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TNFα signaling regulates cystic epithelial cell proliferation through Akt/mTOR and ERK/MAPK/Cdk2 mediated Id2 signaling.

Zhou JX, Fan LX, Li X, Calvet JP, Li X - PLoS ONE (2015)

Bottom Line: However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive.In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation.The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160, United States of America; Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, United States of America.

ABSTRACT
Tumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD.

No MeSH data available.


Related in: MedlinePlus

Inhibition of Cdk2 blocks nuclear translocation of Id2 induced by TNFα and RANKL.Localization of Id2 in Pkd1 wild type MEK cells after TNFα (A) and RANKL (B) treatment in the presence of kinase inhibitors. Pkd1 wild type MEK cells were placed in DMEM F12 containing 1% FBS for 12 hours. Serum starved cells were treated with DMSO or the indicated kinase inhibitors for 3 hours. Inhibitor pre-treated cells were untreated or stimulated with TNFα and RANKL for 3 hours. Id2 was detected with an anti-Id2 antibody, followed by Fluro 488 conjugated anti-rabbit IgG antibody (Green). Nuclear DNA was stained with DAPI (Blue). One result, representative of three independent experiments, is shown. Percentage of cells with nuclear (N) and cystoplasmic (C) localization are indicated below the images. (C) Western blot analysis of the expression of Id2 from cytoplasmic and nuclear fractions of Pkd1 wild-type and  MEK cells treated with TNFα, LY294002 (LY), TNFα plus LY294002, and vehicle control for 3 h. The numbers at the bottom indicate the intensities of Id2 bands relative to actin and Lamin A/C in cytoplasmic fractions and nuclear fractions, respectively.
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pone.0131043.g007: Inhibition of Cdk2 blocks nuclear translocation of Id2 induced by TNFα and RANKL.Localization of Id2 in Pkd1 wild type MEK cells after TNFα (A) and RANKL (B) treatment in the presence of kinase inhibitors. Pkd1 wild type MEK cells were placed in DMEM F12 containing 1% FBS for 12 hours. Serum starved cells were treated with DMSO or the indicated kinase inhibitors for 3 hours. Inhibitor pre-treated cells were untreated or stimulated with TNFα and RANKL for 3 hours. Id2 was detected with an anti-Id2 antibody, followed by Fluro 488 conjugated anti-rabbit IgG antibody (Green). Nuclear DNA was stained with DAPI (Blue). One result, representative of three independent experiments, is shown. Percentage of cells with nuclear (N) and cystoplasmic (C) localization are indicated below the images. (C) Western blot analysis of the expression of Id2 from cytoplasmic and nuclear fractions of Pkd1 wild-type and MEK cells treated with TNFα, LY294002 (LY), TNFα plus LY294002, and vehicle control for 3 h. The numbers at the bottom indicate the intensities of Id2 bands relative to actin and Lamin A/C in cytoplasmic fractions and nuclear fractions, respectively.

Mentions: Previous studies reported that cyclin E/Cdk2 phosphorylates serine 5 of Id2 [32] and phosphorylation of Id2 is required for its nuclear retention in mammary epithelial cells [13]. We found that TNFα or RANKL treatment induced the phosphorylation of Cdk2 (Fig 6E and 6F). Consistent with the report that phosphorylation Cdk2 is regulated by phosphorylation and activation of ERK in mammary epithelial cells [12], we found that treatment with TNFα or RANKL also induced phosphorylation of ERK in renal epithelial cells (Fig 6E and 6F). We further found that treatment with TNFα or RANKL plus Cdk2 inhibitor II or roscovitine significantly blocked Id2 nuclear translocation in renal epithelial cells (Fig 7A and 7B), suggesting that Cdk2 is required for the TNFα or RANKL induced nuclear translocation of Id2 in these cells. It has been reported that PI3K regulates the phosphorylation of Cdk2 [33]. We found that co-treatment with a PI3K inhibitor (LY294002) plus TNFα or RANKL decreased the phosphorylation of Cdk2 in Pkd1 wild type and mutant MEK cells compared to cells treated with TNFα or RANKL alone (Fig 5A and 5B). MAPK signaling has also been reported to regulate the activation of Cdk2 [34]. To further investigate the involvement of PI3K, MAPK, and JNK in TNFα or RANKL induced Id2 nuclear translocation, Pkd1 wild type MEK cells were treated with different inhibitors prior to TNFα or RANKL stimulation, including the PI3K inhibitor LY294002, p38 inhibitor SB202190, ERK inhibitor PD98059 and JNK inhibitor. We found that inhibition of PI3K and MAPK, but not inhibition of JNK, abrogated the nuclear translocation of Id2 induced by TNFα or RANKL stimulation (Fig 7A and 7B). These results suggested that TNFα or RANKL induced nuclear translocation of Id2 might be through PI3K and MAPK mediated phosphorylated Cdk2. In addition, we found that treatment with PI3K inhibitor LY294002 blocked TNFα induced the upregulation of Id2 in nuclear and cytosol fraction in both Pkd1 wild type and MEK cells (Fig 7C).


