The protein phosphatase inhibitor calyculin A stimulates chemokine production by human synovial cells

Abstract

Cultured human synovial fibroblasts express mRNA for the chemotactic cytokines (chemokines) interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and regulated upon activation normal T-cell expressed and presumably secreted (RANTES), when stimulated with IL-1 or tumour necrosis factor alpha (TNF alpha). Calyculin A, a potent type 1/2A protein serine/threonine phosphatase inhibitor, was used to examine the role of protein phosphatases in the regulation of chemokine gene expression. Calyculin A (1 nM) mimicked IL-1 by inducing IL-8 and MCP-1 mRNA expression in synovial cells. IL-8 mRNA was induced over a similar time period (1-6 h) in response to IL-1 or calyculin A, whereas MCP-1 mRNA was induced more rapidly (1-2 h) by calyculin A than by IL-1 (4-6 h). Expression of RANTES mRNA occurred in response to TNF alpha, but could not be induced by stimulation with calyculin A alone. These results suggest that inhibition of protein phosphatase type 1/2A may have a differential role in the regulation of the expression of each of the chemokine genes. Synovial fibroblasts also secreted IL-8 and IL-6 peptide when stimulated with either IL-1/TNF alpha or calyculin A. The amount of IL-8 and IL-6 peptide produced in response to calyculin A was significantly increased above that produced by untreated synovial cells, though it was much less than the amount induced by IL-1 or TNF alpha. Calyculin A also acted synergistically with IL-1 or TNF alpha to cause a 2-fold potentiation of IL-1- or TNF alpha-induced IL-8 mRNA and peptide and RANTES mRNA expression. These results suggest that although inhibition of a protein phosphatase may be able to regulate the magnitude of IL-1-induced chemokine gene expression, the IL-1 signal transduction pathway involves components in addition to phosphatase inhibition, possibly including the activation of a protein kinase, the action of which may be opposed by a protein phosphatase inhibited by calyculin A.