Limits...
Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer.

Brighenti E, Calabrese C, Liguori G, Giannone FA, Trerè D, Montanaro L, Derenzini M - Oncogene (2014)

Bottom Line: The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses.We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development.Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Bologna University, Bologna, Italy.

ABSTRACT
Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

Show MeSH

Related in: MedlinePlus

Stimulation of rRNA transcription by IL-6 downregulates p53 expression and activity. (a) Real-time–PCR evaluation of the TP53 mRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment. (b) Representative western blot and densitometric analysis of p53 expression in NCM460, HepG2, SW1990 and LS174T cells treated with IL-6 for 24 h. (c) Representative western blot and time-course analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. The values relative to p53 expression at 0.5, 1 and 2 h of CHX treatment are significantly higher in control than in IL-6-exposed cells (P<0.01). (d) Western blot evaluation of p53 protein expression and real-time–PCR analysis of 45S rRNA in control and IL-6-treated HepG2 cells exposed to the proteasomal inhibitor MG-132 at a concentration of 10 μm for 2 h (e) Coimmunoprecipitation and densitometric analysis of the amount of p53 and RPL11 protein bound to MDM2 in control and IL-6-stimulated HepG2 cells. The amount of MDM2, p53 and L11 is shown in the first two lanes before immunoprecipitation (input). The third and fourth lanes show the quantity of non-immunoprecipitated proteins. The fifth and sixth lanes show the amount of MDM2, p53 and L11 proteins after immunoprecipitation with anti-MDM2 polyclonal antibody (IP:MDM2). (f) Representative western blot and densitometric analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells treated with Nutlin-3 at a concentration of 5 mM for 16 h. (g) Representative western blot analysis of p53 expression in control (SCR), RPL11-silenced (L11−), MYC-silenced (MYC−) and RPL11− and MYC-silenced HepG2 cells. The cells were exposed, 48 h after the end of the silencing procedure, to IL-6 for 24 h. (h) Real-time–PCR analysis of the mRNA expression of p53 target genes BAX and PUMA in NCM460 and HepG2 cells exposed to IL-6 for 24 h. (i) Representative western blot of p53 expression in NCM460 and HepG2 cells exposed to IL-6 for 24 h and treated with 3.4 μM hydroxyurea or 20 μg/ml 5-FU for 4 h. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01; n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150990&req=5

fig2: Stimulation of rRNA transcription by IL-6 downregulates p53 expression and activity. (a) Real-time–PCR evaluation of the TP53 mRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment. (b) Representative western blot and densitometric analysis of p53 expression in NCM460, HepG2, SW1990 and LS174T cells treated with IL-6 for 24 h. (c) Representative western blot and time-course analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. The values relative to p53 expression at 0.5, 1 and 2 h of CHX treatment are significantly higher in control than in IL-6-exposed cells (P<0.01). (d) Western blot evaluation of p53 protein expression and real-time–PCR analysis of 45S rRNA in control and IL-6-treated HepG2 cells exposed to the proteasomal inhibitor MG-132 at a concentration of 10 μm for 2 h (e) Coimmunoprecipitation and densitometric analysis of the amount of p53 and RPL11 protein bound to MDM2 in control and IL-6-stimulated HepG2 cells. The amount of MDM2, p53 and L11 is shown in the first two lanes before immunoprecipitation (input). The third and fourth lanes show the quantity of non-immunoprecipitated proteins. The fifth and sixth lanes show the amount of MDM2, p53 and L11 proteins after immunoprecipitation with anti-MDM2 polyclonal antibody (IP:MDM2). (f) Representative western blot and densitometric analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells treated with Nutlin-3 at a concentration of 5 mM for 16 h. (g) Representative western blot analysis of p53 expression in control (SCR), RPL11-silenced (L11−), MYC-silenced (MYC−) and RPL11− and MYC-silenced HepG2 cells. The cells were exposed, 48 h after the end of the silencing procedure, to IL-6 for 24 h. (h) Real-time–PCR analysis of the mRNA expression of p53 target genes BAX and PUMA in NCM460 and HepG2 cells exposed to IL-6 for 24 h. (i) Representative western blot of p53 expression in NCM460 and HepG2 cells exposed to IL-6 for 24 h and treated with 3.4 μM hydroxyurea or 20 μg/ml 5-FU for 4 h. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01; n.s., not significant.

