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PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways.

Kveiborg M, Instrell R, Rowlands C, Howell M, Parker PJ - PLoS ONE (2011)

Bottom Line: In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF.However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF.Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom. marie.kveiborg@bric.ku.dk

ABSTRACT
Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

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PMA-induced HB-EGF shedding is ADAM17-dependent.A) AP-HB-EGF release measured as alkaline phosphatase activity (OD 405 nm) in conditioned media and B) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in DMSO control-treated (C; black bars) and 30 min, 400 nM PMA-treated (PMA; white bars) AP-HB-EGF expressing HT1080 cells treated with the ADAM17 inhibitor TAPI-2 (10 µM) or vehicle control. C, D) as in A and B respectively, except that cells were reverse transfected with control (C) or ADAM17 siRNAs for 72 h before treatment. The insert shows western blot analysis of total cell extracts from cells used in C and D, demonstrating efficient siRNA-mediated knockdown of pro and mature forms (arrows) of ADAM17. α-tubulin is used as an internal loading control. All graphs show average values ± standard error of the mean of at least three independent experiments each done in triplicate. *p<0.05; **p<0.01, ***p<0.001 after one-way analysis of variance with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated the comparison is relative to the respective control.
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pone-0017168-g002: PMA-induced HB-EGF shedding is ADAM17-dependent.A) AP-HB-EGF release measured as alkaline phosphatase activity (OD 405 nm) in conditioned media and B) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in DMSO control-treated (C; black bars) and 30 min, 400 nM PMA-treated (PMA; white bars) AP-HB-EGF expressing HT1080 cells treated with the ADAM17 inhibitor TAPI-2 (10 µM) or vehicle control. C, D) as in A and B respectively, except that cells were reverse transfected with control (C) or ADAM17 siRNAs for 72 h before treatment. The insert shows western blot analysis of total cell extracts from cells used in C and D, demonstrating efficient siRNA-mediated knockdown of pro and mature forms (arrows) of ADAM17. α-tubulin is used as an internal loading control. All graphs show average values ± standard error of the mean of at least three independent experiments each done in triplicate. *p<0.05; **p<0.01, ***p<0.001 after one-way analysis of variance with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated the comparison is relative to the respective control.

Mentions: Previous loss-of-function studies demonstrated that at least in mouse embryonic fibroblasts, ADAM17 is the PMA-responsive sheddase of EGFR-ligands [7]. In order to investigate whether this was also the case for HB-EGF shedding in human HT1080 fibrosarcoma cells, we first tested the effect of the small molecule inhibitor TAPI-2 that, while being a non-specific inhibitor of many ADAMs and some other metalloproteases, is a potent inhibitor of ADAM17 protease activity. As shown in Figure 2A, TAPI-2 completely prevented the PMA-induced release of AP-HB-EGF into the conditioned medium. By contrast, the PMA-induced loss of cell surface substrate was only partially inhibited (Figure 2B), suggesting that PMA triggers both ectodomain shedding and an additional loss of cell surface HB-EGF. Next, we used the more specific approach of siRNA-mediated ADAM17 knockdown to examine the PMA-induced AP-HB-EGF shedding. As seen in Figure 2C, similar to the effect of pharmacological ADAM17 protease inhibition, loss of ADAM17 expression caused an almost complete block in the PMA response. Moreover, as for TAPI-2 treatment, ADAM17 knockdown did not completely block the depletion of cell surface substrate (Figure 2D). As shown by western blot analysis of total cell lysates (Figure 2C), a clear reduction in ADAM17 expression was observed after 72hr siRNA-mediated knockdown. The fact that a small stimulatory response of PMA on HB-EGF shedding was still observed after ADAM17 knockdown, while TAPI-2 caused a complete block could be due to the contribution of other ADAMs. However, knockdown of ADAM17 in combination with knockdown of ADAM9 or ADAM12, which have both been implicated in HB-EGF shedding in other cells, caused no further reduction of the PMA response (data not shown), suggesting that the residual HB-EGF shedding is rather due to incomplete knockdown of ADAM17. Together, these data confirm previous reports, showing that ADAM17 is the key PMA-responsive HB-EGF sheddase. Moreover, since measuring the amount of HB-EGF in the conditioned cell media as well as on the cell surface allowed us to distinguish between loss of cell surface substrate due to proteolysis from other changes in substrate availability, the data indicates that 30 min of PMA-treatment not only triggers ADAM17-mediated ectodomain shedding, but also induces significant loss of cell surface HB-EGF by other means, which is insensitive to protease inhibition.


PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways.

