Calyculin A Reveals Serine/Threonine Phosphatase Protein Phosphatase 1 as a Regulatory Nodal Point in Canonical Signal Transducer and Activator of Transcription 3 Signaling of Human Microvascular Endothelial Cells

Department of Pharmacology and Toxicology, Center for Excellence in Cardiovascular-Renal Research, School of Medicine, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research (Impact Factor: 2). 12/2011; 32(2):87-94. DOI: 10.1089/jir.2011.0059
Source: PubMed


Vascular inflammation is initiated by stimuli acting on endothelial cells. A clinical feature of vascular inflammation is increased circulating interleukin 6 (IL-6) type cytokines such as leukemia inhibitory factor (LIF), but their role in vascular inflammation is not fully defined. IL-6 type cytokines activate transcription factor signal transducer and activator of transcription 3 (STAT3), which has a key role in inflammation and the innate immune response. Canonical STAT3 gene induction is due to phosphorylation of (1) Y705, leading to STAT3 dimerization and DNA binding and (2) S727, enhancing homodimerization and DNA binding by recruiting p300/CBP. We asked whether enhancing S727 STAT3 phosphorylation using the protein phosphatase 1 (PP1) inhibitor, calyculin A, would enhance LIF-induced gene expression in human microvascular endothelial cells (HMEC-1). Cotreatment with calyculin A and LIF markedly increased STAT3 S727 phosphorylation, without affecting the increase in the nuclear fraction of STAT3 phosphorylated on Y705. PP2A inhibitors, okadaic acid and fostriecin, did not enhance STAT3 S727 phosphorylation. Surprisingly, calyculin A eliminated LIF-induced gene expression: (1) calyculin A reduced binding of nuclear extracts to a STAT3 consensus site, thereby reducing the overall level of binding observed with LIF; and (2) calyculin A caused p300/CBP phosphorylation, thus resulting in reduced acetylation activity and degradation. Together, these findings reveal a pivotal role of a protein serine/threonine phosphatases that is likely PP1 in HMEC in controlling STAT3 transcriptional activity.

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    • "Cells were pretreated with AS or vehicle (50 µM NaOH) for 15 min. Immunoprecipitations and Western analyses on cell lysates were carried out as described [12], [24]. Cell viability was measured using alamarBlue and cell proliferation was determined by cell count using a NucleoCounter [25]. "
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    • "Sequence homologies of PP1, C subunit of PP2A and PP2B create difficulties to study inhibition of one phosphatase. Calyculin A, isolated from the marine sponge Discodermia calyx, is a combined large phosphates inhibitor which mainly acts on PP1 and PP2A (Ishihara et al., 1989; Resjo et al., 1999; Janssens and Goris, 2001; Edelson and Brautigan, 2012; Heimfarth et al., 2012; Zgheib et al., 2012). The effects of PP1 can only be attributed to PP1 after deduction of the PP2A specific effects solved by the use of OKA inhibition which has a negligible effect on PP1 activity. "
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