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Lactate-modulated induction of THBS-1 activates transforming growth factor (TGF)-beta2 and migration of glioma cells in vitro.

Seliger C, Leukel P, Moeckel S, Jachnik B, Lottaz C, Kreutz M, Brawanski A, Proescholdt M, Bogdahn U, Bosserhoff AK, Vollmann-Zwerenz A, Hau P - PLoS ONE (2013)

Bottom Line: Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide.Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate.Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Wilhelm Sander-NeuroOncology Unit, University Hospital Regensburg, Regensburg, Germany.

ABSTRACT

Background: An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods: Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results: Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion: We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.

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Lactate regulates TGF-beta2 protein.Specific siRNA against LDH-A was used to reduce extracellular lactate levels and to investigate consecutive changes in TGF-beta2 expression. siLDH-A reduces LDH-A (p < 0.01**) and TGF-beta2 (p< 0.05*) mRNA expression significantly 72 hours after treatment (A). In TGF-beta2 Western Blot (B) and ELISAs (C, D) siLDH-A reduces TGF-beta2 protein expression with a maximum reduction 72 hours after treatment (p< 0.001***). Results were normalized to control. Treatment with 20 mM lactic acid (pH 7.1) as well as 20 mM sodium lactate (pH 7.4) increases TGF-beta2 mRNA (E). Expression of TGF-beta2 protein 24 hours after treatment is significantly increased by lactic acid, lactate and HCl (F, lactic acid p < 0.05*; sodium lactate p < 0.01**, HCl p < 0.05*).
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pone-0078935-g004: Lactate regulates TGF-beta2 protein.Specific siRNA against LDH-A was used to reduce extracellular lactate levels and to investigate consecutive changes in TGF-beta2 expression. siLDH-A reduces LDH-A (p < 0.01**) and TGF-beta2 (p< 0.05*) mRNA expression significantly 72 hours after treatment (A). In TGF-beta2 Western Blot (B) and ELISAs (C, D) siLDH-A reduces TGF-beta2 protein expression with a maximum reduction 72 hours after treatment (p< 0.001***). Results were normalized to control. Treatment with 20 mM lactic acid (pH 7.1) as well as 20 mM sodium lactate (pH 7.4) increases TGF-beta2 mRNA (E). Expression of TGF-beta2 protein 24 hours after treatment is significantly increased by lactic acid, lactate and HCl (F, lactic acid p < 0.05*; sodium lactate p < 0.01**, HCl p < 0.05*).

Mentions: As TGF-beta is a known inductor of migration in glioma, we next hypothesized that lactate might induce glioma cell migration by regulation of TGF-beta2. To test this hypothesis, we regulated lactate production by transfecting cells with siLDH-A and monitored for changes of TGF-beta2 levels. Treatment of glioma cells with 0.1 µM siLDH-A specifically suppressed mRNA of LDH-A in HTZ-349 glioma cells to 2% of the initial levels (Figure 4A), leading to decreased levels of lactate and a parallel moderate increase of glucose (Figure S2A). Regulation of LDH-A RNA was followed by regulation of LDH-A but not LDH-B protein (Figure 4B). LDH-A, but not LDH-B knockdown also led to a decrease in TGF-beta2 mRNA (Figure 4A) and TGF-beta2 protein in TGF-beta2 Western blot (Figure 4B) and ELISA (Figure 4C), with a maximum regulation 72 hours after transfection (Figure 4D). Experiments in U87 yielded consensual results (not shown). These results were confirmed in RAV20 and RAV21 brain tumor initiating cells with similar results (Figure S3A).


Lactate-modulated induction of THBS-1 activates transforming growth factor (TGF)-beta2 and migration of glioma cells in vitro.

Seliger C, Leukel P, Moeckel S, Jachnik B, Lottaz C, Kreutz M, Brawanski A, Proescholdt M, Bogdahn U, Bosserhoff AK, Vollmann-Zwerenz A, Hau P - PLoS ONE (2013)

Lactate regulates TGF-beta2 protein.Specific siRNA against LDH-A was used to reduce extracellular lactate levels and to investigate consecutive changes in TGF-beta2 expression. siLDH-A reduces LDH-A (p < 0.01**) and TGF-beta2 (p< 0.05*) mRNA expression significantly 72 hours after treatment (A). In TGF-beta2 Western Blot (B) and ELISAs (C, D) siLDH-A reduces TGF-beta2 protein expression with a maximum reduction 72 hours after treatment (p< 0.001***). Results were normalized to control. Treatment with 20 mM lactic acid (pH 7.1) as well as 20 mM sodium lactate (pH 7.4) increases TGF-beta2 mRNA (E). Expression of TGF-beta2 protein 24 hours after treatment is significantly increased by lactic acid, lactate and HCl (F, lactic acid p < 0.05*; sodium lactate p < 0.01**, HCl p < 0.05*).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815307&req=5

pone-0078935-g004: Lactate regulates TGF-beta2 protein.Specific siRNA against LDH-A was used to reduce extracellular lactate levels and to investigate consecutive changes in TGF-beta2 expression. siLDH-A reduces LDH-A (p < 0.01**) and TGF-beta2 (p< 0.05*) mRNA expression significantly 72 hours after treatment (A). In TGF-beta2 Western Blot (B) and ELISAs (C, D) siLDH-A reduces TGF-beta2 protein expression with a maximum reduction 72 hours after treatment (p< 0.001***). Results were normalized to control. Treatment with 20 mM lactic acid (pH 7.1) as well as 20 mM sodium lactate (pH 7.4) increases TGF-beta2 mRNA (E). Expression of TGF-beta2 protein 24 hours after treatment is significantly increased by lactic acid, lactate and HCl (F, lactic acid p < 0.05*; sodium lactate p < 0.01**, HCl p < 0.05*).
Mentions: As TGF-beta is a known inductor of migration in glioma, we next hypothesized that lactate might induce glioma cell migration by regulation of TGF-beta2. To test this hypothesis, we regulated lactate production by transfecting cells with siLDH-A and monitored for changes of TGF-beta2 levels. Treatment of glioma cells with 0.1 µM siLDH-A specifically suppressed mRNA of LDH-A in HTZ-349 glioma cells to 2% of the initial levels (Figure 4A), leading to decreased levels of lactate and a parallel moderate increase of glucose (Figure S2A). Regulation of LDH-A RNA was followed by regulation of LDH-A but not LDH-B protein (Figure 4B). LDH-A, but not LDH-B knockdown also led to a decrease in TGF-beta2 mRNA (Figure 4A) and TGF-beta2 protein in TGF-beta2 Western blot (Figure 4B) and ELISA (Figure 4C), with a maximum regulation 72 hours after transfection (Figure 4D). Experiments in U87 yielded consensual results (not shown). These results were confirmed in RAV20 and RAV21 brain tumor initiating cells with similar results (Figure S3A).

Bottom Line: Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide.Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate.Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Wilhelm Sander-NeuroOncology Unit, University Hospital Regensburg, Regensburg, Germany.

ABSTRACT

Background: An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods: Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results: Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion: We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.

Show MeSH
Related in: MedlinePlus