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The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis.

Carrick DM, Lai WS, Blackshear PJ - Arthritis Res. Ther. (2004)

Bottom Line: The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine.Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex.The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Clinical Research, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.

ABSTRACT
Tristetraprolin (TTP) is the best-studied member of a small family of three proteins in humans that is characterized by a tandem CCCH zinc finger (TZF) domain with highly conserved sequences and spacing. Although initially discovered as a gene that could be induced rapidly and transiently by the stimulation of fibroblasts with growth factors and mitogens, it is now known that TTP can bind to AU-rich elements in mRNA, leading to the removal of the poly(A) tail from that mRNA and increased rates of mRNA turnover. This activity was discovered after TTP-deficient mice were created and found to have a systemic inflammatory syndrome with severe polyarticular arthritis and autoimmunity, as well as medullary and extramedullary myeloid hyperplasia. The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine. The myeloid hyperplasia might be due in part to increased stability of granulocyte-macrophage colony-stimulating factor (GM-CSF). This review highlights briefly the characteristics of the TTP-deficiency syndrome in mice and its possible genetic modifiers, as well as recent data on the characteristics of the TTP-binding site in the TNF-alpha and GM-CSF mRNAs. Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex. We review the current knowledge of TTP sequence variants in humans and discuss the possible contributions of the TTP-related proteins in mouse physiology and in human monocytes. The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

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Tristetraprolin (TTP), ZFP36L1, and ZFP36L2 expression patterns in human monocytes stimulated with lipopolysaccharide (LPS). Purified monocytes from healthy human subjects (n = 5) were stimulated with LPS (or phosphate-buffered saline as control). Total cellular RNA from the monocytes was converted to cDNA and analyzed by real-time polymerase chain reaction for (a) TTP, (b) ZFP36L1, and (c) ZFP36L2 expression levels. Resulting Ct values were normalized to the geometric mean of four internal control transcripts and then to corresponding samples from PBS-treated cultures at the same time points, then converted to 2-ΔΔCt. The normalized values were then expressed as a fraction of the mean value at which maximum expression occurred (t = 1 hour for TTP and ZFP36L2; t = 1.5 hours for ZFP36L1). These were then expressed as means ± s.e.m.
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Figure 8: Tristetraprolin (TTP), ZFP36L1, and ZFP36L2 expression patterns in human monocytes stimulated with lipopolysaccharide (LPS). Purified monocytes from healthy human subjects (n = 5) were stimulated with LPS (or phosphate-buffered saline as control). Total cellular RNA from the monocytes was converted to cDNA and analyzed by real-time polymerase chain reaction for (a) TTP, (b) ZFP36L1, and (c) ZFP36L2 expression levels. Resulting Ct values were normalized to the geometric mean of four internal control transcripts and then to corresponding samples from PBS-treated cultures at the same time points, then converted to 2-ΔΔCt. The normalized values were then expressed as a fraction of the mean value at which maximum expression occurred (t = 1 hour for TTP and ZFP36L2; t = 1.5 hours for ZFP36L1). These were then expressed as means ± s.e.m.

Mentions: For the data shown in Fig. 8, RNA from LPS-stimulated or PBS-stimulated monocytes from five healthy human subjects was analyzed for expression of TTP, ZFP36L1, and ZFP36L2 transcript levels, along with four internal control transcripts (18S rRNA, PSMB6, HNRPL, and PSMD7). The ΔΔCt method of analysis [57] was used to determine changes in gene expression. The method of normalizing the gene expression data is based on research in [58], in which the geometric mean of several internal controls was used to normalize the gene expression data. The internal controls in the present study were selected on the basis of the previous identification of these genes as being stably expressed in adult and fetal tissue [59] and the absence of evidence from previous work that these genes were affected by LPS. Furthermore, to minimize plate-to-plate variability between real-time PCR assays, the signal from LPS-treated RNA was normalized to the signal from PBS-treated RNA from the same subject (at the same time points), assayed together on the same plate.


The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis.

