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Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function.

Jamali S, Klier M, Ames S, Barros LF, McKenna R, Deitmer JW, Becker HM - Sci Rep (2015)

Bottom Line: Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction.Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1.Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Zoology/Membrane Transport, FB Biologie, TU Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany.

ABSTRACT
The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

No MeSH data available.


Related in: MedlinePlus

CAIX enhances MCT transport activity by facilitating its intramolecular H+ shuttle.(a) Original recordings of intracellular H+-concentration ([H+]i) in Xenopus oocytes expressing MCT1 (black trace), MCT1+CAIX-WT (blue trace), or MCT1+CAIX-H200A (green trace), respectively, during application of 3 and 10 mM lactate and of 5% CO2/10 mM HCO3−. (b,c) Rate of rise in [H+]i, as induced by application of lactate (b) or 5% CO2/10 mM HCO3− (c) in oocytes expressing MCT1, MCT1+CAIX-WT and MCT1+CAIX-H200A, respectively. (d) Original recordings of the log enrichment of 20 native oocytes and 20 oocytes expressing either CAIX-WT or CAIX-H200A. The beginning of the traces shows the rate of degradation of the 18O-labeled substrate in the non-catalysed reaction. The black arrowhead indicates addition of oocytes. (e) Enzymatic activity of native oocytes and oocytes expressing either CAIX-WT or CAIX-H200A. One unit is defined as 100% stimulation of the non-catalysed 18O depletion of doubly labelled 13C18O2. Data are represented as mean ± SEM.
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f4: CAIX enhances MCT transport activity by facilitating its intramolecular H+ shuttle.(a) Original recordings of intracellular H+-concentration ([H+]i) in Xenopus oocytes expressing MCT1 (black trace), MCT1+CAIX-WT (blue trace), or MCT1+CAIX-H200A (green trace), respectively, during application of 3 and 10 mM lactate and of 5% CO2/10 mM HCO3−. (b,c) Rate of rise in [H+]i, as induced by application of lactate (b) or 5% CO2/10 mM HCO3− (c) in oocytes expressing MCT1, MCT1+CAIX-WT and MCT1+CAIX-H200A, respectively. (d) Original recordings of the log enrichment of 20 native oocytes and 20 oocytes expressing either CAIX-WT or CAIX-H200A. The beginning of the traces shows the rate of degradation of the 18O-labeled substrate in the non-catalysed reaction. The black arrowhead indicates addition of oocytes. (e) Enzymatic activity of native oocytes and oocytes expressing either CAIX-WT or CAIX-H200A. One unit is defined as 100% stimulation of the non-catalysed 18O depletion of doubly labelled 13C18O2. Data are represented as mean ± SEM.

Mentions: CAIX facilitates an intramolecular H+ shuttle, with the histidine at position 200, to move H+ between its catalytic centre and the surrounding bulk solution. To test whether CAIX augments MCT1 transport activity by facilitating this H+ shuttle, we replaced His200 by alanine (CAIX-H200A) and coexpressed either CAIX-WT or the mutant CAIX-H200A with MCT1 in Xenopus oocytes (Fig. 4a). Coexpression of MCT1 with CAIX-WT increased Δ[H+]i/Δt to 180% (Fig. 4b), a value quite similar to the augmentation in lactate transport observed in MCF-7 cancer cells. Like in MCF-7 cells, the CAIX-mediated increase in MCT1 transport activity in Xenopus oocytes was independent from CAIX catalytic activity, as indicated by the persistence of the increase in Δ[H+]i/Δt in the presence of 30 μM EZA (Fig. S3a,b). Furthermore, CAIX did not increase the expression level of MCT1, as evaluated by western blot analysis (Fig. S3d). However, coexpression of MCT1 with CAIX-H200A did not increase the rate of lactate-induced acidification (Fig. 4a,b). Compared to CAIX-WT, catalytic activity of CAIX-H200A decreased to 70% and 53%, when determined by pHi measurement during application of CO2/HCO3− (Fig. 4a,c) and by gas analysis mass spectrometry, respectively (Fig. 4d,e). This reduction in catalytic activity corresponds to the reduction in activity determined for CAII-H64A (51%), the equivalent to CAIX-H200A33. The failure of CAIX-H200A to enhance MCT1 transport activity suggests that CAIX functions as a ‘proton-collecting/distributing antenna’ which facilitates its intramolecular H+ shuttle to transfer protons between transporter pore and surrounding protonatable residues. This interaction with MCT1 would require that CAIX is located at the extracellular surface of the plasma membrane. To confirm that MCT1 transport activity is not mediated by intracellular CAIX, we injected 20 ng of purified CAIX protein (which is trapped inside the cytosol) into MCT1-expressing oocytes (Fig. S3a–c). Injection of CAIX protein displayed intracellular catalytic activity, as observed from the increased rate of acidification during application of CO2/HCO3−, but had no effect on MCT1 transport activity. This indicates that the augmentation of transport activity in MCT1+CAIX-coexpressing oocytes is mediated by extracellular CAIX.


Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function.

Jamali S, Klier M, Ames S, Barros LF, McKenna R, Deitmer JW, Becker HM - Sci Rep (2015)

CAIX enhances MCT transport activity by facilitating its intramolecular H+ shuttle.(a) Original recordings of intracellular H+-concentration ([H+]i) in Xenopus oocytes expressing MCT1 (black trace), MCT1+CAIX-WT (blue trace), or MCT1+CAIX-H200A (green trace), respectively, during application of 3 and 10 mM lactate and of 5% CO2/10 mM HCO3−. (b,c) Rate of rise in [H+]i, as induced by application of lactate (b) or 5% CO2/10 mM HCO3− (c) in oocytes expressing MCT1, MCT1+CAIX-WT and MCT1+CAIX-H200A, respectively. (d) Original recordings of the log enrichment of 20 native oocytes and 20 oocytes expressing either CAIX-WT or CAIX-H200A. The beginning of the traces shows the rate of degradation of the 18O-labeled substrate in the non-catalysed reaction. The black arrowhead indicates addition of oocytes. (e) Enzymatic activity of native oocytes and oocytes expressing either CAIX-WT or CAIX-H200A. One unit is defined as 100% stimulation of the non-catalysed 18O depletion of doubly labelled 13C18O2. Data are represented as mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559800&req=5

f4: CAIX enhances MCT transport activity by facilitating its intramolecular H+ shuttle.(a) Original recordings of intracellular H+-concentration ([H+]i) in Xenopus oocytes expressing MCT1 (black trace), MCT1+CAIX-WT (blue trace), or MCT1+CAIX-H200A (green trace), respectively, during application of 3 and 10 mM lactate and of 5% CO2/10 mM HCO3−. (b,c) Rate of rise in [H+]i, as induced by application of lactate (b) or 5% CO2/10 mM HCO3− (c) in oocytes expressing MCT1, MCT1+CAIX-WT and MCT1+CAIX-H200A, respectively. (d) Original recordings of the log enrichment of 20 native oocytes and 20 oocytes expressing either CAIX-WT or CAIX-H200A. The beginning of the traces shows the rate of degradation of the 18O-labeled substrate in the non-catalysed reaction. The black arrowhead indicates addition of oocytes. (e) Enzymatic activity of native oocytes and oocytes expressing either CAIX-WT or CAIX-H200A. One unit is defined as 100% stimulation of the non-catalysed 18O depletion of doubly labelled 13C18O2. Data are represented as mean ± SEM.
Mentions: CAIX facilitates an intramolecular H+ shuttle, with the histidine at position 200, to move H+ between its catalytic centre and the surrounding bulk solution. To test whether CAIX augments MCT1 transport activity by facilitating this H+ shuttle, we replaced His200 by alanine (CAIX-H200A) and coexpressed either CAIX-WT or the mutant CAIX-H200A with MCT1 in Xenopus oocytes (Fig. 4a). Coexpression of MCT1 with CAIX-WT increased Δ[H+]i/Δt to 180% (Fig. 4b), a value quite similar to the augmentation in lactate transport observed in MCF-7 cancer cells. Like in MCF-7 cells, the CAIX-mediated increase in MCT1 transport activity in Xenopus oocytes was independent from CAIX catalytic activity, as indicated by the persistence of the increase in Δ[H+]i/Δt in the presence of 30 μM EZA (Fig. S3a,b). Furthermore, CAIX did not increase the expression level of MCT1, as evaluated by western blot analysis (Fig. S3d). However, coexpression of MCT1 with CAIX-H200A did not increase the rate of lactate-induced acidification (Fig. 4a,b). Compared to CAIX-WT, catalytic activity of CAIX-H200A decreased to 70% and 53%, when determined by pHi measurement during application of CO2/HCO3− (Fig. 4a,c) and by gas analysis mass spectrometry, respectively (Fig. 4d,e). This reduction in catalytic activity corresponds to the reduction in activity determined for CAII-H64A (51%), the equivalent to CAIX-H200A33. The failure of CAIX-H200A to enhance MCT1 transport activity suggests that CAIX functions as a ‘proton-collecting/distributing antenna’ which facilitates its intramolecular H+ shuttle to transfer protons between transporter pore and surrounding protonatable residues. This interaction with MCT1 would require that CAIX is located at the extracellular surface of the plasma membrane. To confirm that MCT1 transport activity is not mediated by intracellular CAIX, we injected 20 ng of purified CAIX protein (which is trapped inside the cytosol) into MCT1-expressing oocytes (Fig. S3a–c). Injection of CAIX protein displayed intracellular catalytic activity, as observed from the increased rate of acidification during application of CO2/HCO3−, but had no effect on MCT1 transport activity. This indicates that the augmentation of transport activity in MCT1+CAIX-coexpressing oocytes is mediated by extracellular CAIX.

Bottom Line: Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction.Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1.Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Zoology/Membrane Transport, FB Biologie, TU Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany.

ABSTRACT
The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

No MeSH data available.


Related in: MedlinePlus