Mentions: The inhibition of rRNA transcription allows a larger amount of ribosomal proteins, no longer used for ribosome building, to bind to MDM2, thus reducing the MDM2-mediated proteasomal degradation of p53 with consequent p53 stabilization.12, 13 Conversely, the upregulation of rRNA synthesis reduces the availability of ribosomal proteins for the binding to MDM2, thus increasing the MDM2-mediated proteasomal p53 digestion.11 Therefore, we wondered whether IL-6 would lower p53 expression and activity through the above-described mechanism. In fact, even though it has been reported that IL-6 activates STAT318 and that activated STAT3 binds to the TP53 gene promoter repressing the transcription of TP53 mRNA,19 we found that no significant change occurred in the transcription level of TP53 mRNA in the NCM460, HepG2, SW1990 and LS174T cell lines (Figure 2a). As western blot analysis demonstrated that IL-6 treatment actually reduced the amount of p53 in the NCM460, HepG2, SW1990 and LS174T cell lines (Figure 2b), we considered the possibility that in IL-6-stimulated cells the reduced availability of ribosomal proteins for MDM2 binding might be responsible for an increased p53 proteasomal degradation. For this reason, we evaluated the half-life of p53 by time-course western blot analysis in control and IL-6-stimulated HepG2 cells after treatment with cycloheximide. We found that the half-life of p53 in IL-6-stimulated cells was shorter than that of control cells (Figure 2c). Also, we treated IL-6-exposed HepG2 cells with the proteasome inhibitor MG-132. We found that the inhibition of proteasomal degradation canceled the difference between the p53 expression of control and stimulated cells without reducing the stimulation of rRNA transcription by IL-6 (Figure 2d). These data indicated that the downregulation of p53 expression was actually due to an increased protein degradation. The increased p53 degradation appeared to be the consequence of a reduced ribosomal protein binding to MDM2, which allowed MDM2 to bind a greater amount of p53 for digestion. In fact, coimmunoprecipitation analysis showed that the quantity of L11 ribosomal protein coimmunoprecipitated with MDM2 was reduced in IL-6-stimulated cells as compared with control cells, whereas the amount of p53 was increased (Figure 2e). In order to define the role of MDM2 in the reduction of p53 stabilization after IL-6 treatment, we evaluated the effect of the MDM2 inhibitor Nutlin-3 on p53 expression in control and IL-6-stimulated HepG2 cells. Nutlin-3 binds MDM2 in the p53-binding pocket, thus hindering the MDM2-mediated degradation of p53, which results in p53 stabilization and activation.20 Western blot analysis showed that Nutlin-3 induced a p53 stabilization that was quite similar in control and IL-6-stimulated cells (Figure 2f). This strongly suggested that the reduction of p53 expression after exposure to IL-6 was actually the consequence of an increased binding of MDM2 to p53. To exclude the possibility that IL-6 might mediate the effect on p53 independent of a reduction in ribosomal protein availability for MDM2 binding, we downregulated the expression of RPL11 by the small-interfering RNA procedure and evaluated the effect of IL-6 stimulation on p53 expression in HepG2 cells. We found that the reduction of RPL11 mRNA and RPL11 protein expression (Supplementary Figure 1) caused a strong reduction of p53 expression and that IL-6 treatment no longer reduced the expression of p53 in RPL11-silenced HepG2 cells (Figure 2g). This observation confirmed the importance of the reduction of ribosomal protein availability for MDM2 binding, in particular of RPL11, in the IL-6-induced p53 downregulation.


Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer.

Brighenti E, Calabrese C, Liguori G, Giannone FA, Trerè D, Montanaro L, Derenzini M - Oncogene (2014)

Stimulation of rRNA transcription by IL-6 downregulates p53 expression and activity. (a) Real-time–PCR evaluation of the TP53 mRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment. (b) Representative western blot and densitometric analysis of p53 expression in NCM460, HepG2, SW1990 and LS174T cells treated with IL-6 for 24 h. (c) Representative western blot and time-course analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. The values relative to p53 expression at 0.5, 1 and 2 h of CHX treatment are significantly higher in control than in IL-6-exposed cells (P<0.01). (d) Western blot evaluation of p53 protein expression and real-time–PCR analysis of 45S rRNA in control and IL-6-treated HepG2 cells exposed to the proteasomal inhibitor MG-132 at a concentration of 10 μm for 2 h (e) Coimmunoprecipitation and densitometric analysis of the amount of p53 and RPL11 protein bound to MDM2 in control and IL-6-stimulated HepG2 cells. The amount of MDM2, p53 and L11 is shown in the first two lanes before immunoprecipitation (input). The third and fourth lanes show the quantity of non-immunoprecipitated proteins. The fifth and sixth lanes show the amount of MDM2, p53 and L11 proteins after immunoprecipitation with anti-MDM2 polyclonal antibody (IP:MDM2). (f) Representative western blot and densitometric analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells treated with Nutlin-3 at a concentration of 5 mM for 16 h. (g) Representative western blot analysis of p53 expression in control (SCR), RPL11-silenced (L11−), MYC-silenced (MYC−) and RPL11− and MYC-silenced HepG2 cells. The cells were exposed, 48 h after the end of the silencing procedure, to IL-6 for 24 h. (h) Real-time–PCR analysis of the mRNA expression of p53 target genes BAX and PUMA in NCM460 and HepG2 cells exposed to IL-6 for 24 h. (i) Representative western blot of p53 expression in NCM460 and HepG2 cells exposed to IL-6 for 24 h and treated with 3.4 μM hydroxyurea or 20 μg/ml 5-FU for 4 h. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01; n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150990&req=5

