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Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane.

Okada T, Lopez-Lago M, Giancotti FG - J. Cell Biol. (2005)

Bottom Line: PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin.Small interference RNA-mediated knockdown of Merlin exerts the same effects.Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. t-okada@ski.mskcc.org

ABSTRACT
Introduction of activated p21-activated kinase (PAK) is sufficient to release primary endothelial cells from contact inhibition of growth. Confluent cells display deficient activation of PAK and translocation of Rac to the plasma membrane at matrix adhesions. Targeting Rac to the plasma membrane rescues these cells from contact inhibition. PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin. Merlin mutants, which are presumed to exert a dominant-negative effect, enable recruitment of Rac to matrix adhesions and promote mitogenesis in confluent cells. Small interference RNA-mediated knockdown of Merlin exerts the same effects. Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway. These results provide a framework for understanding the tumor suppressor function of Merlin and indicate that Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions.

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Merlin mediates contact inhibition of growth by suppressing membrane recruitment of Rac. (A) HUVEC were electroporated with GFP-Rac and then either transfected with a siRNA oligonucleotide targeting human Merlin or mock-transfected as a control. 36 h later, cells were synchronized in G0, detached, and plated on FN under either sparse or confluent conditions, as indicated, and treated with FN beads. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. (B) Cells were transfected with a siRNA oligonucleotide targeting human Merlin (+). G0-synchronized cells were detached, plated on FN under sparse or confluent conditions, and incubated with mitogens and BrdU for 20 h. The graph shows the percentage of cells entering S phase under the indicated conditions. Total lysates were subjected to immunoblotting with the indicated antibodies. (C) Cells were electroporated with a vector encoding Myc-tagged mouse Merlin (mMerlin) or empty vector and transfected with the anti–human siRNA oligonucleotide. After 36 h, cells were synchronized in G0, detached, and plated on FN under confluent conditions for 4 h. The cells were either incubated with mitogens and BrdU for 20 h to measure entry into S phase or treated with mitogens for 12 h and subjected to immunoblotting with the indicated antibodies. (D) siRNA-transfected cells were detached and plated on FN-coated coverslips under confluent conditions in the presence of growth factors. After 20 h, they were subjected to double immunofluorescent staining with anti–VE-cadherin (green) and anti–β-catenin (red). The bottom panels are XZ section views of VE-cadherin and DAPI staining. Error bars represent the mean ± SD.
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fig5: Merlin mediates contact inhibition of growth by suppressing membrane recruitment of Rac. (A) HUVEC were electroporated with GFP-Rac and then either transfected with a siRNA oligonucleotide targeting human Merlin or mock-transfected as a control. 36 h later, cells were synchronized in G0, detached, and plated on FN under either sparse or confluent conditions, as indicated, and treated with FN beads. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. (B) Cells were transfected with a siRNA oligonucleotide targeting human Merlin (+). G0-synchronized cells were detached, plated on FN under sparse or confluent conditions, and incubated with mitogens and BrdU for 20 h. The graph shows the percentage of cells entering S phase under the indicated conditions. Total lysates were subjected to immunoblotting with the indicated antibodies. (C) Cells were electroporated with a vector encoding Myc-tagged mouse Merlin (mMerlin) or empty vector and transfected with the anti–human siRNA oligonucleotide. After 36 h, cells were synchronized in G0, detached, and plated on FN under confluent conditions for 4 h. The cells were either incubated with mitogens and BrdU for 20 h to measure entry into S phase or treated with mitogens for 12 h and subjected to immunoblotting with the indicated antibodies. (D) siRNA-transfected cells were detached and plated on FN-coated coverslips under confluent conditions in the presence of growth factors. After 20 h, they were subjected to double immunofluorescent staining with anti–VE-cadherin (green) and anti–β-catenin (red). The bottom panels are XZ section views of VE-cadherin and DAPI staining. Error bars represent the mean ± SD.

Mentions: The aforementioned experiments are consistent with the model that the dephosphorylated, closed form of Merlin suppresses recruitment of Rac to the membrane, but they do not exclude the possibility that the phosphorylated, “open” form of Merlin exerts an independent and positive effect on cell proliferation. To directly test if Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions, we used RNA interference. Transfection of a small interference RNA (siRNA) targeting human Merlin was sufficient to rescue integrin-dependent recruitment of GFP-Rac to the plasma membrane in confluent HUVEC (Fig. 5 A). Notably, knockdown of Merlin induced a large fraction of confluent HUVEC to progress through G1 and enter into S phase after mitogenic stimulation (Fig. 5 B). Immunoblotting revealed that the siRNA inhibited Merlin expression by >80%. As expected, the Merlin knockdown cells escaping contact inhibition had elevated levels of cyclin D1 and decreased levels of p27 (Fig. 5 B). However, cyclin D1 was not up-regulated and p27 was not down-regulated as effectively as they normally are in sparse cells. We attribute this partial result to the incomplete suppression of Merlin expression after siRNA transfection.


Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane.