Kveiborg M, Instrell R, Rowlands C, Howell M, Parker PJ - PLoS ONE (2011)

PMA-induced HB-EGF shedding is ADAM17-dependent.A) AP-HB-EGF release measured as alkaline phosphatase activity (OD 405 nm) in conditioned media and B) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in DMSO control-treated (C; black bars) and 30 min, 400 nM PMA-treated (PMA; white bars) AP-HB-EGF expressing HT1080 cells treated with the ADAM17 inhibitor TAPI-2 (10 µM) or vehicle control. C, D) as in A and B respectively, except that cells were reverse transfected with control (C) or ADAM17 siRNAs for 72 h before treatment. The insert shows western blot analysis of total cell extracts from cells used in C and D, demonstrating efficient siRNA-mediated knockdown of pro and mature forms (arrows) of ADAM17. α-tubulin is used as an internal loading control. All graphs show average values ± standard error of the mean of at least three independent experiments each done in triplicate. *p<0.05; **p<0.01, ***p<0.001 after one-way analysis of variance with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated the comparison is relative to the respective control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046143&req=5

pone-0017168-g002: PMA-induced HB-EGF shedding is ADAM17-dependent.A) AP-HB-EGF release measured as alkaline phosphatase activity (OD 405 nm) in conditioned media and B) surface AP-HB-EGF measured as cell surface fluorescence intensity per cell (FLU/cell) in DMSO control-treated (C; black bars) and 30 min, 400 nM PMA-treated (PMA; white bars) AP-HB-EGF expressing HT1080 cells treated with the ADAM17 inhibitor TAPI-2 (10 µM) or vehicle control. C, D) as in A and B respectively, except that cells were reverse transfected with control (C) or ADAM17 siRNAs for 72 h before treatment. The insert shows western blot analysis of total cell extracts from cells used in C and D, demonstrating efficient siRNA-mediated knockdown of pro and mature forms (arrows) of ADAM17. α-tubulin is used as an internal loading control. All graphs show average values ± standard error of the mean of at least three independent experiments each done in triplicate. *p<0.05; **p<0.01, ***p<0.001 after one-way analysis of variance with Bonferroni's post tests for multiple comparisons. Unless otherwise indicated the comparison is relative to the respective control.
Mentions: Previous loss-of-function studies demonstrated that at least in mouse embryonic fibroblasts, ADAM17 is the PMA-responsive sheddase of EGFR-ligands [7]. In order to investigate whether this was also the case for HB-EGF shedding in human HT1080 fibrosarcoma cells, we first tested the effect of the small molecule inhibitor TAPI-2 that, while being a non-specific inhibitor of many ADAMs and some other metalloproteases, is a potent inhibitor of ADAM17 protease activity. As shown in Figure 2A, TAPI-2 completely prevented the PMA-induced release of AP-HB-EGF into the conditioned medium. By contrast, the PMA-induced loss of cell surface substrate was only partially inhibited (Figure 2B), suggesting that PMA triggers both ectodomain shedding and an additional loss of cell surface HB-EGF. Next, we used the more specific approach of siRNA-mediated ADAM17 knockdown to examine the PMA-induced AP-HB-EGF shedding. As seen in Figure 2C, similar to the effect of pharmacological ADAM17 protease inhibition, loss of ADAM17 expression caused an almost complete block in the PMA response. Moreover, as for TAPI-2 treatment, ADAM17 knockdown did not completely block the depletion of cell surface substrate (Figure 2D). As shown by western blot analysis of total cell lysates (Figure 2C), a clear reduction in ADAM17 expression was observed after 72hr siRNA-mediated knockdown. The fact that a small stimulatory response of PMA on HB-EGF shedding was still observed after ADAM17 knockdown, while TAPI-2 caused a complete block could be due to the contribution of other ADAMs. However, knockdown of ADAM17 in combination with knockdown of ADAM9 or ADAM12, which have both been implicated in HB-EGF shedding in other cells, caused no further reduction of the PMA response (data not shown), suggesting that the residual HB-EGF shedding is rather due to incomplete knockdown of ADAM17. Together, these data confirm previous reports, showing that ADAM17 is the key PMA-responsive HB-EGF sheddase. Moreover, since measuring the amount of HB-EGF in the conditioned cell media as well as on the cell surface allowed us to distinguish between loss of cell surface substrate due to proteolysis from other changes in substrate availability, the data indicates that 30 min of PMA-treatment not only triggers ADAM17-mediated ectodomain shedding, but also induces significant loss of cell surface HB-EGF by other means, which is insensitive to protease inhibition.

Bottom Line: In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF.However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF.Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom. marie.kveiborg@bric.ku.dk

ABSTRACT
Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.

Show MeSH
Related in: MedlinePlus