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

Migration and adhesion of Nf1+/− MSPCs was mediated by mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3-K) pathways. (A) Representative high power fields (20× objective lens) of wound healing assays for WT and Nf1+/− MSPCs cultured with serum free media or 10% FBS in the presence or absence of either LY294002 or PD0325901. Nf1+/− MSPCs have enhanced migration in comparison to WT in serum free or 10% FBS supplemented media, which was significantly decreased by LY294002 and PD0325901 (** p < 0.01 for Nf1+/− MSPCs vs. WT MSPCs cultured in media; *** p < 0.001 for 10% FBS treated Nf1+/− MSPCs vs. 10% FBS treated WT MSPCs; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS); (B) Representative high power fields (20× objective lens) from CH296 adhesion assays for WT and Nf1+/− MSPCs performed in serum free or 10% FBS supplemented media in the presence or absence of LY294002 or PD0325901. The adhesion of Nf1+/− MSPCs was significantly increased in comparison to WT MSPCs in either serum free or 10% FBS supplemented media. Adhesions were significantly reduced in the presence of LY294002 and PD0325901 (* p < 0.05 for Nf1+/−vs. WT MSPCs in serum free media; *** p < 0.001 for Nf1+/−vs. WT MSPCs in 10% FBS supplemented media; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS). Data are represented as mean ± SD from three individual 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-f005: Migration and adhesion of Nf1+/− MSPCs was mediated by mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3-K) pathways. (A) Representative high power fields (20× objective lens) of wound healing assays for WT and Nf1+/− MSPCs cultured with serum free media or 10% FBS in the presence or absence of either LY294002 or PD0325901. Nf1+/− MSPCs have enhanced migration in comparison to WT in serum free or 10% FBS supplemented media, which was significantly decreased by LY294002 and PD0325901 (** p < 0.01 for Nf1+/− MSPCs vs. WT MSPCs cultured in media; *** p < 0.001 for 10% FBS treated Nf1+/− MSPCs vs. 10% FBS treated WT MSPCs; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS); (B) Representative high power fields (20× objective lens) from CH296 adhesion assays for WT and Nf1+/− MSPCs performed in serum free or 10% FBS supplemented media in the presence or absence of LY294002 or PD0325901. The adhesion of Nf1+/− MSPCs was significantly increased in comparison to WT MSPCs in either serum free or 10% FBS supplemented media. Adhesions were significantly reduced in the presence of LY294002 and PD0325901 (* p < 0.05 for Nf1+/−vs. WT MSPCs in serum free media; *** p < 0.001 for Nf1+/−vs. WT MSPCs in 10% FBS supplemented media; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS). Data are represented as mean ± SD from three individual experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.

Mentions: In order to determine the functional implications of pharmacologically inhibiting hyperactivated PI3-K and MAPK pathways in Nf1+/− MSPCs, wound healing and adhesion assays were performed to assess migration and adhesion of MSPCs in the presence of LY294002 or PD0325901 inhibitors. In comparison to WT, Nf1+/− MSPCs demonstrate increased migration in response to either media alone or media supplemented with 10% FBS, which was significantly attenuated by LY294002 or PD0325901 (p < 0.001, Figure 5A). Migration of Nf1+/− MSPCs vs. WT MSPCs responded to different concentrations of PD0325901, as shown in Figure S3. A similar trend was noted in the adhesion assay where Nf1+/− MSPCs were significantly more adhesive in comparison to WT in response to either media alone or 10% FBS stimulation. Likewise, adhesion was markedly reduced in the presence of LY294002 and PD0325901 (p < 0.001, Figure 5B). The efficacy of PI3-K and MAPK inhibitors to significantly attenuate the enhanced signal transduction, migration, and adhesion of MSPCs with Nf1 haploinsufficiency suggests that these two pathways may play important role in mediating Nf1+/− MSPC gain-in-functions.