Okada T, Lopez-Lago M, Giancotti FG - J. Cell Biol. (2005)

Merlin mediates contact inhibition of growth by suppressing membrane recruitment of Rac. (A) HUVEC were electroporated with GFP-Rac and then either transfected with a siRNA oligonucleotide targeting human Merlin or mock-transfected as a control. 36 h later, cells were synchronized in G0, detached, and plated on FN under either sparse or confluent conditions, as indicated, and treated with FN beads. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. (B) Cells were transfected with a siRNA oligonucleotide targeting human Merlin (+). G0-synchronized cells were detached, plated on FN under sparse or confluent conditions, and incubated with mitogens and BrdU for 20 h. The graph shows the percentage of cells entering S phase under the indicated conditions. Total lysates were subjected to immunoblotting with the indicated antibodies. (C) Cells were electroporated with a vector encoding Myc-tagged mouse Merlin (mMerlin) or empty vector and transfected with the anti–human siRNA oligonucleotide. After 36 h, cells were synchronized in G0, detached, and plated on FN under confluent conditions for 4 h. The cells were either incubated with mitogens and BrdU for 20 h to measure entry into S phase or treated with mitogens for 12 h and subjected to immunoblotting with the indicated antibodies. (D) siRNA-transfected cells were detached and plated on FN-coated coverslips under confluent conditions in the presence of growth factors. After 20 h, they were subjected to double immunofluorescent staining with anti–VE-cadherin (green) and anti–β-catenin (red). The bottom panels are XZ section views of VE-cadherin and DAPI staining. Error bars represent the mean ± SD.
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Related In: Results  -  Collection

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fig5: Merlin mediates contact inhibition of growth by suppressing membrane recruitment of Rac. (A) HUVEC were electroporated with GFP-Rac and then either transfected with a siRNA oligonucleotide targeting human Merlin or mock-transfected as a control. 36 h later, cells were synchronized in G0, detached, and plated on FN under either sparse or confluent conditions, as indicated, and treated with FN beads. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. (B) Cells were transfected with a siRNA oligonucleotide targeting human Merlin (+). G0-synchronized cells were detached, plated on FN under sparse or confluent conditions, and incubated with mitogens and BrdU for 20 h. The graph shows the percentage of cells entering S phase under the indicated conditions. Total lysates were subjected to immunoblotting with the indicated antibodies. (C) Cells were electroporated with a vector encoding Myc-tagged mouse Merlin (mMerlin) or empty vector and transfected with the anti–human siRNA oligonucleotide. After 36 h, cells were synchronized in G0, detached, and plated on FN under confluent conditions for 4 h. The cells were either incubated with mitogens and BrdU for 20 h to measure entry into S phase or treated with mitogens for 12 h and subjected to immunoblotting with the indicated antibodies. (D) siRNA-transfected cells were detached and plated on FN-coated coverslips under confluent conditions in the presence of growth factors. After 20 h, they were subjected to double immunofluorescent staining with anti–VE-cadherin (green) and anti–β-catenin (red). The bottom panels are XZ section views of VE-cadherin and DAPI staining. Error bars represent the mean ± SD.
Mentions: The aforementioned experiments are consistent with the model that the dephosphorylated, closed form of Merlin suppresses recruitment of Rac to the membrane, but they do not exclude the possibility that the phosphorylated, “open” form of Merlin exerts an independent and positive effect on cell proliferation. To directly test if Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions, we used RNA interference. Transfection of a small interference RNA (siRNA) targeting human Merlin was sufficient to rescue integrin-dependent recruitment of GFP-Rac to the plasma membrane in confluent HUVEC (Fig. 5 A). Notably, knockdown of Merlin induced a large fraction of confluent HUVEC to progress through G1 and enter into S phase after mitogenic stimulation (Fig. 5 B). Immunoblotting revealed that the siRNA inhibited Merlin expression by >80%. As expected, the Merlin knockdown cells escaping contact inhibition had elevated levels of cyclin D1 and decreased levels of p27 (Fig. 5 B). However, cyclin D1 was not up-regulated and p27 was not down-regulated as effectively as they normally are in sparse cells. We attribute this partial result to the incomplete suppression of Merlin expression after siRNA transfection.

Bottom Line: PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin.Small interference RNA-mediated knockdown of Merlin exerts the same effects.Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. t-okada@ski.mskcc.org

ABSTRACT
Introduction of activated p21-activated kinase (PAK) is sufficient to release primary endothelial cells from contact inhibition of growth. Confluent cells display deficient activation of PAK and translocation of Rac to the plasma membrane at matrix adhesions. Targeting Rac to the plasma membrane rescues these cells from contact inhibition. PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin. Merlin mutants, which are presumed to exert a dominant-negative effect, enable recruitment of Rac to matrix adhesions and promote mitogenesis in confluent cells. Small interference RNA-mediated knockdown of Merlin exerts the same effects. Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway. These results provide a framework for understanding the tumor suppressor function of Merlin and indicate that Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions.

Show MeSH
Related in: MedlinePlus