Limits...
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

Enhancement of cellular adhesion in Nf1+/− MSPCs. (A) MSPCs were plated into wells pre-coated with either 8 µg/mL CH296 (recombinant fibronectin fragment) or 0.1% bovine serum albumin (BSA). Following a 30 min incubation period at 37 °C, the plates were washed and adherent cells were counted on five representative fields/well from six replicate wells, (original magnification ×200); (B) Nf1+/− MSPCs had significantly increased adhesion to CH296 coated plates in comparison to WT MSPCs (*** p < 0.001 for CH296 coated Nf1+/− MSPCs vs. CH296 coated WT MSPCs); (C) Preferential adhesion to fibronectin binding sites, H296 and CH271, was assessed. Nf1+/− MSPCs exhibited significantly greater adhesion to CH271 as compared to H296. (*** p < 0.001 for CH271 coated Nf1+/− MSPCs vs. CH271 coated WT MSPCs); (D) Expression of CH271 receptor, CD49e, was quantified by flow cytometry, demonstrating significantly increased expression of CD49e in Nf1+/− MSPCs in comparison to WT. The green lines represent isotype controls while the green solid areas represent the experimental samples. Data are one representative result of three independent experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490447&req=5

ijms-16-12345-f003: Enhancement of cellular adhesion in Nf1+/− MSPCs. (A) MSPCs were plated into wells pre-coated with either 8 µg/mL CH296 (recombinant fibronectin fragment) or 0.1% bovine serum albumin (BSA). Following a 30 min incubation period at 37 °C, the plates were washed and adherent cells were counted on five representative fields/well from six replicate wells, (original magnification ×200); (B) Nf1+/− MSPCs had significantly increased adhesion to CH296 coated plates in comparison to WT MSPCs (*** p < 0.001 for CH296 coated Nf1+/− MSPCs vs. CH296 coated WT MSPCs); (C) Preferential adhesion to fibronectin binding sites, H296 and CH271, was assessed. Nf1+/− MSPCs exhibited significantly greater adhesion to CH271 as compared to H296. (*** p < 0.001 for CH271 coated Nf1+/− MSPCs vs. CH271 coated WT MSPCs); (D) Expression of CH271 receptor, CD49e, was quantified by flow cytometry, demonstrating significantly increased expression of CD49e in Nf1+/− MSPCs in comparison to WT. The green lines represent isotype controls while the green solid areas represent the experimental samples. Data are one representative result of three independent experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.

Mentions: Nf1+/− MSPCs showed significantly increased adhesion to recombinant fibronectin fragment, CH296, pre-coated plates as compared to WT controls (Figure 3A; p < 0.001, Figure 3B). Fibronectin’s binding sites, H296 and CH271, were analyzed for preferential affinity in Nf1+/− MSPCs. H296 and CH271 specific adhesion assays demonstrated a significant increase in Nf1+/− MSPC adhesion in CH271 assays but not H296 assays as compared to WT (p < 0.001, Figure 3C). By contrast, WT MSPCs did not show preference to either CH271 or H296. To further characterize the increased affinity of Nf1+/− MSPCs for CH271, expression of its receptor, CD49e (also known as integrin α5 or fibronectin receptor α), was quantified by flow cytometry. The expression level of CD49e in Nf1+/− MSPCs was significantly increased as compared to WT MSPCs (Figure 3D). We also analyzed CD49d, the H296 receptor, and found no statistical difference in expression between WT and Nf1+/− MSPCs (data not shown).


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)

Enhancement of cellular adhesion in Nf1+/− MSPCs. (A) MSPCs were plated into wells pre-coated with either 8 µg/mL CH296 (recombinant fibronectin fragment) or 0.1% bovine serum albumin (BSA). Following a 30 min incubation period at 37 °C, the plates were washed and adherent cells were counted on five representative fields/well from six replicate wells, (original magnification ×200); (B) Nf1+/− MSPCs had significantly increased adhesion to CH296 coated plates in comparison to WT MSPCs (*** p < 0.001 for CH296 coated Nf1+/− MSPCs vs. CH296 coated WT MSPCs); (C) Preferential adhesion to fibronectin binding sites, H296 and CH271, was assessed. Nf1+/− MSPCs exhibited significantly greater adhesion to CH271 as compared to H296. (*** p < 0.001 for CH271 coated Nf1+/− MSPCs vs. CH271 coated WT MSPCs); (D) Expression of CH271 receptor, CD49e, was quantified by flow cytometry, demonstrating significantly increased expression of CD49e in Nf1+/− MSPCs in comparison to WT. The green lines represent isotype controls while the green solid areas represent the experimental samples. Data are one representative result 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-f003: Enhancement of cellular adhesion in Nf1+/− MSPCs. (A) MSPCs were plated into wells pre-coated with either 8 µg/mL CH296 (recombinant fibronectin fragment) or 0.1% bovine serum albumin (BSA). Following a 30 min incubation period at 37 °C, the plates were washed and adherent cells were counted on five representative fields/well from six replicate wells, (original magnification ×200); (B) Nf1+/− MSPCs had significantly increased adhesion to CH296 coated plates in comparison to WT MSPCs (*** p < 0.001 for CH296 coated Nf1+/− MSPCs vs. CH296 coated WT MSPCs); (C) Preferential adhesion to fibronectin binding sites, H296 and CH271, was assessed. Nf1+/− MSPCs exhibited significantly greater adhesion to CH271 as compared to H296. (*** p < 0.001 for CH271 coated Nf1+/− MSPCs vs. CH271 coated WT MSPCs); (D) Expression of CH271 receptor, CD49e, was quantified by flow cytometry, demonstrating significantly increased expression of CD49e in Nf1+/− MSPCs in comparison to WT. The green lines represent isotype controls while the green solid areas represent the experimental samples. Data are one representative result of three independent experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.
Mentions: Nf1+/− MSPCs showed significantly increased adhesion to recombinant fibronectin fragment, CH296, pre-coated plates as compared to WT controls (Figure 3A; p < 0.001, Figure 3B). Fibronectin’s binding sites, H296 and CH271, were analyzed for preferential affinity in Nf1+/− MSPCs. H296 and CH271 specific adhesion assays demonstrated a significant increase in Nf1+/− MSPC adhesion in CH271 assays but not H296 assays as compared to WT (p < 0.001, Figure 3C). By contrast, WT MSPCs did not show preference to either CH271 or H296. To further characterize the increased affinity of Nf1+/− MSPCs for CH271, expression of its receptor, CD49e (also known as integrin α5 or fibronectin receptor α), was quantified by flow cytometry. The expression level of CD49e in Nf1+/− MSPCs was significantly increased as compared to WT MSPCs (Figure 3D). We also analyzed CD49d, the H296 receptor, and found no statistical difference in expression between WT and Nf1+/− MSPCs (data not shown).

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