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Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells.

Zhou Y, He Y, Sharma R, Xing W, Estwick SA, Wu X, Rhodes SD, Xu M, Yang FC - Int J Mol Sci (2015)

Bottom Line: We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients.Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs.Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China. yuanzhou@ihcams.ac.cn.

ABSTRACT
Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1(+/-)) mice. Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1(+/-) MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.

No MeSH data available.


Related in: MedlinePlus

Migration and actin polymerization were significantly enhanced in Nf1+/− MSPCs. (A) Wound healing assays were performed by incubating WT and Nf1+/− MSPCs in 10 µg/mL of mitomycin C for one hour, after which a linear wound (marked by the white dotted lines) was created as shown. Wound healing was allowed to proceed in fresh media for 24 h, (original magnification ×200); (B) The number of cells migrating into the wound field were quantified, revealing an increased migration in Nf1+/− MSPCs compared with WT MSPCs (F = 75.76, Df = 1, *** p < 0.001; *** p < 0.001 for Nf1+/− MSPCs vs. WT MSPCs in the presence of 10% FBS, * p < 0.05 for untreated Nf1+/− MSPCs vs. untreated WT MSPCs). Data are represented as mean ± SD from duplicate wells from three independent experiments, each experiment was performed with different MSPCs culture isolated from individual mice; (C) Actin polymerization was measured following 2 h starvation and subsequent treatment with 10% FBS for different time periods. Flow cytometry analysis was performed following 400 nM FITC-phalloidin staining. An increased F-actin content was observed in Nf1+/− MSPCs comparison to WT MSPCs. A representative result of one of three independent experiments is shown; each experiment was performed with different MSPCs culture isolated from individual mice.
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ijms-16-12345-f002: Migration and actin polymerization were significantly enhanced in Nf1+/− MSPCs. (A) Wound healing assays were performed by incubating WT and Nf1+/− MSPCs in 10 µg/mL of mitomycin C for one hour, after which a linear wound (marked by the white dotted lines) was created as shown. Wound healing was allowed to proceed in fresh media for 24 h, (original magnification ×200); (B) The number of cells migrating into the wound field were quantified, revealing an increased migration in Nf1+/− MSPCs compared with WT MSPCs (F = 75.76, Df = 1, *** p < 0.001; *** p < 0.001 for Nf1+/− MSPCs vs. WT MSPCs in the presence of 10% FBS, * p < 0.05 for untreated Nf1+/− MSPCs vs. untreated WT MSPCs). Data are represented as mean ± SD from duplicate wells from three independent experiments, each experiment was performed with different MSPCs culture isolated from individual mice; (C) Actin polymerization was measured following 2 h starvation and subsequent treatment with 10% FBS for different time periods. Flow cytometry analysis was performed following 400 nM FITC-phalloidin staining. An increased F-actin content was observed in Nf1+/− MSPCs comparison to WT MSPCs. A representative result of one of three independent experiments is shown; each experiment was performed with different MSPCs culture isolated from individual mice.

Mentions: Wound healing assays was performed to assess migration of MSPCs. Nf1+/− MSPCs were noted to occupy an increased proportion of the wound space as compared to WT MSPCs 24 h after wound formation and continued culture in medium containing 10% fetal bovine serum (FBS) (Figure 2A). Quantification of the wound healing assay revealed a significant increase in the number of migrating Nf1+/− MSPCs (F = 75.76, Df = 1, p < 0.001; Figure 2B). Actin polymerization was further assessed to determine a molecular basis for the increased migration noted in Nf1+/− MSPCs. Nf1+/− MSPCs, as compared to WT controls, demonstrated significantly increased F-actin (polymerized actin) content after FBS stimulation at 30 s (Figure 2C, Figure S1). Taken together, these results suggested an increase in the migratory capacity of Nf1+/−MSPCs secondary to enhanced F-actin polymerization.


Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells.

Zhou Y, He Y, Sharma R, Xing W, Estwick SA, Wu X, Rhodes SD, Xu M, Yang FC - Int J Mol Sci (2015)

Migration and actin polymerization were significantly enhanced in Nf1+/− MSPCs. (A) Wound healing assays were performed by incubating WT and Nf1+/− MSPCs in 10 µg/mL of mitomycin C for one hour, after which a linear wound (marked by the white dotted lines) was created as shown. Wound healing was allowed to proceed in fresh media for 24 h, (original magnification ×200); (B) The number of cells migrating into the wound field were quantified, revealing an increased migration in Nf1+/− MSPCs compared with WT MSPCs (F = 75.76, Df = 1, *** p < 0.001; *** p < 0.001 for Nf1+/− MSPCs vs. WT MSPCs in the presence of 10% FBS, * p < 0.05 for untreated Nf1+/− MSPCs vs. untreated WT MSPCs). Data are represented as mean ± SD from duplicate wells from three independent experiments, each experiment was performed with different MSPCs culture isolated from individual mice; (C) Actin polymerization was measured following 2 h starvation and subsequent treatment with 10% FBS for different time periods. Flow cytometry analysis was performed following 400 nM FITC-phalloidin staining. An increased F-actin content was observed in Nf1+/− MSPCs comparison to WT MSPCs. A representative result of one of three independent experiments is shown; each experiment was performed with different MSPCs culture isolated from individual mice.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490447&req=5

ijms-16-12345-f002: Migration and actin polymerization were significantly enhanced in Nf1+/− MSPCs. (A) Wound healing assays were performed by incubating WT and Nf1+/− MSPCs in 10 µg/mL of mitomycin C for one hour, after which a linear wound (marked by the white dotted lines) was created as shown. Wound healing was allowed to proceed in fresh media for 24 h, (original magnification ×200); (B) The number of cells migrating into the wound field were quantified, revealing an increased migration in Nf1+/− MSPCs compared with WT MSPCs (F = 75.76, Df = 1, *** p < 0.001; *** p < 0.001 for Nf1+/− MSPCs vs. WT MSPCs in the presence of 10% FBS, * p < 0.05 for untreated Nf1+/− MSPCs vs. untreated WT MSPCs). Data are represented as mean ± SD from duplicate wells from three independent experiments, each experiment was performed with different MSPCs culture isolated from individual mice; (C) Actin polymerization was measured following 2 h starvation and subsequent treatment with 10% FBS for different time periods. Flow cytometry analysis was performed following 400 nM FITC-phalloidin staining. An increased F-actin content was observed in Nf1+/− MSPCs comparison to WT MSPCs. A representative result of one of three independent experiments is shown; each experiment was performed with different MSPCs culture isolated from individual mice.
Mentions: Wound healing assays was performed to assess migration of MSPCs. Nf1+/− MSPCs were noted to occupy an increased proportion of the wound space as compared to WT MSPCs 24 h after wound formation and continued culture in medium containing 10% fetal bovine serum (FBS) (Figure 2A). Quantification of the wound healing assay revealed a significant increase in the number of migrating Nf1+/− MSPCs (F = 75.76, Df = 1, p < 0.001; Figure 2B). Actin polymerization was further assessed to determine a molecular basis for the increased migration noted in Nf1+/− MSPCs. Nf1+/− MSPCs, as compared to WT controls, demonstrated significantly increased F-actin (polymerized actin) content after FBS stimulation at 30 s (Figure 2C, Figure S1). Taken together, these results suggested an increase in the migratory capacity of Nf1+/−MSPCs secondary to enhanced F-actin polymerization.

Bottom Line: We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients.Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs.Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China. yuanzhou@ihcams.ac.cn.

ABSTRACT
Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1(+/-)) mice. Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1(+/-) MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.

No MeSH data available.


Related in: MedlinePlus