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Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

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Post-translational changes associated with conditional reprogramming.A, Heatmap of the post-translational changes occurring in HFKs after 2 days in culture with J2 cells (J2), Y or J2 cells and Y (J2+Y). Shown are proteins that were modified ≥50% by each condition as determined by RPPA analysis. B, Summary of RPPA analysis. Co-culture of HFKs with J2 cells and Y preferentially increased pAkt, and reduced phosphorylation of 13 proteins, including Smad2. C, Western analysis of conditionally reprogrammed HFKs. J2 cells prevented the reduction of pSmad1/5, pSmad2, TGF-β receptor II (TβRII), nuclear β-catenin, c-Myc, EGFR, ERBB2, VEGFR2, pSrc, pERK, pGSK3β, eIF4G and p4EFBP by Y.
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pone.0116755.g004: Post-translational changes associated with conditional reprogramming.A, Heatmap of the post-translational changes occurring in HFKs after 2 days in culture with J2 cells (J2), Y or J2 cells and Y (J2+Y). Shown are proteins that were modified ≥50% by each condition as determined by RPPA analysis. B, Summary of RPPA analysis. Co-culture of HFKs with J2 cells and Y preferentially increased pAkt, and reduced phosphorylation of 13 proteins, including Smad2. C, Western analysis of conditionally reprogrammed HFKs. J2 cells prevented the reduction of pSmad1/5, pSmad2, TGF-β receptor II (TβRII), nuclear β-catenin, c-Myc, EGFR, ERBB2, VEGFR2, pSrc, pERK, pGSK3β, eIF4G and p4EFBP by Y.

Mentions: To obtain a better picture of signaling networks associated with conditional reprogramming, posttranslational changes were assessed by RPPA (Fig. 4A, B; Table D in S1 File). J2 cells in combination with Y increased the level of pT308Akt and reduced the levels of 14 phosphoproteins. Y alone increased expression of pY1353Ron, pT187p27, pY754PDGFRα and pS83ASK1; J2 cells increased expression of 15 phosphoproteins, particularly pMARCKS and pAdducin (Table D in S1 File). The most notable change in common among the three conditions was reduced Smad1/2/5/8 phosphorylation (Fig. 4C, Fig. E in S1 File), and was consistent with the functional siRNA screening data from J2 cells (Fig. 3) as well as the gene expression profiling of conditionally reprogrammed HFKs (Fig. 2). Western blotting confirmed that Smad phosphorylation was reduced by Y, and that the levels of c-Myc, EGFR, ERBB2, pY416Src, pS9GSK3β, eIF4G and pT37/414EFBP were increased by J2 cells (Fig. 4C, Fig. E in S1 File).


Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Post-translational changes associated with conditional reprogramming.A, Heatmap of the post-translational changes occurring in HFKs after 2 days in culture with J2 cells (J2), Y or J2 cells and Y (J2+Y). Shown are proteins that were modified ≥50% by each condition as determined by RPPA analysis. B, Summary of RPPA analysis. Co-culture of HFKs with J2 cells and Y preferentially increased pAkt, and reduced phosphorylation of 13 proteins, including Smad2. C, Western analysis of conditionally reprogrammed HFKs. J2 cells prevented the reduction of pSmad1/5, pSmad2, TGF-β receptor II (TβRII), nuclear β-catenin, c-Myc, EGFR, ERBB2, VEGFR2, pSrc, pERK, pGSK3β, eIF4G and p4EFBP by Y.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340869&req=5

pone.0116755.g004: Post-translational changes associated with conditional reprogramming.A, Heatmap of the post-translational changes occurring in HFKs after 2 days in culture with J2 cells (J2), Y or J2 cells and Y (J2+Y). Shown are proteins that were modified ≥50% by each condition as determined by RPPA analysis. B, Summary of RPPA analysis. Co-culture of HFKs with J2 cells and Y preferentially increased pAkt, and reduced phosphorylation of 13 proteins, including Smad2. C, Western analysis of conditionally reprogrammed HFKs. J2 cells prevented the reduction of pSmad1/5, pSmad2, TGF-β receptor II (TβRII), nuclear β-catenin, c-Myc, EGFR, ERBB2, VEGFR2, pSrc, pERK, pGSK3β, eIF4G and p4EFBP by Y.
Mentions: To obtain a better picture of signaling networks associated with conditional reprogramming, posttranslational changes were assessed by RPPA (Fig. 4A, B; Table D in S1 File). J2 cells in combination with Y increased the level of pT308Akt and reduced the levels of 14 phosphoproteins. Y alone increased expression of pY1353Ron, pT187p27, pY754PDGFRα and pS83ASK1; J2 cells increased expression of 15 phosphoproteins, particularly pMARCKS and pAdducin (Table D in S1 File). The most notable change in common among the three conditions was reduced Smad1/2/5/8 phosphorylation (Fig. 4C, Fig. E in S1 File), and was consistent with the functional siRNA screening data from J2 cells (Fig. 3) as well as the gene expression profiling of conditionally reprogrammed HFKs (Fig. 2). Western blotting confirmed that Smad phosphorylation was reduced by Y, and that the levels of c-Myc, EGFR, ERBB2, pY416Src, pS9GSK3β, eIF4G and pT37/414EFBP were increased by J2 cells (Fig. 4C, Fig. E in S1 File).

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

Show MeSH
Related in: MedlinePlus