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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.

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CHC regulates NF-κB activation independently of endocytosis and CLCa.(A) Uptake of Alexa 594-transferrin (Alexa 594-Tf) in cells transfected with control (left panels), AP2M1 (middle panels) or CHC (right panels) siRNAs; Scale bars, 10 µm. (B) Quantification of transferrin uptake by automated image analysis (results are expressed as the mean ± SD of 12 images; graph representative of 2 independent experiments). (C) AP2M1 knockdown fails to enhance IκBα degradation. Cell lysates from control, AP2M1 or CHC siRNA-transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as a loading control. (D) Densitometric quantification of the levels of IκBα shown in Figure 4C (graph representative of 2 independent experiments). (E) AP2M1 knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, AP2M1 or CHC siRNAs for 72 hours. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (F) Inhibition of transferrin uptake after dynasore and PAO treatment. HeLa cells were left untreated (Ctrl) or treated with dynasore (80 µM) (Dyn) or PAO (5 µM) 10 minutes before and during the transferrin uptake assay (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (G) Dynasore and PAO fail to enhance basal degradation of IκBα. HeLa cells were pretreated for 10 minutes with dynasore (80 µM) or PAO (5 µM) and analyzed by western immunoblotting using an IκBα antibody. Actin is shown as a loading control (results representative of 2 independent experiments). (H) Long-term inhibition of endocytosis in dynasore-treated HeLa cells. Transferrin uptake in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (I) Long-term inhibition of endocytosis fails to enhance the basal degradation of IκBα. Basal degradation of IκBα in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours. As positive control of the degradation of IκBα, cells were stimulated for 20 minutes with TNFα (results representative of 2 independent experiments). (J) CLCa knockdown fails to enhance IκBα degradation. Cell lysates from control, CLCa or CHC siRNA transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as loading control. (K) Densitometric quantification of IκBα levels shown in Figure 4F (Graph representative of 2 independent experiments). (L) CLCa knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, CLCa or CHC siRNAs for 72 hours and supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).
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pone-0017158-g004: CHC regulates NF-κB activation independently of endocytosis and CLCa.(A) Uptake of Alexa 594-transferrin (Alexa 594-Tf) in cells transfected with control (left panels), AP2M1 (middle panels) or CHC (right panels) siRNAs; Scale bars, 10 µm. (B) Quantification of transferrin uptake by automated image analysis (results are expressed as the mean ± SD of 12 images; graph representative of 2 independent experiments). (C) AP2M1 knockdown fails to enhance IκBα degradation. Cell lysates from control, AP2M1 or CHC siRNA-transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as a loading control. (D) Densitometric quantification of the levels of IκBα shown in Figure 4C (graph representative of 2 independent experiments). (E) AP2M1 knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, AP2M1 or CHC siRNAs for 72 hours. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (F) Inhibition of transferrin uptake after dynasore and PAO treatment. HeLa cells were left untreated (Ctrl) or treated with dynasore (80 µM) (Dyn) or PAO (5 µM) 10 minutes before and during the transferrin uptake assay (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (G) Dynasore and PAO fail to enhance basal degradation of IκBα. HeLa cells were pretreated for 10 minutes with dynasore (80 µM) or PAO (5 µM) and analyzed by western immunoblotting using an IκBα antibody. Actin is shown as a loading control (results representative of 2 independent experiments). (H) Long-term inhibition of endocytosis in dynasore-treated HeLa cells. Transferrin uptake in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (I) Long-term inhibition of endocytosis fails to enhance the basal degradation of IκBα. Basal degradation of IκBα in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours. As positive control of the degradation of IκBα, cells were stimulated for 20 minutes with TNFα (results representative of 2 independent experiments). (J) CLCa knockdown fails to enhance IκBα degradation. Cell lysates from control, CLCa or CHC siRNA transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as loading control. (K) Densitometric quantification of IκBα levels shown in Figure 4F (Graph representative of 2 independent experiments). (L) CLCa knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, CLCa or CHC siRNAs for 72 hours and supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).

