Limits...
Endocytosis-independent function of clathrin heavy chain in the control of basal NF-κB activation.

Kim ML, Sorg I, Arrieumerlou C - PLoS ONE (2011)

Bottom Line: Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression.The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown.We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT

Background: Nuclear factor-κB (NF-κB) is a transcription factor that regulates the transcription of genes involved in a variety of biological processes, including innate and adaptive immunity, stress responses and cell proliferation. Constitutive or excessive NF-κB activity has been associated with inflammatory disorders and higher risk of cancer. In contrast to the mechanisms controlling inducible activation, the regulation of basal NF-κB activation is not well understood. Here we test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-κB activity in epithelial cells.

Methodology: Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression. Immunofluorescence staining showed constitutive nuclear localization of the NF-κB subunit p65 in absence of stimulation after CHC knockdown. Elevated basal p65 nuclear localization is caused by constitutive phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) through an IκB kinase α (IKKα)-dependent mechanism. The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown. Disruption of clathrin-mediated endocytosis by chemical inhibition or depletion of the μ2-subunit of the endocytosis adaptor protein AP-2, and knockdown of clathrin light chain a (CHLa), failed to induce constitutive NF-κB activation and IL-8 expression, showing that CHC acts on NF-κB independently of endocytosis and CLCa.

Conclusions: We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells. Furthermore, our data suggest a potential link between a defect in CHC expression and chronic inflammation disorder and cancer.

Show MeSH

Related in: MedlinePlus

CHC prevents constitutive IL-8 expression in unstimulated epithelial cells.(A) Constitutive IL-8 expression after knockdown of CHC in HeLa cells. Cells were transfected with control or CHC siRNAs. After 72 hours, supernatants were collected and analyzed for their content in IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (B) Constitutive IL-8 expression after knockdown of CHC in MCF-7 cells. MCF-7 cells were treated as described in (A) (results are expressed as the mean ± SD of 3 independent experiments). (C) IKKα-depletion abolishes the constitutive secretion of IL-8 induced by CHC knockdown. HeLa cells were transfected with different combinations of IKKα and CHC siRNAs for 72 hours. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3045402&req=5

pone-0017158-g003: CHC prevents constitutive IL-8 expression in unstimulated epithelial cells.(A) Constitutive IL-8 expression after knockdown of CHC in HeLa cells. Cells were transfected with control or CHC siRNAs. After 72 hours, supernatants were collected and analyzed for their content in IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (B) Constitutive IL-8 expression after knockdown of CHC in MCF-7 cells. MCF-7 cells were treated as described in (A) (results are expressed as the mean ± SD of 3 independent experiments). (C) IKKα-depletion abolishes the constitutive secretion of IL-8 induced by CHC knockdown. HeLa cells were transfected with different combinations of IKKα and CHC siRNAs for 72 hours. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).

Mentions: Previous results indicated that CHC was required to prevent constitutive p65 nuclear translocation. Because this process directly contributes to the regulation of gene expression, we tested whether the presence of CHC was also necessary to prevent constitutive expression of genes regulated by NF-κB. In particular, we investigated the expression of the proinflammatory chemokine IL-8. IL-8 secretion was measured by ELISA in the supernatant of CHC and control siRNA transfected HeLa cells. In line with the results obtained on NF-κB activation, knocking down CHC strongly enhanced basal IL-8 secretion (Figure 3A), showing that, indeed, the expression of CHC was critical to prevent constitutive IL-8 expression in HeLa cells. The same result was obtained in the breast cancer cell line MCF-7 (Figure 3B). Furthermore, consistent with the results obtained on IκBα degradation, constitutive expression of IL-8 was massively reduced when IKKα was knocked down (Figure 3C). Taken together, these results showed that CHC prevents constitutive expression of IL-8, and that this new function of CHC in NF-κB signaling depends on IKKα and corresponds to a general mechanism taking place in different cells lines.


