Corticosteroids modulate the expression of the PKC-anchoring protein RACK-1 and cytokine release in THP-1 cells.
We demonstrated that cortisol reduces the expression of RACK-1 (Receptor for Activated C Kinase-1), a protein required for immune cell activation. The aim of this study was to evaluate whether and to what extent other clinically relevant corticosteroids may modulate RACK-1 expression. We used the human promyelocytic cell line THP-1 to investigate the effects of cortisol, prednisone, prednisolone, budesonide, betamethasone and methylprednisolone on RACK-1 expression and cytokine production. As anticipated, all corticosteroids inhibited at non-cytotoxic concentrations in a dose and time related manner LPS-induced TNF-α and IL-8 release, with budesonide, betamethasone and methylprednisolone being the most active followed by prednisolone, cortisol and prednisone. To a similar extent, all corticosteroids also reduced RACK-1 mRNA expression and RACK-1 protein levels as assessed by Real Time PCR and Western blot, respectively. Prednisone was the least potent compound while betamethasone and methylprednisolone where the most active. A good correlation was observed between RACK-1 mRNA or protein levels and cytokine release (Pearson r=0.7376, p=0.0471 for RACK-1 mRNA and TNF-α release, and Pearson r=0.8108, p=0.0252 for RACK-1 protein and IL-8 release). Mifepristone, a potent glucocorticoid receptor (GR) antagonist, completely prevented the effect of cortisol, demonstrating that RACK-1 downregulation is via GR. Furthermore, to by-pass the defective PKC activation due to the decrease in RACK-1, we used a RACK-1 pseudosubstrate, that directly activates PKC-beta. RACK-1 pseudosubstrate was able to restore LPS-induced cytokine production affected by cortisol, supporting the role of RACK-1 in the anti-inflammatory effect of corticosteroids. These results confirm the involvement of RACK-1 in immune cell activation and identify this protein as a novel transcriptional target of corticosteroid-induced anti-inflammatory effects.
Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano, Milan, Italy. Electronic address: emanuela.corsini@unimi.it.
Emanuela
E
Corsini
Department of Drug Sciences - Pharmacology, University of Pavia, Pavia, Italy.
Antonella
A
Pinto
Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano, Milan, Italy.
Valentina
V
Galbiati
Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano, Milan, Italy.
Barbara
B
Viviani
Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano, Milan, Italy.
Corrado L
CL
Galli
Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano, Milan, Italy.
Marina
M
Marinovich
Department of Drug Sciences - Pharmacology, University of Pavia, Pavia, Italy.
Marco
M
Racchi
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D013256:Steroids / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000893:Anti-Inflammatory Agents; C508606:CXCL8 protein, human; D005938:Glucocorticoids; D016209:Interleukin-8; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D013256:Steroids; D014409:Tumor Necrosis Factor-alpha; D019204:GTP-Binding Proteins
Corticosteroids; Cytokines; Protein kinase C; Signal transduction