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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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Levels of SphK1 mRNA expression and immunofluorescence intensity and  the number of SphK1 positive cells are reduced after LPS and DMS treatments. RT-PCR analysis demonstrates that SphK1 mRNA expression is increased more than 25 fold in corpus callosum of rats injected with LPS, and the concomitant injection of DMS prevented this increase of SphK1 (A). Injection of DMS alone did not have any effect on SphK1 mRNA expression. Quantitative analysis shows that the number of SphK1-positive AMC decreased in rats injected with DMS+LPS, when compared to that in rats injected with LPS alone (B), Further, the intensity of SphK1 immunofluorescence was also decreased in rats injected with DMS+LPS when compared to that in rats injected with LPS alone (C). Data are presented as mean ± S.E. * vs control, * P < 0.05, ** P < 0.01, *** P < 0.001 Scale bar = 50 μm.
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Figure 8: Levels of SphK1 mRNA expression and immunofluorescence intensity and the number of SphK1 positive cells are reduced after LPS and DMS treatments. RT-PCR analysis demonstrates that SphK1 mRNA expression is increased more than 25 fold in corpus callosum of rats injected with LPS, and the concomitant injection of DMS prevented this increase of SphK1 (A). Injection of DMS alone did not have any effect on SphK1 mRNA expression. Quantitative analysis shows that the number of SphK1-positive AMC decreased in rats injected with DMS+LPS, when compared to that in rats injected with LPS alone (B), Further, the intensity of SphK1 immunofluorescence was also decreased in rats injected with DMS+LPS when compared to that in rats injected with LPS alone (C). Data are presented as mean ± S.E. * vs control, * P < 0.05, ** P < 0.01, *** P < 0.001 Scale bar = 50 μm.

Mentions: As mentioned previously, LPS upregulated SphK1 mRNA and protein expression in AMC of corpus callosum (Figure 7). However, injection of DMS prior to LPS evidently suppressed LPS-induced SphK1 expression in AMC as manifested by its reduced immunofluorescence staining and mRNA expression (Figure 7A-D, Figure 8A-C). A similar phenomenon was observed in microglial cell cultures treated with LPS and DMS + LPS when compared with corresponding controls (Figure 9A-D, Figure 10A, B). The suppression of SphK1 by DMS pretreatment was corroborated by western blot analysis (Figure 10C,D) and RT-PCR (Figure 8A). DMS alone had no significant effect on the expression of SphK1 (Figure 7B, 9C) and this was confirmed by RT-PCR (Figure 8A). LPS-induced TNF-α immunofluorescence intensity (Figure 11C, Figure 12A,B) was found to be reduced in AMC after DMS pretreatment and there was no marked change in LPS-induced IL-1β (Figure 13C, Figure 14A,B) immunofluorescence intensity in those cells. However, real time RT-PCR analysis showed that LPS-induced TNF-α and IL-1β mRNA expression was suppressed in primary culture microglia treated with DMS prior to LPS (Figure 12C and Figure 14C). On the other hand, the intensity of NFκB immunoexpression in AMC found in corpus callosum was significantly increased at 1 h after LPS injection, and NFκB immunofluorescence colocalized with OX-42 (Figure 15Aa-Cc, Figure 16B). Moreover, nuclear translocation of NFκB appeared to be more evident in those cells (Figure 15Bb, Figure 16A). Pretreatment of rats with DMS reduced the incidence of OX-42-labeled, NFκB-immunopositive AMC and NFκB nuclear translocation (Figure 15C). In vitro analysis using primary cultures of microglia also showed that LPS treatment induced NFκB translocation from cytoplasm to the nucleus (Figure 17Ba-Bc). This translocation of NFκB was reversed in activated cells pretreated with DMS (Figure 17Da-Dc).


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)

Levels of SphK1 mRNA expression and immunofluorescence intensity and  the number of SphK1 positive cells are reduced after LPS and DMS treatments. RT-PCR analysis demonstrates that SphK1 mRNA expression is increased more than 25 fold in corpus callosum of rats injected with LPS, and the concomitant injection of DMS prevented this increase of SphK1 (A). Injection of DMS alone did not have any effect on SphK1 mRNA expression. Quantitative analysis shows that the number of SphK1-positive AMC decreased in rats injected with DMS+LPS, when compared to that in rats injected with LPS alone (B), Further, the intensity of SphK1 immunofluorescence was also decreased in rats injected with DMS+LPS when compared to that in rats injected with LPS alone (C). Data are presented as mean ± S.E. * vs control, * P < 0.05, ** P < 0.01, *** P < 0.001 Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 8: Levels of SphK1 mRNA expression and immunofluorescence intensity and the number of SphK1 positive cells are reduced after LPS and DMS treatments. RT-PCR analysis demonstrates that SphK1 mRNA expression is increased more than 25 fold in corpus callosum of rats injected with LPS, and the concomitant injection of DMS prevented this increase of SphK1 (A). Injection of DMS alone did not have any effect on SphK1 mRNA expression. Quantitative analysis shows that the number of SphK1-positive AMC decreased in rats injected with DMS+LPS, when compared to that in rats injected with LPS alone (B), Further, the intensity of SphK1 immunofluorescence was also decreased in rats injected with DMS+LPS when compared to that in rats injected with LPS alone (C). Data are presented as mean ± S.E. * vs control, * P < 0.05, ** P < 0.01, *** P < 0.001 Scale bar = 50 μm.
Mentions: As mentioned previously, LPS upregulated SphK1 mRNA and protein expression in AMC of corpus callosum (Figure 7). However, injection of DMS prior to LPS evidently suppressed LPS-induced SphK1 expression in AMC as manifested by its reduced immunofluorescence staining and mRNA expression (Figure 7A-D, Figure 8A-C). A similar phenomenon was observed in microglial cell cultures treated with LPS and DMS + LPS when compared with corresponding controls (Figure 9A-D, Figure 10A, B). The suppression of SphK1 by DMS pretreatment was corroborated by western blot analysis (Figure 10C,D) and RT-PCR (Figure 8A). DMS alone had no significant effect on the expression of SphK1 (Figure 7B, 9C) and this was confirmed by RT-PCR (Figure 8A). LPS-induced TNF-α immunofluorescence intensity (Figure 11C, Figure 12A,B) was found to be reduced in AMC after DMS pretreatment and there was no marked change in LPS-induced IL-1β (Figure 13C, Figure 14A,B) immunofluorescence intensity in those cells. However, real time RT-PCR analysis showed that LPS-induced TNF-α and IL-1β mRNA expression was suppressed in primary culture microglia treated with DMS prior to LPS (Figure 12C and Figure 14C). On the other hand, the intensity of NFκB immunoexpression in AMC found in corpus callosum was significantly increased at 1 h after LPS injection, and NFκB immunofluorescence colocalized with OX-42 (Figure 15Aa-Cc, Figure 16B). Moreover, nuclear translocation of NFκB appeared to be more evident in those cells (Figure 15Bb, Figure 16A). Pretreatment of rats with DMS reduced the incidence of OX-42-labeled, NFκB-immunopositive AMC and NFκB nuclear translocation (Figure 15C). In vitro analysis using primary cultures of microglia also showed that LPS treatment induced NFκB translocation from cytoplasm to the nucleus (Figure 17Ba-Bc). This translocation of NFκB was reversed in activated cells pretreated with DMS (Figure 17Da-Dc).

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