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Sphingosine kinase-1 mediates gene expression regulation of a monocyte chemoattractant protein-1 gene

A technology of sphingosine kinase and monocytes, applied in the field of gene expression regulation of monocyte chemoattractant protein-1 gene mediated by sphingosine kinase-1, can solve the problem of no small molecule antagonist

Inactive Publication Date: 2008-01-09
JANSSEN PHARMA NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, despite intensive screening, there are still no clinically available small molecule antagonists of the CCR2 receptor (Daly et al. Microcirculation. 2003 Jun;10(3-4):247-57)

Method used

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  • Sphingosine kinase-1 mediates gene expression regulation of a monocyte chemoattractant protein-1 gene
  • Sphingosine kinase-1 mediates gene expression regulation of a monocyte chemoattractant protein-1 gene
  • Sphingosine kinase-1 mediates gene expression regulation of a monocyte chemoattractant protein-1 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0165] cDNA microarray study of gene expression in HMVEC

[0166] In an effort to rapidly evaluate the potential global role of SK1 in endothelial cell function, cDNA microarray analysis was performed in HMVECs in the absence and presence of the SK1 inhibitor DMS. Cells are stimulated by two receptor subtypes: G protein-coupled receptors, the protease-activated receptor (PAR) family with thrombin as an agonist; and cytokine receptors with TNF-α as an agonist.

[0167] A total of 138 genes were identified that were either induced or repressed under one of the experimental conditions. Fold change results for stimulated cells without and with DMS were plotted along two axes, thus generating dot blots to visualize genes detected above a selected threshold (Figure 1). These analyzes revealed that sphingosine kinase is linked to signaling through the thrombin receptor, an event that had never been observed before. However, this is the first example of previous studies demonstratin...

Embodiment 2

[0174] Specific inhibition of SK-1 abolishes induction of MCP-1 in HMVECs

[0175] Although DMS has been shown to inhibit SK activity at lower concentrations, it can also affect the activity of protein kinase C family members as well as casein kinases at higher concentrations. Therefore, small interfering RNA (siRNA) was used to selectively inhibit SK by specifically targeting SK family members, specifically human SK1 or SK2. With the addition of fluorescein to the 3' end of the siRNA, we were able to observe nearly 100% siRNA transfection efficiency. The designed siRNAs showed specificity to their respective human SK (hSK) isoforms as detected by Taqman quantitative RT-PCR (Figure 2). When hSK1 transcripts were examined, it was observed that only hSK1-specific siRNA inhibited hSK1 expression, while hSK2-specific siRNA and control non-silencing siRNA had no effect on hSK1 expression pattern. Similarly, when detecting hSK2 transcript levels, only hSK2-specific siRNA affected ...

Embodiment 3

[0180] PAR expression in HMVEC is restricted by PAR-1, PAR-2 ​​and PAR-4

[0181] Thrombin, a serine protease, can act on many substrates, but we were interested in PAR-activated thrombin activity. With 4 human PARs identified so far, we wanted to determine the PAR expression profile in HMVEC by performing RT-PCR analysis. RT-PCR results showed that HMVEC expressed thrombin-sensitive PAR-1 and PAR-4, and thrombin-insensitive and trypsin-sensitive PAR-2. Under our assay conditions, RT-PCR could not amplify the expected band of the third thrombin-sensitive receptor, PAR-3, suggesting that HMVEC do not express PAR-3 or express PAR-3 at very low levels.

[0182] RT-PCR - HMVEC were cultured and RNA was isolated (TriReagent-BioMol) followed by DNase treatment and washing using the RNeasy Maxi kit (Qiagen). RT-PCR was performed using GC-rich PCR reagent (Invitrogen) and Superscript II reverse transcriptase (Invitrogen). Results were visualized by UV gel electrophoresis and imag...

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Abstract

The present invention relates to methods of identifying, monitoring, and using compounds that regulate the biological activity of Sphingosine kinase-1.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to Application Serial No. 60 / 628,390, filed November 16,2004. field of invention [0003] The present invention relates to methods of identifying, monitoring and using compounds that modulate the biological activity of sphingosine kinase-1. In particular, the methods of the invention involve signal transduction involving sphingosine kinase-1, thrombin, or monocyte chemoattractant protein-1. Background of the invention [0004] Once thought to provide a passive barrier function between the vessel-tissue compartment, the vascular endothelium is now recognized as having a major role in maintaining normal hemostasis and coordinating tissue responses to injury. Increased exposure of the endothelium to broad-spectrum irritants occurs during the acute injury phase, as in mild inflammatory or thrombotic events, when the vasculature comes into contact with cellular components of blood and prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/50
CPCG01N2333/91215G01N33/5064G01N2500/00C12Q1/485G01N33/6863
Inventor A·伯纳尔C·K·德里安
Owner JANSSEN PHARMA NV
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