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 adhesion of Nf1+/− MSPCs was mediated by mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3-K) pathways. (A) Representative high power fields (20× objective lens) of wound healing assays for WT and Nf1+/− MSPCs cultured with serum free media or 10% FBS in the presence or absence of either LY294002 or PD0325901. Nf1+/− MSPCs have enhanced migration in comparison to WT in serum free or 10% FBS supplemented media, which was significantly decreased by LY294002 and PD0325901 (** p < 0.01 for Nf1+/− MSPCs vs. WT MSPCs cultured in media; *** p < 0.001 for 10% FBS treated Nf1+/− MSPCs vs. 10% FBS treated WT MSPCs; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS); (B) Representative high power fields (20× objective lens) from CH296 adhesion assays for WT and Nf1+/− MSPCs performed in serum free or 10% FBS supplemented media in the presence or absence of LY294002 or PD0325901. The adhesion of Nf1+/− MSPCs was significantly increased in comparison to WT MSPCs in either serum free or 10% FBS supplemented media. Adhesions were significantly reduced in the presence of LY294002 and PD0325901 (* p < 0.05 for Nf1+/−vs. WT MSPCs in serum free media; *** p < 0.001 for Nf1+/−vs. WT MSPCs in 10% FBS supplemented media; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS). Data are represented as mean ± SD from three individual 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-f005: Migration and adhesion of Nf1+/− MSPCs was mediated by mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3-K) pathways. (A) Representative high power fields (20× objective lens) of wound healing assays for WT and Nf1+/− MSPCs cultured with serum free media or 10% FBS in the presence or absence of either LY294002 or PD0325901. Nf1+/− MSPCs have enhanced migration in comparison to WT in serum free or 10% FBS supplemented media, which was significantly decreased by LY294002 and PD0325901 (** p < 0.01 for Nf1+/− MSPCs vs. WT MSPCs cultured in media; *** p < 0.001 for 10% FBS treated Nf1+/− MSPCs vs. 10% FBS treated WT MSPCs; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS); (B) Representative high power fields (20× objective lens) from CH296 adhesion assays for WT and Nf1+/− MSPCs performed in serum free or 10% FBS supplemented media in the presence or absence of LY294002 or PD0325901. The adhesion of Nf1+/− MSPCs was significantly increased in comparison to WT MSPCs in either serum free or 10% FBS supplemented media. Adhesions were significantly reduced in the presence of LY294002 and PD0325901 (* p < 0.05 for Nf1+/−vs. WT MSPCs in serum free media; *** p < 0.001 for Nf1+/−vs. WT MSPCs in 10% FBS supplemented media; *** p < 0.001 for LY294002 or PD0325901 treated and untreated Nf1+/− MSPCs in the presence of 10% FBS). Data are represented as mean ± SD from three individual experiments, and each experiment was performed with different MSPCs culture isolated from individual mice.
Mentions: In order to determine the functional implications of pharmacologically inhibiting hyperactivated PI3-K and MAPK pathways in Nf1+/− MSPCs, wound healing and adhesion assays were performed to assess migration and adhesion of MSPCs in the presence of LY294002 or PD0325901 inhibitors. In comparison to WT, Nf1+/− MSPCs demonstrate increased migration in response to either media alone or media supplemented with 10% FBS, which was significantly attenuated by LY294002 or PD0325901 (p < 0.001, Figure 5A). Migration of Nf1+/− MSPCs vs. WT MSPCs responded to different concentrations of PD0325901, as shown in Figure S3. A similar trend was noted in the adhesion assay where Nf1+/− MSPCs were significantly more adhesive in comparison to WT in response to either media alone or 10% FBS stimulation. Likewise, adhesion was markedly reduced in the presence of LY294002 and PD0325901 (p < 0.001, Figure 5B). The efficacy of PI3-K and MAPK inhibitors to significantly attenuate the enhanced signal transduction, migration, and adhesion of MSPCs with Nf1 haploinsufficiency suggests that these two pathways may play important role in mediating Nf1+/− MSPC gain-in-functions.

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