fig2: Stimulation of rRNA transcription by IL-6 downregulates p53 expression and activity. (a) Real-time–PCR evaluation of the TP53 mRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment. (b) Representative western blot and densitometric analysis of p53 expression in NCM460, HepG2, SW1990 and LS174T cells treated with IL-6 for 24 h. (c) Representative western blot and time-course analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. The values relative to p53 expression at 0.5, 1 and 2 h of CHX treatment are significantly higher in control than in IL-6-exposed cells (P<0.01). (d) Western blot evaluation of p53 protein expression and real-time–PCR analysis of 45S rRNA in control and IL-6-treated HepG2 cells exposed to the proteasomal inhibitor MG-132 at a concentration of 10 μm for 2 h (e) Coimmunoprecipitation and densitometric analysis of the amount of p53 and RPL11 protein bound to MDM2 in control and IL-6-stimulated HepG2 cells. The amount of MDM2, p53 and L11 is shown in the first two lanes before immunoprecipitation (input). The third and fourth lanes show the quantity of non-immunoprecipitated proteins. The fifth and sixth lanes show the amount of MDM2, p53 and L11 proteins after immunoprecipitation with anti-MDM2 polyclonal antibody (IP:MDM2). (f) Representative western blot and densitometric analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells treated with Nutlin-3 at a concentration of 5 mM for 16 h. (g) Representative western blot analysis of p53 expression in control (SCR), RPL11-silenced (L11−), MYC-silenced (MYC−) and RPL11− and MYC-silenced HepG2 cells. The cells were exposed, 48 h after the end of the silencing procedure, to IL-6 for 24 h. (h) Real-time–PCR analysis of the mRNA expression of p53 target genes BAX and PUMA in NCM460 and HepG2 cells exposed to IL-6 for 24 h. (i) Representative western blot of p53 expression in NCM460 and HepG2 cells exposed to IL-6 for 24 h and treated with 3.4 μM hydroxyurea or 20 μg/ml 5-FU for 4 h. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01; n.s., not significant.
Mentions: The inhibition of rRNA transcription allows a larger amount of ribosomal proteins, no longer used for ribosome building, to bind to MDM2, thus reducing the MDM2-mediated proteasomal degradation of p53 with consequent p53 stabilization.12, 13 Conversely, the upregulation of rRNA synthesis reduces the availability of ribosomal proteins for the binding to MDM2, thus increasing the MDM2-mediated proteasomal p53 digestion.11 Therefore, we wondered whether IL-6 would lower p53 expression and activity through the above-described mechanism. In fact, even though it has been reported that IL-6 activates STAT318 and that activated STAT3 binds to the TP53 gene promoter repressing the transcription of TP53 mRNA,19 we found that no significant change occurred in the transcription level of TP53 mRNA in the NCM460, HepG2, SW1990 and LS174T cell lines (Figure 2a). As western blot analysis demonstrated that IL-6 treatment actually reduced the amount of p53 in the NCM460, HepG2, SW1990 and LS174T cell lines (Figure 2b), we considered the possibility that in IL-6-stimulated cells the reduced availability of ribosomal proteins for MDM2 binding might be responsible for an increased p53 proteasomal degradation. For this reason, we evaluated the half-life of p53 by time-course western blot analysis in control and IL-6-stimulated HepG2 cells after treatment with cycloheximide. We found that the half-life of p53 in IL-6-stimulated cells was shorter than that of control cells (Figure 2c). Also, we treated IL-6-exposed HepG2 cells with the proteasome inhibitor MG-132. We found that the inhibition of proteasomal degradation canceled the difference between the p53 expression of control and stimulated cells without reducing the stimulation of rRNA transcription by IL-6 (Figure 2d). These data indicated that the downregulation of p53 expression was actually due to an increased protein degradation. The increased p53 degradation appeared to be the consequence of a reduced ribosomal protein binding to MDM2, which allowed MDM2 to bind a greater amount of p53 for digestion. In fact, coimmunoprecipitation analysis showed that the quantity of L11 ribosomal protein coimmunoprecipitated with MDM2 was reduced in IL-6-stimulated cells as compared with control cells, whereas the amount of p53 was increased (Figure 2e). In order to define the role of MDM2 in the reduction of p53 stabilization after IL-6 treatment, we evaluated the effect of the MDM2 inhibitor Nutlin-3 on p53 expression in control and IL-6-stimulated HepG2 cells. Nutlin-3 binds MDM2 in the p53-binding pocket, thus hindering the MDM2-mediated degradation of p53, which results in p53 stabilization and activation.20 Western blot analysis showed that Nutlin-3 induced a p53 stabilization that was quite similar in control and IL-6-stimulated cells (Figure 2f). This strongly suggested that the reduction of p53 expression after exposure to IL-6 was actually the consequence of an increased binding of MDM2 to p53. To exclude the possibility that IL-6 might mediate the effect on p53 independent of a reduction in ribosomal protein availability for MDM2 binding, we downregulated the expression of RPL11 by the small-interfering RNA procedure and evaluated the effect of IL-6 stimulation on p53 expression in HepG2 cells. We found that the reduction of RPL11 mRNA and RPL11 protein expression (Supplementary Figure 1) caused a strong reduction of p53 expression and that IL-6 treatment no longer reduced the expression of p53 in RPL11-silenced HepG2 cells (Figure 2g). This observation confirmed the importance of the reduction of ribosomal protein availability for MDM2 binding, in particular of RPL11, in the IL-6-induced p53 downregulation.

Bottom Line: The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses.We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development.Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Bologna University, Bologna, Italy.

ABSTRACT
Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

Show MeSH
Related in: MedlinePlus