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.

Show MeSH

Related in: MedlinePlus

LIMKs are overexpressed in human vestibular schwannomas. Lysed cultured human Schwann cells (HSC) cultured in 100-mm dishes and homogenized frozen human sporadic vestibular schwannomas (VS) were resolved by SDS-PAGE and analyzed by western blot. Quantitation of the western blots was done by densitometry analysis with ImageJ software, normalized to β-actin and is plotted below. Western blots for (a) LIMK1, (b) phospho-Thr508-LIMK1, (c) LIMK2, (d) phospho-Thr505-LIMK2 and (e) phospho-Ser3-cofilin and cofilin. β-actin was used as a loading control. *P<0.05 determined using unpaired t-test of HSC vs. VS populations, two-tailed. (f) HSCs from normal individuals (control) and HEI193 cells at P43 and P52 were cultured in 60-mm dishes and analyzed by western blotting for phospho-Thr508/505-LIMK1/2. β-actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4016185&req=5

Figure 9: LIMKs are overexpressed in human vestibular schwannomas. Lysed cultured human Schwann cells (HSC) cultured in 100-mm dishes and homogenized frozen human sporadic vestibular schwannomas (VS) were resolved by SDS-PAGE and analyzed by western blot. Quantitation of the western blots was done by densitometry analysis with ImageJ software, normalized to β-actin and is plotted below. Western blots for (a) LIMK1, (b) phospho-Thr508-LIMK1, (c) LIMK2, (d) phospho-Thr505-LIMK2 and (e) phospho-Ser3-cofilin and cofilin. β-actin was used as a loading control. *P<0.05 determined using unpaired t-test of HSC vs. VS populations, two-tailed. (f) HSCs from normal individuals (control) and HEI193 cells at P43 and P52 were cultured in 60-mm dishes and analyzed by western blotting for phospho-Thr508/505-LIMK1/2. β-actin was used as a loading control.

Mentions: To examine the relevance of LIMK inhibition to NF2, we compared LIMK1 and LIMK2 protein and phosphorylation levels in a small number of samples of HSCs from normal individuals and sporadic human vestibular schwannomas (VSs). We observed a significant increase (P<0.05 determined using unpaired t- test of HSC vs. VS populations, two-tailed) in both the protein and phospho-Thre508 levels of LIMK1 in VSs compared to HSCs (Figure 9a, b). Similarly, both LIMK2 and phospho-Thr505-LIMK2 levels showed a tendency to be higher in VSs compared to HSCs (Figure 9 c, d). Interestingly, the proportion of phospho-Ser3-cofilin to the total cofilin was higher in VSs than in HSCs (Figure 9 e).


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)

LIMKs are overexpressed in human vestibular schwannomas. Lysed cultured human Schwann cells (HSC) cultured in 100-mm dishes and homogenized frozen human sporadic vestibular schwannomas (VS) were resolved by SDS-PAGE and analyzed by western blot. Quantitation of the western blots was done by densitometry analysis with ImageJ software, normalized to β-actin and is plotted below. Western blots for (a) LIMK1, (b) phospho-Thr508-LIMK1, (c) LIMK2, (d) phospho-Thr505-LIMK2 and (e) phospho-Ser3-cofilin and cofilin. β-actin was used as a loading control. *P<0.05 determined using unpaired t-test of HSC vs. VS populations, two-tailed. (f) HSCs from normal individuals (control) and HEI193 cells at P43 and P52 were cultured in 60-mm dishes and analyzed by western blotting for phospho-Thr508/505-LIMK1/2. β-actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4016185&req=5

Figure 9: LIMKs are overexpressed in human vestibular schwannomas. Lysed cultured human Schwann cells (HSC) cultured in 100-mm dishes and homogenized frozen human sporadic vestibular schwannomas (VS) were resolved by SDS-PAGE and analyzed by western blot. Quantitation of the western blots was done by densitometry analysis with ImageJ software, normalized to β-actin and is plotted below. Western blots for (a) LIMK1, (b) phospho-Thr508-LIMK1, (c) LIMK2, (d) phospho-Thr505-LIMK2 and (e) phospho-Ser3-cofilin and cofilin. β-actin was used as a loading control. *P<0.05 determined using unpaired t-test of HSC vs. VS populations, two-tailed. (f) HSCs from normal individuals (control) and HEI193 cells at P43 and P52 were cultured in 60-mm dishes and analyzed by western blotting for phospho-Thr508/505-LIMK1/2. β-actin was used as a loading control.
Mentions: To examine the relevance of LIMK inhibition to NF2, we compared LIMK1 and LIMK2 protein and phosphorylation levels in a small number of samples of HSCs from normal individuals and sporadic human vestibular schwannomas (VSs). We observed a significant increase (P<0.05 determined using unpaired t- test of HSC vs. VS populations, two-tailed) in both the protein and phospho-Thre508 levels of LIMK1 in VSs compared to HSCs (Figure 9a, b). Similarly, both LIMK2 and phospho-Thr505-LIMK2 levels showed a tendency to be higher in VSs compared to HSCs (Figure 9 c, d). Interestingly, the proportion of phospho-Ser3-cofilin to the total cofilin was higher in VSs than in HSCs (Figure 9 e).

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