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PPARβ/δ promotes HRAS-induced senescence and tumor suppression by potentiating p-ERK and repressing p-AKT signaling.

Zhu B, Ferry CH, Blazanin N, Bility MT, Khozoie C, Kang BH, Glick AB, Gonzalez FJ, Peters JM - Oncogene (2013)

Bottom Line: PPARβ/δ expression increased p-ERK and decreased p-AKT activity.Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ.Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Biomedical Sciences, The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA, USA.

ABSTRACT
Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits skin tumorigenesis through mechanisms that may be dependent on HRAS signaling. The present study examined the hypothesis that PPARβ/δ promotes HRAS-induced senescence resulting in suppression of tumorigenesis. PPARβ/δ expression increased p-ERK and decreased p-AKT activity. Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ. Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression. Decreased p-AKT activity in turn promotes cellular senescence through upregulation of p53 and p27 expression. Both over-expression of RASGRP1 and shRNA-mediated knockdown of ILK partially restore cellular senescence in Pparβ/δ- cells. Higher PPARβ/δ expression is also correlated with increased senescence observed in human benign neurofibromas and colon adenoma lesions in vivo. These results demonstrate that PPARβ/δ promotes senescence to inhibit tumorigenesis and provide new mechanistic insights into HRAS-induced cellular senescence.

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PPARβ/δ promotes HRAS-induced senescence in fibroblasts. (a–c) Wild-type (+/+) or Pparβ/δ- (−/−) dermal fibroblasts were infected with HRAS virus for 3 days. (a) Representative photomicrographs of mock and HRAS-expressing dermal fibroblasts and (b) quantification of β-gal-positive cells. (c) qPCR of Hras, p16, p21, Rasgrp1, Rasgap120 and Ilk in dermal fibroblasts. (d–g) Mutant HRAS (G12V) was introduced into BJ cells using a previously described retroviral approach 12. (d) Representative photomicrographs of control (BJ) and PPARβ/δ knockdown cells (BJPPARβ/δ shRNA) and (e) quantification of β-gal-positive cells. (f) qPCR of PPARβ/δ, RASGRP1, ILK, DcR2, p16 and p21 in both cell types. (g) Western blot analysis of p-AKT, p-ERK, senescence markers p16, p21, and HRAS in both cell types. Relative expression level of proteins was normalized to that of LDH and is shown as the relative fold change as compared to control. Values represent the mean ± S.E.M.. *significantly different than control (P ≤ 0.05).
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Figure 5: PPARβ/δ promotes HRAS-induced senescence in fibroblasts. (a–c) Wild-type (+/+) or Pparβ/δ- (−/−) dermal fibroblasts were infected with HRAS virus for 3 days. (a) Representative photomicrographs of mock and HRAS-expressing dermal fibroblasts and (b) quantification of β-gal-positive cells. (c) qPCR of Hras, p16, p21, Rasgrp1, Rasgap120 and Ilk in dermal fibroblasts. (d–g) Mutant HRAS (G12V) was introduced into BJ cells using a previously described retroviral approach 12. (d) Representative photomicrographs of control (BJ) and PPARβ/δ knockdown cells (BJPPARβ/δ shRNA) and (e) quantification of β-gal-positive cells. (f) qPCR of PPARβ/δ, RASGRP1, ILK, DcR2, p16 and p21 in both cell types. (g) Western blot analysis of p-AKT, p-ERK, senescence markers p16, p21, and HRAS in both cell types. Relative expression level of proteins was normalized to that of LDH and is shown as the relative fold change as compared to control. Values represent the mean ± S.E.M.. *significantly different than control (P ≤ 0.05).

Mentions: To confirm that the observed decreased HRAS-induced senescence in Pparβ/δ- keratinocytes is also observed in other cell types, mouse and human fibroblasts expressing oncogenic HRAS were examined. Decreased HRAS-induced senescence was observed in Pparβ/δ- dermal fibroblasts as compared to wild-type cells (Fig 5a, b). Decreased mRNA expression of the senescence markers p16 and p21 was observed in HRAS-expressing Pparβ/δ- dermal fibroblasts as compared to controls (Fig 5c). In addition, Rasgrp1 mRNA was lower and Ilk and Rasgap120 mRNA were higher in HRAS-expressing Pparβ/δ- dermal fibroblasts (Fig 5c), all of which recapitulates the changes found in primary keratinocytes. The human BJ fibroblast cell line was also examined to confirm the PPARβ/δ-dependent effects observed in mouse cells. Decreased HRAS-induced senescence was observed following knockdown of PPARβ/δ by shRNA in BJ cells expressing mutant HRAS (Figure 5d, e). Further, in BJ cells expressing mutant HRAS following knockdown of PPARβ/δ, RASGRP1 mRNA was decreased, ILK mRNA was increased, and HRAS-induced expression of p16, p21, and DcR2 was reduced (Figure 5f, g). Additionally, HRAS-induced expression of p-ERK and p-AKT was repressed and enhanced, respectively, following knockdown of PPARβ/δ in BJ cells expressing mutant HRAS (Figure 5f, g).


