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Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.

Zatovicova M, Sedlakova O, Svastova E, Ohradanova A, Ciampor F, Arribas J, Pastorek J, Pastorekova S - Br. J. Cancer (2005)

Bottom Line: It is a highly active enzyme functionally involved in both pH control and cell adhesion.Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels.Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent.

View Article: PubMed Central - PubMed

Affiliation: Center of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 845 05, Slovak Republic.

ABSTRACT
Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.

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Inhibition of the basal carbonic anhydrase IX (CA IX) shedding with BB-94 metalloprotease inhibitor. (A) CGL3 cells were biotinylated and then allowed to shed CA IX ECD in the presence and absence of 1 μM BB-94. The media (m) were collected, the cells were extracted (ex) and the corresponding aliquots of the materials were analysed by immunoprecipitation–immunoblotting as described in Figure 1c. (B) ELISA was used to determine the inhibition of CA IX shedding with 1 μM BB-94. The cells were treated for 24 h throughout their incubation in normoxia or hypoxia. Extent of inhibition was illustrated as a ratio of the absorbance values of the materials from BB-94-treated cells vs their nontreated counterparts normalised according to total protein concentrations. (C) ELISA assessment of a BB-94 concentration-dependent inhibition of CA IX ECD release in MDCK+CA IX cells. The results are expressed as a ratio between the BB-94-inhibited and control samples. *P<0.01 and **P<0.001 by Student's t-test.
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fig3: Inhibition of the basal carbonic anhydrase IX (CA IX) shedding with BB-94 metalloprotease inhibitor. (A) CGL3 cells were biotinylated and then allowed to shed CA IX ECD in the presence and absence of 1 μM BB-94. The media (m) were collected, the cells were extracted (ex) and the corresponding aliquots of the materials were analysed by immunoprecipitation–immunoblotting as described in Figure 1c. (B) ELISA was used to determine the inhibition of CA IX shedding with 1 μM BB-94. The cells were treated for 24 h throughout their incubation in normoxia or hypoxia. Extent of inhibition was illustrated as a ratio of the absorbance values of the materials from BB-94-treated cells vs their nontreated counterparts normalised according to total protein concentrations. (C) ELISA assessment of a BB-94 concentration-dependent inhibition of CA IX ECD release in MDCK+CA IX cells. The results are expressed as a ratio between the BB-94-inhibited and control samples. *P<0.01 and **P<0.001 by Student's t-test.

Mentions: Numerous cell-surface proteins are shed by a mechanism that involves metalloproteases, particularly the matrix metalloproteases and members of the ADAM family of proteins. The activity of these proteases is sensitive to BB-94 inhibitor (batimastat). Therefore, we used this inhibitor to treat the cells and see whether it can influence the shedding of CA IX. Immunoblotting of CA IX protein immunoprecipitated from CGL3 cells grown in the presence of BB-94 revealed a considerably reduced production of the CA IX ECD (Figure 3A). This finding was confirmed by sandwich ELISA, which showed that 1 μM BB-94 inhibited CA IX ECD shedding to about half of the control value. Similar reduction was observed in other three cell lines, namely CGL1 and transfected C33a+CA IX as well as MDCK+CA IX cells, and appeared comparable under hypoxia (Figure 3B). Moreover, ECD shedding was inhibited by BB-94 in a concentration-dependent manner in CA IX-transfected MDCK cells where 10 μM BB-94 decreased the level of ECD to less than a tenth of a control value (Figure 3C).


Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.

Zatovicova M, Sedlakova O, Svastova E, Ohradanova A, Ciampor F, Arribas J, Pastorek J, Pastorekova S - Br. J. Cancer (2005)

Inhibition of the basal carbonic anhydrase IX (CA IX) shedding with BB-94 metalloprotease inhibitor. (A) CGL3 cells were biotinylated and then allowed to shed CA IX ECD in the presence and absence of 1 μM BB-94. The media (m) were collected, the cells were extracted (ex) and the corresponding aliquots of the materials were analysed by immunoprecipitation–immunoblotting as described in Figure 1c. (B) ELISA was used to determine the inhibition of CA IX shedding with 1 μM BB-94. The cells were treated for 24 h throughout their incubation in normoxia or hypoxia. Extent of inhibition was illustrated as a ratio of the absorbance values of the materials from BB-94-treated cells vs their nontreated counterparts normalised according to total protein concentrations. (C) ELISA assessment of a BB-94 concentration-dependent inhibition of CA IX ECD release in MDCK+CA IX cells. The results are expressed as a ratio between the BB-94-inhibited and control samples. *P<0.01 and **P<0.001 by Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361518&req=5

fig3: Inhibition of the basal carbonic anhydrase IX (CA IX) shedding with BB-94 metalloprotease inhibitor. (A) CGL3 cells were biotinylated and then allowed to shed CA IX ECD in the presence and absence of 1 μM BB-94. The media (m) were collected, the cells were extracted (ex) and the corresponding aliquots of the materials were analysed by immunoprecipitation–immunoblotting as described in Figure 1c. (B) ELISA was used to determine the inhibition of CA IX shedding with 1 μM BB-94. The cells were treated for 24 h throughout their incubation in normoxia or hypoxia. Extent of inhibition was illustrated as a ratio of the absorbance values of the materials from BB-94-treated cells vs their nontreated counterparts normalised according to total protein concentrations. (C) ELISA assessment of a BB-94 concentration-dependent inhibition of CA IX ECD release in MDCK+CA IX cells. The results are expressed as a ratio between the BB-94-inhibited and control samples. *P<0.01 and **P<0.001 by Student's t-test.
Mentions: Numerous cell-surface proteins are shed by a mechanism that involves metalloproteases, particularly the matrix metalloproteases and members of the ADAM family of proteins. The activity of these proteases is sensitive to BB-94 inhibitor (batimastat). Therefore, we used this inhibitor to treat the cells and see whether it can influence the shedding of CA IX. Immunoblotting of CA IX protein immunoprecipitated from CGL3 cells grown in the presence of BB-94 revealed a considerably reduced production of the CA IX ECD (Figure 3A). This finding was confirmed by sandwich ELISA, which showed that 1 μM BB-94 inhibited CA IX ECD shedding to about half of the control value. Similar reduction was observed in other three cell lines, namely CGL1 and transfected C33a+CA IX as well as MDCK+CA IX cells, and appeared comparable under hypoxia (Figure 3B). Moreover, ECD shedding was inhibited by BB-94 in a concentration-dependent manner in CA IX-transfected MDCK cells where 10 μM BB-94 decreased the level of ECD to less than a tenth of a control value (Figure 3C).

Bottom Line: It is a highly active enzyme functionally involved in both pH control and cell adhesion.Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels.Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent.

View Article: PubMed Central - PubMed

Affiliation: Center of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 845 05, Slovak Republic.

ABSTRACT
Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.

Show MeSH
Related in: MedlinePlus