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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

Nf1+/− MSPCs exhibit increased Akt (also known as protein kinase B) and extracellular-signal-regulated protein kinase (Erk)1/2 phosphorylation, which can be inhibited by LY294002 and PD0325901, respectively. Phosphorylation of Akt and Erk1/2 was determined by Western blot in WT and Nf1+/− MSPCs following 10% FBS stimulation in the presence or absence of PI3-K inhibitor, LY294002, or MAPK inhibitor, PD0325901. Data represents one of three independent experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.
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ijms-16-12345-f004: Nf1+/− MSPCs exhibit increased Akt (also known as protein kinase B) and extracellular-signal-regulated protein kinase (Erk)1/2 phosphorylation, which can be inhibited by LY294002 and PD0325901, respectively. Phosphorylation of Akt and Erk1/2 was determined by Western blot in WT and Nf1+/− MSPCs following 10% FBS stimulation in the presence or absence of PI3-K inhibitor, LY294002, or MAPK inhibitor, PD0325901. Data represents one of three independent experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.

Mentions: Serum starved WT and Nf1+/− MSPCs were stimulated with 10% FBS to assess activation of the PI3-K and MAPK pathways downstream of Ras. Baseline expression of phosphorylated (p) Akt (also known as protein kinase B) and extracellular-signal-regulated protein kinase (Erk) 1/2 in Nf1+/− and WT MSPCs were undetectable. However, stimulation with 10% FBS for 2 and 5 min showed significantly enhanced expression of pAkt and pErk1/2 in Nf1+/− MSPCs compared to WT MSPCs. Increased pAkt levels were restored to baseline by 30 min pretreatment of a PI3-K inhibitor, LY294002, in both WT and Nf1+/− MSPCs. Likewise, pErk1/2 expression was significantly reduced in the presence of a MEK inhibitor, PD0325901, in both WT and Nf1+/− MSPCs (Figure 4). The pretreatment of LY294002 and PD0325901 has also been prolonged to 4 h, and the results did not show a significant pathway cross talk occurred between these two inhibitors (Figure S2).


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)

Nf1+/− MSPCs exhibit increased Akt (also known as protein kinase B) and extracellular-signal-regulated protein kinase (Erk)1/2 phosphorylation, which can be inhibited by LY294002 and PD0325901, respectively. Phosphorylation of Akt and Erk1/2 was determined by Western blot in WT and Nf1+/− MSPCs following 10% FBS stimulation in the presence or absence of PI3-K inhibitor, LY294002, or MAPK inhibitor, PD0325901. Data represents one of three independent experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12345-f004: Nf1+/− MSPCs exhibit increased Akt (also known as protein kinase B) and extracellular-signal-regulated protein kinase (Erk)1/2 phosphorylation, which can be inhibited by LY294002 and PD0325901, respectively. Phosphorylation of Akt and Erk1/2 was determined by Western blot in WT and Nf1+/− MSPCs following 10% FBS stimulation in the presence or absence of PI3-K inhibitor, LY294002, or MAPK inhibitor, PD0325901. Data represents one of three independent experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.
Mentions: Serum starved WT and Nf1+/− MSPCs were stimulated with 10% FBS to assess activation of the PI3-K and MAPK pathways downstream of Ras. Baseline expression of phosphorylated (p) Akt (also known as protein kinase B) and extracellular-signal-regulated protein kinase (Erk) 1/2 in Nf1+/− and WT MSPCs were undetectable. However, stimulation with 10% FBS for 2 and 5 min showed significantly enhanced expression of pAkt and pErk1/2 in Nf1+/− MSPCs compared to WT MSPCs. Increased pAkt levels were restored to baseline by 30 min pretreatment of a PI3-K inhibitor, LY294002, in both WT and Nf1+/− MSPCs. Likewise, pErk1/2 expression was significantly reduced in the presence of a MEK inhibitor, PD0325901, in both WT and Nf1+/− MSPCs (Figure 4). The pretreatment of LY294002 and PD0325901 has also been prolonged to 4 h, and the results did not show a significant pathway cross talk occurred between these two inhibitors (Figure S2).

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