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Regulatory mechanism of endothelin receptor B in the cerebral arteries after focal cerebral ischemia.

Grell AS, Thigarajah R, Edvinsson L, Samraj AK - PLoS ONE (2014)

Bottom Line: Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels.It also significantly reduced the ETBR mediated cerebrovascular contractility.The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Experimental Research, Glostrup research institute, University of Copenhagen, Glostrup, Denmark.

ABSTRACT

Background and purpose: Increased expression of endothelin receptor type B (ETBR), a vasoactive receptor, has recently been implied in the reduced cerebral blood flow and exacerbated neuronal damage after ischemia-reperfusion (I/R). The study explores the regulatory mechanisms of ETBR to identify drug targets to restore normal cerebral artery contractile function as part of successful neuroprotective therapy.

Methods: We have employed in vitro methods on human and rat cerebral arteries to study the regulatory mechanisms and the efficacy of target selective inhibitor, Mithramycin A (MitA), to block the ETBR mediated contractile properties. Later, middle cerebral artery occluded (MCAO) rats were used to substantiate the observations. Quantative PCR, immunohistochemistry, western blot and wire myograph methods were employed to study the expression and contractile properties of cerebral arteries.

Results: Increased expression of specificity protein (Sp1) was observed in human and rat cerebral arteries after organ culture, strongly correlating with the ETBR upregulation. Similar observations were made in MCAO rats. Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels. It also significantly reduced the ETBR mediated cerebrovascular contractility. Detailed analysis indicated that ERK1/2 mediated phosphorylation of Sp1 might be essential for ETBR transcription.

Conclusion: Transcription factor Sp1 regulates the ETBR mediated vasoconstriction in focal cerebral ischemia via MEK-ERK signaling, which is also conserved in humans. The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

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ERK1/2 mediated phosphorylation of Sp1 is essential for ETBR upregulation.A. and B. Bar graphs show the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 based on the intensity measurements of western blots (n = 4 per group). No statistical difference between groups was observed. C. Representative western blot showing the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 in culture conditions. D. Representative immunohistochemical staining of MCA sections showing the phosphorylation status of Sp1 (T739) after 120 min of culturing (n = 4 per group). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Bonferronis multiple comparison test was performed for statistical significance.
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pone-0113624-g003: ERK1/2 mediated phosphorylation of Sp1 is essential for ETBR upregulation.A. and B. Bar graphs show the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 based on the intensity measurements of western blots (n = 4 per group). No statistical difference between groups was observed. C. Representative western blot showing the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 in culture conditions. D. Representative immunohistochemical staining of MCA sections showing the phosphorylation status of Sp1 (T739) after 120 min of culturing (n = 4 per group). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Bonferronis multiple comparison test was performed for statistical significance.

Mentions: Transcription factor Sp1 mediates signal transduction either by multiple protein interaction or by posttranslational modification such as phosphorylation [19]. Studies have demonstrated that ERK1/2 directly phosphorylates Sp1 on threonine 453 (T453) and 739 (T739) in vitro and in vivo[20]. Hence, the status of phosphorylated Sp1 (p-Sp1) and ERK1/2 (p-ERK1/2) was evaluated at various time points in culture conditions. Western blot analysis showed that vascular ERK1/2 is activated as early as 5 min after initiating the incubation. When the blots were probed for phosphorylated Sp1 at T453, a known ERK1/2 target, we found that Sp1 could be phosphorylated within 5 minutes of ERK1/2 activation (Fig. 3A, B and C). Intensity measurements of those blots showed time dependent increase of p-Sp1 levels correlating with the increased p-ERK1/2 but not statistically significant (Fig. 3A and B). To substantiate the phosphorylated status of Sp1, sections of organ cultured MCAs were immunohistochemically analyzed for phospho-T739, another ERK1/2 target. It showed an increased immunoreactivity for phospho-T739. These observations suggest that the phosphorylation of T453 and T739 could be essential for the nuclear translocation and DNA binding function of Sp1 (Fig. 3D). Even though the intensity measurements of immunoblots indicated that p-Sp1 levels slowly decrease over time, we still observed sustained basal level of p-Sp1 up to six hours in culturing conditions (data not shown). The observation of ERK1/2 activation is in line with the previous studies indicating that MEK-ERK signaling is essential for ETBR upregulation [10], [21]. Taken together these observations suggest that ERK1/2 mediated phosphorylation of Sp1 could be essential for the ETBR transcription.


Regulatory mechanism of endothelin receptor B in the cerebral arteries after focal cerebral ischemia.

