Limits...
LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

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Elevated levels of LIMK and phospho-Ser3-cofilin in Nf2ΔEx2 MSCs compared to control MSCs(a) Representative confocal images of Nf2ΔEx2 MSCs and Nf2flox2/flox2 MSCs grown overnight on glass coverslips, fixed and immunostained with the indicated antibodies (green). F-actin was visualized with phaloidin-Alexa633 (white) and the nucleus was visualized by DAPI stain (blue). Scale bar: 20 µm. (b) Quantitation of the immunofluorescence for the indicated proteins in three independent experiments was performed with Volocity software. ***P<0.001; *P<0.05 determined by two-way ANOVA using Bonferroni post-tests. (c) Characterization of Nf2flox2/flox2 and Nf2ΔEx2 MSCs. Control Nf2 with the exon2 flanked by loxP sites, Nf2flox2/flox2 and Nf2ΔEx2 MSCs analyzed by western blotting for N-terminus merlin and (d) PCR analysis of genomic DNA. Primers P4/P5 amplified a 305-bp band for wild type Nf2 FVB/N and a 442-bp band for Nf2flox2/flox2 and primers P6/P5 amplified a 338-bp band for Nf2ΔEx2. (e)Nf2ΔEx2 MSCs and control Nf2flox2/flox2 MSCs analyzed by western blotting for LIMK1, LIMK2, phospho-LIMK1/2 (Thr508/505), and (f) phospho-Ser3-cofilin and cofilin. Anti-β-actin was used as a loading control.
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Figure 1: Elevated levels of LIMK and phospho-Ser3-cofilin in Nf2ΔEx2 MSCs compared to control MSCs(a) Representative confocal images of Nf2ΔEx2 MSCs and Nf2flox2/flox2 MSCs grown overnight on glass coverslips, fixed and immunostained with the indicated antibodies (green). F-actin was visualized with phaloidin-Alexa633 (white) and the nucleus was visualized by DAPI stain (blue). Scale bar: 20 µm. (b) Quantitation of the immunofluorescence for the indicated proteins in three independent experiments was performed with Volocity software. ***P<0.001; *P<0.05 determined by two-way ANOVA using Bonferroni post-tests. (c) Characterization of Nf2flox2/flox2 and Nf2ΔEx2 MSCs. Control Nf2 with the exon2 flanked by loxP sites, Nf2flox2/flox2 and Nf2ΔEx2 MSCs analyzed by western blotting for N-terminus merlin and (d) PCR analysis of genomic DNA. Primers P4/P5 amplified a 305-bp band for wild type Nf2 FVB/N and a 442-bp band for Nf2flox2/flox2 and primers P6/P5 amplified a 338-bp band for Nf2ΔEx2. (e)Nf2ΔEx2 MSCs and control Nf2flox2/flox2 MSCs analyzed by western blotting for LIMK1, LIMK2, phospho-LIMK1/2 (Thr508/505), and (f) phospho-Ser3-cofilin and cofilin. Anti-β-actin was used as a loading control.

Mentions: Using two complementary techniques, we assessed the levels of LIMK and phospho-Ser3-cofilin in Nf2- deficient (Nf2ΔEx2) MSC lines developed by in vitro ad-Cre deletion of Nf2 exon2 from Nf2flox2/flox2 MSCs (34). By immunostaining Nf2ΔEx2 MSCs were confirmed to be merlin-deficient and expressed the SC marker S100 (Figure 1a). Merlin-deficient Schwann cells were larger than control Nf2flox2/flox2 MSCs and had increased levels of F-actin revealed by phalloidin staining. The intensity of LIMK1 and LIMK2 immunofluorescence was higher in Nf2ΔEx2 MSCs than in controls and was detected throughout the cell. Consistent with increased LIMK activity, the intensity of phospho-Ser3-cofilin immunofluorescence was also higher in Nf2ΔEx2 MSCs than in controls (Figure 1 a, b).


LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Elevated levels of LIMK and phospho-Ser3-cofilin in Nf2ΔEx2 MSCs compared to control MSCs(a) Representative confocal images of Nf2ΔEx2 MSCs and Nf2flox2/flox2 MSCs grown overnight on glass coverslips, fixed and immunostained with the indicated antibodies (green). F-actin was visualized with phaloidin-Alexa633 (white) and the nucleus was visualized by DAPI stain (blue). Scale bar: 20 µm. (b) Quantitation of the immunofluorescence for the indicated proteins in three independent experiments was performed with Volocity software. ***P<0.001; *P<0.05 determined by two-way ANOVA using Bonferroni post-tests. (c) Characterization of Nf2flox2/flox2 and Nf2ΔEx2 MSCs. Control Nf2 with the exon2 flanked by loxP sites, Nf2flox2/flox2 and Nf2ΔEx2 MSCs analyzed by western blotting for N-terminus merlin and (d) PCR analysis of genomic DNA. Primers P4/P5 amplified a 305-bp band for wild type Nf2 FVB/N and a 442-bp band for Nf2flox2/flox2 and primers P6/P5 amplified a 338-bp band for Nf2ΔEx2. (e)Nf2ΔEx2 MSCs and control Nf2flox2/flox2 MSCs analyzed by western blotting for LIMK1, LIMK2, phospho-LIMK1/2 (Thr508/505), and (f) phospho-Ser3-cofilin and cofilin. Anti-β-actin was used as a loading control.
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Figure 1: Elevated levels of LIMK and phospho-Ser3-cofilin in Nf2ΔEx2 MSCs compared to control MSCs(a) Representative confocal images of Nf2ΔEx2 MSCs and Nf2flox2/flox2 MSCs grown overnight on glass coverslips, fixed and immunostained with the indicated antibodies (green). F-actin was visualized with phaloidin-Alexa633 (white) and the nucleus was visualized by DAPI stain (blue). Scale bar: 20 µm. (b) Quantitation of the immunofluorescence for the indicated proteins in three independent experiments was performed with Volocity software. ***P<0.001; *P<0.05 determined by two-way ANOVA using Bonferroni post-tests. (c) Characterization of Nf2flox2/flox2 and Nf2ΔEx2 MSCs. Control Nf2 with the exon2 flanked by loxP sites, Nf2flox2/flox2 and Nf2ΔEx2 MSCs analyzed by western blotting for N-terminus merlin and (d) PCR analysis of genomic DNA. Primers P4/P5 amplified a 305-bp band for wild type Nf2 FVB/N and a 442-bp band for Nf2flox2/flox2 and primers P6/P5 amplified a 338-bp band for Nf2ΔEx2. (e)Nf2ΔEx2 MSCs and control Nf2flox2/flox2 MSCs analyzed by western blotting for LIMK1, LIMK2, phospho-LIMK1/2 (Thr508/505), and (f) phospho-Ser3-cofilin and cofilin. Anti-β-actin was used as a loading control.
Mentions: Using two complementary techniques, we assessed the levels of LIMK and phospho-Ser3-cofilin in Nf2- deficient (Nf2ΔEx2) MSC lines developed by in vitro ad-Cre deletion of Nf2 exon2 from Nf2flox2/flox2 MSCs (34). By immunostaining Nf2ΔEx2 MSCs were confirmed to be merlin-deficient and expressed the SC marker S100 (Figure 1a). Merlin-deficient Schwann cells were larger than control Nf2flox2/flox2 MSCs and had increased levels of F-actin revealed by phalloidin staining. The intensity of LIMK1 and LIMK2 immunofluorescence was higher in Nf2ΔEx2 MSCs than in controls and was detected throughout the cell. Consistent with increased LIMK activity, the intensity of phospho-Ser3-cofilin immunofluorescence was also higher in Nf2ΔEx2 MSCs than in controls (Figure 1 a, b).

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Show MeSH
Related in: MedlinePlus