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Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats.

Lin H, Baby N, Lu J, Kaur C, Zhang C, Xu J, Ling EA, Dheen ST - J Neuroinflammation (2011)

Bottom Line: Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro.This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment.We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, Second Military Medical University, 800 Xiangyin Road, Shanghai, 200433, PR China.

ABSTRACT
Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.

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Quantitative analysis of number, intensity and mRNA expression of SphK1 positive AMC.  The number of SphK1-positive AMC (co-expressing OX-42) is increased 1 h after LPS injection (A). The intensity of SphK1 immunofluorescence is also increased significantly at 30 min to 1 h compared with that 6 h after LPS injection and that of control (B). SphK1 mRNA expression (C) is detected in primary microglial cells in vitro and its expression was induced significantly in cells exposed to LPS for 1 h, as revealed by RT-PCR (D) The data are normalized with the housekeeping gene, β-actin (C) and presented as mean ± S.E. Control vs LPS, * P < 0.05, ** P < 0.01.,*** P < 0.001
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Figure 4: Quantitative analysis of number, intensity and mRNA expression of SphK1 positive AMC. The number of SphK1-positive AMC (co-expressing OX-42) is increased 1 h after LPS injection (A). The intensity of SphK1 immunofluorescence is also increased significantly at 30 min to 1 h compared with that 6 h after LPS injection and that of control (B). SphK1 mRNA expression (C) is detected in primary microglial cells in vitro and its expression was induced significantly in cells exposed to LPS for 1 h, as revealed by RT-PCR (D) The data are normalized with the housekeeping gene, β-actin (C) and presented as mean ± S.E. Control vs LPS, * P < 0.05, ** P < 0.01.,*** P < 0.001

Mentions: In 5-d-old rats injected with LPS, the incidence of SphK1-immunopositive AMC as well as SphK1 immunoreactivity in corpus callosum was increased compared to that of corresponding control rats with saline injection (Figure 3Aa-Dc, Figure 4A, B). The number of SphK1-positive AMC (co-expressing OX-42) was found to be significantly increased at 1 h compared with that at 30 min or 6 h after LPS injection and that of controls (Figure 3Ba-c, Ca-c, Da-c, Figure 4A). The intensity of SphK1 immunofluorescence was found to be increased significantly at 30 min to 1 h compared with that 6 h after LPS injection and that of controls (Figure 4B). Further, mRNA expression of SphK1 (Figure 4C) was detected in primary microglial cells in vitro and its expression was induced significantly in cells exposed to LPS for 1 h (Figure 4D).


Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats.

Lin H, Baby N, Lu J, Kaur C, Zhang C, Xu J, Ling EA, Dheen ST - J Neuroinflammation (2011)

Quantitative analysis of number, intensity and mRNA expression of SphK1 positive AMC.  The number of SphK1-positive AMC (co-expressing OX-42) is increased 1 h after LPS injection (A). The intensity of SphK1 immunofluorescence is also increased significantly at 30 min to 1 h compared with that 6 h after LPS injection and that of control (B). SphK1 mRNA expression (C) is detected in primary microglial cells in vitro and its expression was induced significantly in cells exposed to LPS for 1 h, as revealed by RT-PCR (D) The data are normalized with the housekeeping gene, β-actin (C) and presented as mean ± S.E. Control vs LPS, * P < 0.05, ** P < 0.01.,*** P < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050721&req=5

Figure 4: Quantitative analysis of number, intensity and mRNA expression of SphK1 positive AMC. The number of SphK1-positive AMC (co-expressing OX-42) is increased 1 h after LPS injection (A). The intensity of SphK1 immunofluorescence is also increased significantly at 30 min to 1 h compared with that 6 h after LPS injection and that of control (B). SphK1 mRNA expression (C) is detected in primary microglial cells in vitro and its expression was induced significantly in cells exposed to LPS for 1 h, as revealed by RT-PCR (D) The data are normalized with the housekeeping gene, β-actin (C) and presented as mean ± S.E. Control vs LPS, * P < 0.05, ** P < 0.01.,*** P < 0.001
Mentions: In 5-d-old rats injected with LPS, the incidence of SphK1-immunopositive AMC as well as SphK1 immunoreactivity in corpus callosum was increased compared to that of corresponding control rats with saline injection (Figure 3Aa-Dc, Figure 4A, B). The number of SphK1-positive AMC (co-expressing OX-42) was found to be significantly increased at 1 h compared with that at 30 min or 6 h after LPS injection and that of controls (Figure 3Ba-c, Ca-c, Da-c, Figure 4A). The intensity of SphK1 immunofluorescence was found to be increased significantly at 30 min to 1 h compared with that 6 h after LPS injection and that of controls (Figure 4B). Further, mRNA expression of SphK1 (Figure 4C) was detected in primary microglial cells in vitro and its expression was induced significantly in cells exposed to LPS for 1 h (Figure 4D).

Bottom Line: Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro.This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment.We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, Second Military Medical University, 800 Xiangyin Road, Shanghai, 200433, PR China.

ABSTRACT
Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.

Show MeSH
Related in: MedlinePlus