PPARβ/δ promotes HRAS-induced senescence and tumor suppression by potentiating p-ERK and repressing p-AKT signaling.

Zhu B, Ferry CH, Blazanin N, Bility MT, Khozoie C, Kang BH, Glick AB, Gonzalez FJ, Peters JM - Oncogene (2013)

PPARβ/δ promotes HRAS-induced senescence in fibroblasts. (a–c) Wild-type (+/+) or Pparβ/δ- (−/−) dermal fibroblasts were infected with HRAS virus for 3 days. (a) Representative photomicrographs of mock and HRAS-expressing dermal fibroblasts and (b) quantification of β-gal-positive cells. (c) qPCR of Hras, p16, p21, Rasgrp1, Rasgap120 and Ilk in dermal fibroblasts. (d–g) Mutant HRAS (G12V) was introduced into BJ cells using a previously described retroviral approach 12. (d) Representative photomicrographs of control (BJ) and PPARβ/δ knockdown cells (BJPPARβ/δ shRNA) and (e) quantification of β-gal-positive cells. (f) qPCR of PPARβ/δ, RASGRP1, ILK, DcR2, p16 and p21 in both cell types. (g) Western blot analysis of p-AKT, p-ERK, senescence markers p16, p21, and HRAS in both cell types. Relative expression level of proteins was normalized to that of LDH and is shown as the relative fold change as compared to control. Values represent the mean ± S.E.M.. *significantly different than control (P ≤ 0.05).
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Figure 5: PPARβ/δ promotes HRAS-induced senescence in fibroblasts. (a–c) Wild-type (+/+) or Pparβ/δ- (−/−) dermal fibroblasts were infected with HRAS virus for 3 days. (a) Representative photomicrographs of mock and HRAS-expressing dermal fibroblasts and (b) quantification of β-gal-positive cells. (c) qPCR of Hras, p16, p21, Rasgrp1, Rasgap120 and Ilk in dermal fibroblasts. (d–g) Mutant HRAS (G12V) was introduced into BJ cells using a previously described retroviral approach 12. (d) Representative photomicrographs of control (BJ) and PPARβ/δ knockdown cells (BJPPARβ/δ shRNA) and (e) quantification of β-gal-positive cells. (f) qPCR of PPARβ/δ, RASGRP1, ILK, DcR2, p16 and p21 in both cell types. (g) Western blot analysis of p-AKT, p-ERK, senescence markers p16, p21, and HRAS in both cell types. Relative expression level of proteins was normalized to that of LDH and is shown as the relative fold change as compared to control. Values represent the mean ± S.E.M.. *significantly different than control (P ≤ 0.05).
Mentions: To confirm that the observed decreased HRAS-induced senescence in Pparβ/δ- keratinocytes is also observed in other cell types, mouse and human fibroblasts expressing oncogenic HRAS were examined. Decreased HRAS-induced senescence was observed in Pparβ/δ- dermal fibroblasts as compared to wild-type cells (Fig 5a, b). Decreased mRNA expression of the senescence markers p16 and p21 was observed in HRAS-expressing Pparβ/δ- dermal fibroblasts as compared to controls (Fig 5c). In addition, Rasgrp1 mRNA was lower and Ilk and Rasgap120 mRNA were higher in HRAS-expressing Pparβ/δ- dermal fibroblasts (Fig 5c), all of which recapitulates the changes found in primary keratinocytes. The human BJ fibroblast cell line was also examined to confirm the PPARβ/δ-dependent effects observed in mouse cells. Decreased HRAS-induced senescence was observed following knockdown of PPARβ/δ by shRNA in BJ cells expressing mutant HRAS (Figure 5d, e). Further, in BJ cells expressing mutant HRAS following knockdown of PPARβ/δ, RASGRP1 mRNA was decreased, ILK mRNA was increased, and HRAS-induced expression of p16, p21, and DcR2 was reduced (Figure 5f, g). Additionally, HRAS-induced expression of p-ERK and p-AKT was repressed and enhanced, respectively, following knockdown of PPARβ/δ in BJ cells expressing mutant HRAS (Figure 5f, g).

Bottom Line: PPARβ/δ expression increased p-ERK and decreased p-AKT activity.Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ.Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Biomedical Sciences, The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA, USA.

ABSTRACT
Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits skin tumorigenesis through mechanisms that may be dependent on HRAS signaling. The present study examined the hypothesis that PPARβ/δ promotes HRAS-induced senescence resulting in suppression of tumorigenesis. PPARβ/δ expression increased p-ERK and decreased p-AKT activity. Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ. Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression. Decreased p-AKT activity in turn promotes cellular senescence through upregulation of p53 and p27 expression. Both over-expression of RASGRP1 and shRNA-mediated knockdown of ILK partially restore cellular senescence in Pparβ/δ- cells. Higher PPARβ/δ expression is also correlated with increased senescence observed in human benign neurofibromas and colon adenoma lesions in vivo. These results demonstrate that PPARβ/δ promotes senescence to inhibit tumorigenesis and provide new mechanistic insights into HRAS-induced cellular senescence.

Show MeSH
Related in: MedlinePlus