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Br J Pharmacol. 2007 Feb;150(3):271-80. Epub 2007 Jan 2.

Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase-1 expression and activity.

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  • 1Pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.

Abstract

BACKGROUND AND PURPOSE:

Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved.

EXPERIMENTAL APPROACH:

MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate.

KEY RESULTS:

Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration.

CONCLUSIONS AND IMPLICATIONS:

These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.

PMID:
17200676
PMCID:
PMC2013899
DOI:
10.1038/sj.bjp.0706983
[PubMed - indexed for MEDLINE]
Free PMC Article
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