Endocytosis-independent function of clathrin heavy chain in the control of basal NF-κB activation.

Kim ML, Sorg I, Arrieumerlou C - PLoS ONE (2011)

CHC prevents constitutive IL-8 expression in unstimulated epithelial cells.(A) Constitutive IL-8 expression after knockdown of CHC in HeLa cells. Cells were transfected with control or CHC siRNAs. After 72 hours, supernatants were collected and analyzed for their content in IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (B) Constitutive IL-8 expression after knockdown of CHC in MCF-7 cells. MCF-7 cells were treated as described in (A) (results are expressed as the mean ± SD of 3 independent experiments). (C) IKKα-depletion abolishes the constitutive secretion of IL-8 induced by CHC knockdown. HeLa cells were transfected with different combinations of IKKα and CHC siRNAs for 72 hours. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045402&req=5

pone-0017158-g003: CHC prevents constitutive IL-8 expression in unstimulated epithelial cells.(A) Constitutive IL-8 expression after knockdown of CHC in HeLa cells. Cells were transfected with control or CHC siRNAs. After 72 hours, supernatants were collected and analyzed for their content in IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (B) Constitutive IL-8 expression after knockdown of CHC in MCF-7 cells. MCF-7 cells were treated as described in (A) (results are expressed as the mean ± SD of 3 independent experiments). (C) IKKα-depletion abolishes the constitutive secretion of IL-8 induced by CHC knockdown. HeLa cells were transfected with different combinations of IKKα and CHC siRNAs for 72 hours. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).
Mentions: Previous results indicated that CHC was required to prevent constitutive p65 nuclear translocation. Because this process directly contributes to the regulation of gene expression, we tested whether the presence of CHC was also necessary to prevent constitutive expression of genes regulated by NF-κB. In particular, we investigated the expression of the proinflammatory chemokine IL-8. IL-8 secretion was measured by ELISA in the supernatant of CHC and control siRNA transfected HeLa cells. In line with the results obtained on NF-κB activation, knocking down CHC strongly enhanced basal IL-8 secretion (Figure 3A), showing that, indeed, the expression of CHC was critical to prevent constitutive IL-8 expression in HeLa cells. The same result was obtained in the breast cancer cell line MCF-7 (Figure 3B). Furthermore, consistent with the results obtained on IκBα degradation, constitutive expression of IL-8 was massively reduced when IKKα was knocked down (Figure 3C). Taken together, these results showed that CHC prevents constitutive expression of IL-8, and that this new function of CHC in NF-κB signaling depends on IKKα and corresponds to a general mechanism taking place in different cells lines.

Bottom Line: Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression.The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown.We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT

Background: Nuclear factor-κB (NF-κB) is a transcription factor that regulates the transcription of genes involved in a variety of biological processes, including innate and adaptive immunity, stress responses and cell proliferation. Constitutive or excessive NF-κB activity has been associated with inflammatory disorders and higher risk of cancer. In contrast to the mechanisms controlling inducible activation, the regulation of basal NF-κB activation is not well understood. Here we test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-κB activity in epithelial cells.

Methodology: Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression. Immunofluorescence staining showed constitutive nuclear localization of the NF-κB subunit p65 in absence of stimulation after CHC knockdown. Elevated basal p65 nuclear localization is caused by constitutive phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) through an IκB kinase α (IKKα)-dependent mechanism. The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown. Disruption of clathrin-mediated endocytosis by chemical inhibition or depletion of the μ2-subunit of the endocytosis adaptor protein AP-2, and knockdown of clathrin light chain a (CHLa), failed to induce constitutive NF-κB activation and IL-8 expression, showing that CHC acts on NF-κB independently of endocytosis and CLCa.

Conclusions: We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells. Furthermore, our data suggest a potential link between a defect in CHC expression and chronic inflammation disorder and cancer.

Show MeSH
Related in: MedlinePlus