interaction by the P300 inhibitor A-485 decreased STAT3 acetylation and H3K27ac. Finally, we confirmed that the P300 inhibitor A-485 inhibited the binding of STAT3 with P300, which inhibited its transcriptional activity by reducing the expression of Ccnd1 (Cyclin D1).

Conclusions: Targeting the P300 protein interaction with STAT3 may alleviate STAT3-mediated fibrotic signaling in humans and other species.

Keywords: EGFR; EP300; PIAS3; STAT3 acetylation; STRING; histone acetylation; pericytes; protein–protein interaction.

Figure 1

The boxplot shows interaction score distribution (X-axis) for STAT3-interacting proteins based on sources of evidence (Y-axis). We found that the aggregated score (combined_score) is mainly driven by text mining, which is known to have noise and false positive results. In addition, there is almost no evidence for homology. This suggests that major sources of identifying STAT3-interacting proteins are experimentally determined or appear in similar pathways. Some of these proteins also show coexpression statistically derived from RNA-seq data.

Figure 2

The heatmap shows the confidence score of every protein (shown as individual columns) estimated from each data source (shown as individual rows). The color represents the confidence score for each element in the metrics that ranges between [0, 1]. Clustering is performed on STAT3-associated proteins to group them based on scores from similar sources. Three groups of proteins are identified based on their association with STAT3 estimated from various sources. These sets of proteins are described in the heatmap.

Figure 3

A protein–protein interaction network is created for STAT3-associated proteins from the STRING database. (A–C) Three different organisms (humans, mice, and rabbits) were selected to create the PPI network, where nodes are colored based on their cluster membership. Red represents the cluster with the largest number of proteins, green the cluster with a medium number, and blue the cluster with the smallest number of proteins. For each network, the annotation includes the total number of proteins interacting with STAT3, the total number of interactions amongst these proteins, and the total number of clusters identified. (D) The Venn diagram shows the overlap between these STAT3-interacting proteins in humans, mice, and rabbits. (E) The list of the top 25 proteins that are common in all three species is shown.

Figure 4

The inhibition of P300 inhibits IL-6-induced STAT3 acetylation and its binding. Immunofluorescence staining shows that (A) IL-6 treatment increased the Histone 3 Lysine acetylation (H3K27ac), which was decreased by the P300 inhibitor (A-485). The right panels show the quantitation of image intensity as log corrected total cell fluorescence (CTCF). Data are represented as ±SEM. (B) IL-6 treatment increased the acetylation of STAT3 on the Lysine 685 residue, which was inhibited by A-485 in pericytes, 10T1/2. Scale bar = 10 μm. The right panels show the quantitation of image intensity as log CTCF. The data are represented as ±SEM. (C) RT-PCR to detect Ccnd1 (Cyclin D1) gene expression using RT-PCR. Fold changes relative to Ctrl were plotted after normalizing them to Gapdh. Data are represented as ±SD. (D) The immunoprecipitation of STAT3 and immunoblotting for P300 showed the binding of STAT3 with P300 and a decrease in P300 binding following A-485 treatment in pericytes, 10T1/2. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.