A tumor protein p63 isoform that induces rheumatoid arthritis and is targeted by methotrexate

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint deterioration. The clinical outcomes of patients with active RA can be improved using anti-rheumatic medications, such as methotrexate (MTX). Many patients rely on MTX to limit the destructive joint damage and functional disability typical of RA. Although the drug is a folic acid antagonist, its precise mechanisms in RA patients are largely unknown.

Previous research suggests that MTX also affects a type of white blood cell called CD4+ T cells. These cells are believed to play a role in the development of RA—specifically, the balance between the activation of interleukin-17-producing helper T (Th17) cells and CD4+ regulatory T (Treg). Researchers suspect that MTX affects CD4+ T cells by suppressing T cell activity and increasing Treg cells, but its specific effects besides folate metabolism remain unclear.

Recently, a group of researchers discovered that MTX targets tumor protein p63 (TP63) in CD4+ T cells. Their findings were published the JCI Insight journal.

"We were keen to profile gene expression before and after MTX treatment since the drug likely targets CD4T cells, and little is known about its influence on gene expression in patients with active RA," explains a team author.

The researchers employed DNA microarray profiling of human CD4+ T cells from RA patients to understand how MTX influences gene expression. They also used gene knockdown—a molecular technique to suppress a target gene—and RNA sequencing (RNA-Seq) to validate gene function. The researchers found that TAp63, a protein isoform of TP63, was highly expressed in human and mouse Th17 cells.

 The author says, "Patients who received MTX treatment had significantly lower TAp63 messenger RNA expression in their CD4T cells. MTX also suppressed TAp63 proteins in human and mouse Th17 cells. TAp63 suppression in mouse Th17 cells resulted in the improvement of autoimmune arthritis in mice."

The RNA-Seq and gene knockdown data revealed that another gene, FOXP3, the master regulator of Treg cells, was targeted by TAp63. When TAp63 was "knocked down" in Treg cells, Foxp3 protein expression increased. By performing a reporter assay, the researchers confirmed that TAp63 was bound to the FOXP3 enhancer and suppressed it.

Together, these findings suggest that TAp63 is intricately linked to the balance of Th17 and Treg cell differentiation. Thus, inhibiting TAp63 could enhance the suppressive function of Treg cells and limit autoimmune RA.