Carrick DM, Lai WS, Blackshear PJ - Arthritis Res. Ther. (2004)

Tristetraprolin (TTP), ZFP36L1, and ZFP36L2 expression patterns in human monocytes stimulated with lipopolysaccharide (LPS). Purified monocytes from healthy human subjects (n = 5) were stimulated with LPS (or phosphate-buffered saline as control). Total cellular RNA from the monocytes was converted to cDNA and analyzed by real-time polymerase chain reaction for (a) TTP, (b) ZFP36L1, and (c) ZFP36L2 expression levels. Resulting Ct values were normalized to the geometric mean of four internal control transcripts and then to corresponding samples from PBS-treated cultures at the same time points, then converted to 2-ΔΔCt. The normalized values were then expressed as a fraction of the mean value at which maximum expression occurred (t = 1 hour for TTP and ZFP36L2; t = 1.5 hours for ZFP36L1). These were then expressed as means ± s.e.m.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1064869&req=5

Figure 8: Tristetraprolin (TTP), ZFP36L1, and ZFP36L2 expression patterns in human monocytes stimulated with lipopolysaccharide (LPS). Purified monocytes from healthy human subjects (n = 5) were stimulated with LPS (or phosphate-buffered saline as control). Total cellular RNA from the monocytes was converted to cDNA and analyzed by real-time polymerase chain reaction for (a) TTP, (b) ZFP36L1, and (c) ZFP36L2 expression levels. Resulting Ct values were normalized to the geometric mean of four internal control transcripts and then to corresponding samples from PBS-treated cultures at the same time points, then converted to 2-ΔΔCt. The normalized values were then expressed as a fraction of the mean value at which maximum expression occurred (t = 1 hour for TTP and ZFP36L2; t = 1.5 hours for ZFP36L1). These were then expressed as means ± s.e.m.
Mentions: For the data shown in Fig. 8, RNA from LPS-stimulated or PBS-stimulated monocytes from five healthy human subjects was analyzed for expression of TTP, ZFP36L1, and ZFP36L2 transcript levels, along with four internal control transcripts (18S rRNA, PSMB6, HNRPL, and PSMD7). The ΔΔCt method of analysis [57] was used to determine changes in gene expression. The method of normalizing the gene expression data is based on research in [58], in which the geometric mean of several internal controls was used to normalize the gene expression data. The internal controls in the present study were selected on the basis of the previous identification of these genes as being stably expressed in adult and fetal tissue [59] and the absence of evidence from previous work that these genes were affected by LPS. Furthermore, to minimize plate-to-plate variability between real-time PCR assays, the signal from LPS-treated RNA was normalized to the signal from PBS-treated RNA from the same subject (at the same time points), assayed together on the same plate.

Bottom Line: The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine.Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex.The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Clinical Research, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.

ABSTRACT
Tristetraprolin (TTP) is the best-studied member of a small family of three proteins in humans that is characterized by a tandem CCCH zinc finger (TZF) domain with highly conserved sequences and spacing. Although initially discovered as a gene that could be induced rapidly and transiently by the stimulation of fibroblasts with growth factors and mitogens, it is now known that TTP can bind to AU-rich elements in mRNA, leading to the removal of the poly(A) tail from that mRNA and increased rates of mRNA turnover. This activity was discovered after TTP-deficient mice were created and found to have a systemic inflammatory syndrome with severe polyarticular arthritis and autoimmunity, as well as medullary and extramedullary myeloid hyperplasia. The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine. The myeloid hyperplasia might be due in part to increased stability of granulocyte-macrophage colony-stimulating factor (GM-CSF). This review highlights briefly the characteristics of the TTP-deficiency syndrome in mice and its possible genetic modifiers, as well as recent data on the characteristics of the TTP-binding site in the TNF-alpha and GM-CSF mRNAs. Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex. We review the current knowledge of TTP sequence variants in humans and discuss the possible contributions of the TTP-related proteins in mouse physiology and in human monocytes. The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

Show MeSH
Related in: MedlinePlus