TNFα signaling regulates cystic epithelial cell proliferation through Akt/mTOR and ERK/MAPK/Cdk2 mediated Id2 signaling.

Zhou JX, Fan LX, Li X, Calvet JP, Li X - PLoS ONE (2015)

Inhibition of Cdk2 blocks nuclear translocation of Id2 induced by TNFα and RANKL.Localization of Id2 in Pkd1 wild type MEK cells after TNFα (A) and RANKL (B) treatment in the presence of kinase inhibitors. Pkd1 wild type MEK cells were placed in DMEM F12 containing 1% FBS for 12 hours. Serum starved cells were treated with DMSO or the indicated kinase inhibitors for 3 hours. Inhibitor pre-treated cells were untreated or stimulated with TNFα and RANKL for 3 hours. Id2 was detected with an anti-Id2 antibody, followed by Fluro 488 conjugated anti-rabbit IgG antibody (Green). Nuclear DNA was stained with DAPI (Blue). One result, representative of three independent experiments, is shown. Percentage of cells with nuclear (N) and cystoplasmic (C) localization are indicated below the images. (C) Western blot analysis of the expression of Id2 from cytoplasmic and nuclear fractions of Pkd1 wild-type and  MEK cells treated with TNFα, LY294002 (LY), TNFα plus LY294002, and vehicle control for 3 h. The numbers at the bottom indicate the intensities of Id2 bands relative to actin and Lamin A/C in cytoplasmic fractions and nuclear fractions, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482222&req=5

pone.0131043.g007: Inhibition of Cdk2 blocks nuclear translocation of Id2 induced by TNFα and RANKL.Localization of Id2 in Pkd1 wild type MEK cells after TNFα (A) and RANKL (B) treatment in the presence of kinase inhibitors. Pkd1 wild type MEK cells were placed in DMEM F12 containing 1% FBS for 12 hours. Serum starved cells were treated with DMSO or the indicated kinase inhibitors for 3 hours. Inhibitor pre-treated cells were untreated or stimulated with TNFα and RANKL for 3 hours. Id2 was detected with an anti-Id2 antibody, followed by Fluro 488 conjugated anti-rabbit IgG antibody (Green). Nuclear DNA was stained with DAPI (Blue). One result, representative of three independent experiments, is shown. Percentage of cells with nuclear (N) and cystoplasmic (C) localization are indicated below the images. (C) Western blot analysis of the expression of Id2 from cytoplasmic and nuclear fractions of Pkd1 wild-type and MEK cells treated with TNFα, LY294002 (LY), TNFα plus LY294002, and vehicle control for 3 h. The numbers at the bottom indicate the intensities of Id2 bands relative to actin and Lamin A/C in cytoplasmic fractions and nuclear fractions, respectively.
Mentions: Previous studies reported that cyclin E/Cdk2 phosphorylates serine 5 of Id2 [32] and phosphorylation of Id2 is required for its nuclear retention in mammary epithelial cells [13]. We found that TNFα or RANKL treatment induced the phosphorylation of Cdk2 (Fig 6E and 6F). Consistent with the report that phosphorylation Cdk2 is regulated by phosphorylation and activation of ERK in mammary epithelial cells [12], we found that treatment with TNFα or RANKL also induced phosphorylation of ERK in renal epithelial cells (Fig 6E and 6F). We further found that treatment with TNFα or RANKL plus Cdk2 inhibitor II or roscovitine significantly blocked Id2 nuclear translocation in renal epithelial cells (Fig 7A and 7B), suggesting that Cdk2 is required for the TNFα or RANKL induced nuclear translocation of Id2 in these cells. It has been reported that PI3K regulates the phosphorylation of Cdk2 [33]. We found that co-treatment with a PI3K inhibitor (LY294002) plus TNFα or RANKL decreased the phosphorylation of Cdk2 in Pkd1 wild type and mutant MEK cells compared to cells treated with TNFα or RANKL alone (Fig 5A and 5B). MAPK signaling has also been reported to regulate the activation of Cdk2 [34]. To further investigate the involvement of PI3K, MAPK, and JNK in TNFα or RANKL induced Id2 nuclear translocation, Pkd1 wild type MEK cells were treated with different inhibitors prior to TNFα or RANKL stimulation, including the PI3K inhibitor LY294002, p38 inhibitor SB202190, ERK inhibitor PD98059 and JNK inhibitor. We found that inhibition of PI3K and MAPK, but not inhibition of JNK, abrogated the nuclear translocation of Id2 induced by TNFα or RANKL stimulation (Fig 7A and 7B). These results suggested that TNFα or RANKL induced nuclear translocation of Id2 might be through PI3K and MAPK mediated phosphorylated Cdk2. In addition, we found that treatment with PI3K inhibitor LY294002 blocked TNFα induced the upregulation of Id2 in nuclear and cytosol fraction in both Pkd1 wild type and MEK cells (Fig 7C).

Bottom Line: However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive.In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation.The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160, United States of America; Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, United States of America.

ABSTRACT
Tumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD.

No MeSH data available.


Related in: MedlinePlus