Grell AS, Thigarajah R, Edvinsson L, Samraj AK - PLoS ONE (2014)

ERK1/2 mediated phosphorylation of Sp1 is essential for ETBR upregulation.A. and B. Bar graphs show the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 based on the intensity measurements of western blots (n = 4 per group). No statistical difference between groups was observed. C. Representative western blot showing the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 in culture conditions. D. Representative immunohistochemical staining of MCA sections showing the phosphorylation status of Sp1 (T739) after 120 min of culturing (n = 4 per group). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Bonferronis multiple comparison test was performed for statistical significance.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257531&req=5

pone-0113624-g003: ERK1/2 mediated phosphorylation of Sp1 is essential for ETBR upregulation.A. and B. Bar graphs show the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 based on the intensity measurements of western blots (n = 4 per group). No statistical difference between groups was observed. C. Representative western blot showing the time-dependent changes in the phosphorylation status of Sp1 (T453) and ERK1/2 in culture conditions. D. Representative immunohistochemical staining of MCA sections showing the phosphorylation status of Sp1 (T739) after 120 min of culturing (n = 4 per group). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Bonferronis multiple comparison test was performed for statistical significance.
Mentions: Transcription factor Sp1 mediates signal transduction either by multiple protein interaction or by posttranslational modification such as phosphorylation [19]. Studies have demonstrated that ERK1/2 directly phosphorylates Sp1 on threonine 453 (T453) and 739 (T739) in vitro and in vivo[20]. Hence, the status of phosphorylated Sp1 (p-Sp1) and ERK1/2 (p-ERK1/2) was evaluated at various time points in culture conditions. Western blot analysis showed that vascular ERK1/2 is activated as early as 5 min after initiating the incubation. When the blots were probed for phosphorylated Sp1 at T453, a known ERK1/2 target, we found that Sp1 could be phosphorylated within 5 minutes of ERK1/2 activation (Fig. 3A, B and C). Intensity measurements of those blots showed time dependent increase of p-Sp1 levels correlating with the increased p-ERK1/2 but not statistically significant (Fig. 3A and B). To substantiate the phosphorylated status of Sp1, sections of organ cultured MCAs were immunohistochemically analyzed for phospho-T739, another ERK1/2 target. It showed an increased immunoreactivity for phospho-T739. These observations suggest that the phosphorylation of T453 and T739 could be essential for the nuclear translocation and DNA binding function of Sp1 (Fig. 3D). Even though the intensity measurements of immunoblots indicated that p-Sp1 levels slowly decrease over time, we still observed sustained basal level of p-Sp1 up to six hours in culturing conditions (data not shown). The observation of ERK1/2 activation is in line with the previous studies indicating that MEK-ERK signaling is essential for ETBR upregulation [10], [21]. Taken together these observations suggest that ERK1/2 mediated phosphorylation of Sp1 could be essential for the ETBR transcription.

Bottom Line: Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels.It also significantly reduced the ETBR mediated cerebrovascular contractility.The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Experimental Research, Glostrup research institute, University of Copenhagen, Glostrup, Denmark.

ABSTRACT

Background and purpose: Increased expression of endothelin receptor type B (ETBR), a vasoactive receptor, has recently been implied in the reduced cerebral blood flow and exacerbated neuronal damage after ischemia-reperfusion (I/R). The study explores the regulatory mechanisms of ETBR to identify drug targets to restore normal cerebral artery contractile function as part of successful neuroprotective therapy.

Methods: We have employed in vitro methods on human and rat cerebral arteries to study the regulatory mechanisms and the efficacy of target selective inhibitor, Mithramycin A (MitA), to block the ETBR mediated contractile properties. Later, middle cerebral artery occluded (MCAO) rats were used to substantiate the observations. Quantative PCR, immunohistochemistry, western blot and wire myograph methods were employed to study the expression and contractile properties of cerebral arteries.

Results: Increased expression of specificity protein (Sp1) was observed in human and rat cerebral arteries after organ culture, strongly correlating with the ETBR upregulation. Similar observations were made in MCAO rats. Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels. It also significantly reduced the ETBR mediated cerebrovascular contractility. Detailed analysis indicated that ERK1/2 mediated phosphorylation of Sp1 might be essential for ETBR transcription.

Conclusion: Transcription factor Sp1 regulates the ETBR mediated vasoconstriction in focal cerebral ischemia via MEK-ERK signaling, which is also conserved in humans. The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

Show MeSH
Related in: MedlinePlus