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HIF-1 and c-Src mediate increased glucose uptake induced by endothelin-1 and connexin43 in astrocytes.

Valle-Casuso JC, González-Sánchez A, Medina JM, Tabernero A - PLoS ONE (2012)

Bottom Line: Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43.In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α.In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
In previous work we showed that endothelin-1 (ET-1) increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α) in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43), the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.

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Effect of silencing Cx43 on the expression of HIF-1α in astrocytes.Astrocytes were transfected with NT-siRNA or with Cx43-siRNA. At the indicated times the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that the decrease in Cx43 expression was concomitant with HIF-1α up-regulation. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the NT-siRNA condition at time 0. ***p<0.001 versus the corresponding NT-siRNA values.
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pone-0032448-g002: Effect of silencing Cx43 on the expression of HIF-1α in astrocytes.Astrocytes were transfected with NT-siRNA or with Cx43-siRNA. At the indicated times the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that the decrease in Cx43 expression was concomitant with HIF-1α up-regulation. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the NT-siRNA condition at time 0. ***p<0.001 versus the corresponding NT-siRNA values.

Mentions: The inhibition of gap junctional communication promoted by ET-1 in astrocytes is well documented [12], [16], [17], [44], [45], [46], [47]. Thus, ET-1 triggers very fast changes (within 3–10 minutes) in the gap junctional communication and in the phosphorylation status of Cx43 [48]. Changes in gap junctional communication and in the phosphorylation status of Cx43 are associated with changes in Cx43 interaction with other proteins and with Cx43 endocytosis [41], [42], [48]. Thus, a prolonged (24 hours) exposure to ET-1 reduces Cx43 expression in astrocytes [24], [25]. Interestingly, the down-regulation of Cx43 promoted by ET-1 coincided with the up-regulation of HIF-1α (Figure 1C). Since our previous work indicated that the effect of ET-1 on glucose uptake was due to the reduction in Cx43 expression, we investigated the effect of decreasing Cx43 expression on HIF-1α levels. Thus, by using specific siRNA against Cx43 [26], [43] we found that 48 h after the transfection with Cx43-siRNA the level of Cx43 was reduced by about 50% (Figures 2A and 2C) and the expression of HIF-1α was increased by about 80% (Figure 2B), when compared to astrocytes transfected with a non-targeting siRNA (NT-siRNA). Consequently, our results show that silencing Cx43 up-regulated HIF-1α. In addition, Figure 3 shows that the difference in HIF-1α levels between ET-1 and the control was not statistically significant in cells transfected with Cx43-siRNA, indicating that ET-1 was not able to further increase significantly the levels of HIF-1α in Cx43-silenced astrocytes.


HIF-1 and c-Src mediate increased glucose uptake induced by endothelin-1 and connexin43 in astrocytes.

Valle-Casuso JC, González-Sánchez A, Medina JM, Tabernero A - PLoS ONE (2012)

Effect of silencing Cx43 on the expression of HIF-1α in astrocytes.Astrocytes were transfected with NT-siRNA or with Cx43-siRNA. At the indicated times the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that the decrease in Cx43 expression was concomitant with HIF-1α up-regulation. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the NT-siRNA condition at time 0. ***p<0.001 versus the corresponding NT-siRNA values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285680&req=5

pone-0032448-g002: Effect of silencing Cx43 on the expression of HIF-1α in astrocytes.Astrocytes were transfected with NT-siRNA or with Cx43-siRNA. At the indicated times the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that the decrease in Cx43 expression was concomitant with HIF-1α up-regulation. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the NT-siRNA condition at time 0. ***p<0.001 versus the corresponding NT-siRNA values.
Mentions: The inhibition of gap junctional communication promoted by ET-1 in astrocytes is well documented [12], [16], [17], [44], [45], [46], [47]. Thus, ET-1 triggers very fast changes (within 3–10 minutes) in the gap junctional communication and in the phosphorylation status of Cx43 [48]. Changes in gap junctional communication and in the phosphorylation status of Cx43 are associated with changes in Cx43 interaction with other proteins and with Cx43 endocytosis [41], [42], [48]. Thus, a prolonged (24 hours) exposure to ET-1 reduces Cx43 expression in astrocytes [24], [25]. Interestingly, the down-regulation of Cx43 promoted by ET-1 coincided with the up-regulation of HIF-1α (Figure 1C). Since our previous work indicated that the effect of ET-1 on glucose uptake was due to the reduction in Cx43 expression, we investigated the effect of decreasing Cx43 expression on HIF-1α levels. Thus, by using specific siRNA against Cx43 [26], [43] we found that 48 h after the transfection with Cx43-siRNA the level of Cx43 was reduced by about 50% (Figures 2A and 2C) and the expression of HIF-1α was increased by about 80% (Figure 2B), when compared to astrocytes transfected with a non-targeting siRNA (NT-siRNA). Consequently, our results show that silencing Cx43 up-regulated HIF-1α. In addition, Figure 3 shows that the difference in HIF-1α levels between ET-1 and the control was not statistically significant in cells transfected with Cx43-siRNA, indicating that ET-1 was not able to further increase significantly the levels of HIF-1α in Cx43-silenced astrocytes.

Bottom Line: Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43.In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α.In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
In previous work we showed that endothelin-1 (ET-1) increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α) in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43), the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.

Show MeSH
Related in: MedlinePlus