ABSTRACT
OBJECTIVE: To identify the site(s) in tumor necrosis factor (TNFalpha), interleukin-6 (IL-6), and macrophage inflammatory protein-1alpha (MIP-1alpha) biosynthesis that is blocked by SB202190, a selective inhibitor of p38-mitogen activated protein kinase (p38). MATERIALS: Human blood monocytes isolated by centrifugal elutriation. METHODS: Monocytes were stimulated with lipopolysaccharide in the presence of 0, 0.3, 1 and 3 microM SB202190. Induced TNFalpha, IL-6, and MIP-1alpha protein and mRNA were measured by ELISA and quantitative RT-PCR, respectively. The half-lives of cytokine mRNA levels were determined following treatment of cells with actinomycin D or SB202190. RESULTS: SB202190 suppressed >60% of lipopolysaccharide-induced TNFalpha, IL-6, and MIP-1alpha protein and mRNA expression. Suppressed mRNA levels could be attributed to a >2 to 7-fold reduction in cytokine mRNA half-lives. In contrast, SB202190 did not destabilize mRNAs encoding interferon-induced gene 15 protein and glyceraldehyde-3-phosphate dehydrogenase. CONCLUSIONS: Specific mRNA destabilization represents an important and novel site of action for the cytokine suppressive effects of p38 inhibitors.
Subject(s)
Cytokines/genetics , Mitogen-Activated Protein Kinases/antagonists & inhibitors , RNA, Messenger/drug effects , Cells, Cultured , Chemokine CCL3 , Chemokine CCL4 , Cytokines/blood , Enzyme Inhibitors/blood , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Humans , Imidazoles/blood , Imidazoles/pharmacology , Interleukin-6/antagonists & inhibitors , Interleukin-6/biosynthesis , Interleukin-6/blood , Interleukin-6/genetics , Kinetics , Lipopolysaccharides/pharmacology , Macrophage Inflammatory Proteins/antagonists & inhibitors , Macrophage Inflammatory Proteins/biosynthesis , Macrophage Inflammatory Proteins/blood , Macrophage Inflammatory Proteins/genetics , Mitogen-Activated Protein Kinases/blood , Monocytes/drug effects , Monocytes/metabolism , Pyridines/blood , Pyridines/pharmacology , RNA Stability/drug effects , RNA, Messenger/blood , RNA, Messenger/chemistry , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , p38 Mitogen-Activated Protein KinasesABSTRACT
STCP-1 stimulated T cell chemoattractant protein-1 (STCP-1) (macrophage-derived chemokine; MDC), a recently described CC chemokine for chronically activated T lymphocytes, was found to act specifically on a subset of memory CD4 lymphocytes that displayed a Th2 cytokine profile. Also, STCP-1, thymus and activation regulated chemokine (TARC), eotaxin, and eotaxin-2 acted specifically on in vitro derived Th2 lymphocytes, while IP-10 (IFN-gamma-inducible 10-kDa protein) showed some preference for Th1 lymphocytes. The corresponding receptors for eotaxin, TARC, and IP-10 are also differentially expressed on Th1 and Th2 lymphocytes. In desensitization Ca flux experiments, TARC and STCP-1 bound to a common receptor and therefore at least one chemokine receptor for STCP-1 is CCR4. STCP-1 expression is restricted to immune cells. Dendritic cells, B cells, and macrophages produce STCP-1 constitutively, while NK cells, monocytes, and CD4 lymphocytes produce STCP-1 upon appropriate stimulation. Production of STCP-1 is positively modulated by Th2 cytokines IL-4 and IL-13 but inhibited by IL-10.
Subject(s)
Chemokines, CC/physiology , Chemokines/pharmacology , Chemotaxis, Leukocyte/drug effects , Immunologic Memory , Interleukin-13/physiology , Interleukin-4/physiology , Lymphocyte Activation , Monocytes/drug effects , Receptors, Chemokine/drug effects , Animals , B-Lymphocytes/metabolism , Calcium Signaling , Chemokine CCL11 , Chemokine CCL17 , Chemokine CCL22 , Chemokine CCL24 , Chemokine CCL5/pharmacology , Chemokine CXCL10 , Chemokines, CC/biosynthesis , Chemokines, CC/pharmacology , Chemokines, CXC/pharmacology , Cytokines/pharmacology , Dendritic Cells/metabolism , Feedback , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-10/pharmacology , Interleukin-13/pharmacology , Interleukin-4/pharmacology , Killer Cells, Natural/metabolism , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Monocytes/metabolism , Receptors, CCR3 , Receptors, CCR4 , Receptors, CXCR3 , Receptors, Chemokine/analysis , Receptors, Chemokine/physiologyABSTRACT
RNA aptamers were selected against an affinity column containing a farnesylated peptide modeled after the carboxyl terminus of K ras, the major oncogenic form of this small G protein family. After 10-rounds of selection, 25% of the RNA applied to the column could be specifically eluted. Sequence analysis of the binding RNA aptamers revealed two consensus sequences--GGGUGGG and GGGAGG. Quantitative fluorescence binding studies on two of the high-affinity aptamers, showed a binding affinities of 139 nM and 0.93 microM, respectively for the farnesylated peptide. Binding to the nonfarnesylated peptide was at least 10-fold weaker, showing that the aptamers can recognize the hydrophobic farnesyl moiety. High affinity aptamers could be useful in specifically interfering with oncogenic ras function in particular, and G proteins in general.