Mentions: Through its activity in CME, CHC is involved in the internalization of nutrients, pathogens, antigens, growth factors and receptors [13], [15], [16]. To test whether CHC regulated indirectly the NF-κB pathway via its function in CME, we measured p65 nuclear translocation and IL-8 secretion in cells where CME was disrupted by RNAi-mediated depletion of the μ2-subunit of the main CME adaptor protein AP-2 (AP2M1). The recruitment of AP-2 at the plasma membrane is critical for the initiation of CME [23]. AP-2 interacts with sorting signals present in the cytoplasmic domains of membrane proteins destined to become cargo in the coated vesicles. In addition, AP-2 recruits clathrin onto the membrane, where it functions as a scaffold for vesicle budding. First, in order to demonstrate that CME was impaired in AP2M1 and CHC-depleted cells, the CME-dependent mechanism of transferrin uptake was monitored in HeLa cells. As previously reported [23], depletion of both CHC and AP2M1 impaired the uptake of fluorescently labeled transferrin (Figures 4A and 4B). However, although the depletion of AP2M1 blocked transferrin uptake to the same extent as CHC knockdown, it failed to increase basal IκBα degradation (Figures 4C and 4D) and IL-8 secretion (Figure 4E), suggesting that CHC controls basal NF-κB activation and gene expression independently of its activity in CME. In order to further validate this result, we tested whether chemical inhibition of endocytosis by the drugs phenylarsine oxide (PAO) and dynasore had an effect on NF-κB signaling. PAO is a chemical compound that, at low micromolar concentrations, blocks CME [24]. Dynasore is a cell-permeable small molecule that inhibits the GTPase activity of dynamin and blocks the formation of clathrin-coated vesicles [25]. Whereas a short term incubation with these drugs effectively blocked transferrin uptake (Figure 4F), neither of them had an effect on the degradation of IκBα (Figure 4G). HeLa cells stimulated with the inflammatory cytokine tumor necrosis factor α (TNFα) that rapidly activates NF-κB, were used as positive control for the degradation of IκBα. To better mimic the long-lasting effect of CHC knockdown on endocytosis, endocytosis and IκBα degradation were examined after 48 hours of dynasore treatment. Although endocytosis was still effectively blocked (Figure 4H), the degradation of IκBα was unchanged compared to untreated cells (Figure 4I), showing that inhibition of endocytosis had no effect on the activation of NF-κB. Taken together, these results strongly indicated that CHC regulates basal NF-κB activation independently of its function in endocytosis.


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 regulates NF-κB activation independently of endocytosis and CLCa.(A) Uptake of Alexa 594-transferrin (Alexa 594-Tf) in cells transfected with control (left panels), AP2M1 (middle panels) or CHC (right panels) siRNAs; Scale bars, 10 µm. (B) Quantification of transferrin uptake by automated image analysis (results are expressed as the mean ± SD of 12 images; graph representative of 2 independent experiments). (C) AP2M1 knockdown fails to enhance IκBα degradation. Cell lysates from control, AP2M1 or CHC siRNA-transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as a loading control. (D) Densitometric quantification of the levels of IκBα shown in Figure 4C (graph representative of 2 independent experiments). (E) AP2M1 knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, AP2M1 or CHC siRNAs for 72 hours. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (F) Inhibition of transferrin uptake after dynasore and PAO treatment. HeLa cells were left untreated (Ctrl) or treated with dynasore (80 µM) (Dyn) or PAO (5 µM) 10 minutes before and during the transferrin uptake assay (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (G) Dynasore and PAO fail to enhance basal degradation of IκBα. HeLa cells were pretreated for 10 minutes with dynasore (80 µM) or PAO (5 µM) and analyzed by western immunoblotting using an IκBα antibody. Actin is shown as a loading control (results representative of 2 independent experiments). (H) Long-term inhibition of endocytosis in dynasore-treated HeLa cells. Transferrin uptake in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (I) Long-term inhibition of endocytosis fails to enhance the basal degradation of IκBα. Basal degradation of IκBα in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours. As positive control of the degradation of IκBα, cells were stimulated for 20 minutes with TNFα (results representative of 2 independent experiments). (J) CLCa knockdown fails to enhance IκBα degradation. Cell lysates from control, CLCa or CHC siRNA transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as loading control. (K) Densitometric quantification of IκBα levels shown in Figure 4F (Graph representative of 2 independent experiments). (L) CLCa knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, CLCa or CHC siRNAs for 72 hours and supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).
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pone-0017158-g004: CHC regulates NF-κB activation independently of endocytosis and CLCa.(A) Uptake of Alexa 594-transferrin (Alexa 594-Tf) in cells transfected with control (left panels), AP2M1 (middle panels) or CHC (right panels) siRNAs; Scale bars, 10 µm. (B) Quantification of transferrin uptake by automated image analysis (results are expressed as the mean ± SD of 12 images; graph representative of 2 independent experiments). (C) AP2M1 knockdown fails to enhance IκBα degradation. Cell lysates from control, AP2M1 or CHC siRNA-transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as a loading control. (D) Densitometric quantification of the levels of IκBα shown in Figure 4C (graph representative of 2 independent experiments). (E) AP2M1 knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, AP2M1 or CHC siRNAs for 72 hours. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (F) Inhibition of transferrin uptake after dynasore and PAO treatment. HeLa cells were left untreated (Ctrl) or treated with dynasore (80 µM) (Dyn) or PAO (5 µM) 10 minutes before and during the transferrin uptake assay (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (G) Dynasore and PAO fail to enhance basal degradation of IκBα. HeLa cells were pretreated for 10 minutes with dynasore (80 µM) or PAO (5 µM) and analyzed by western immunoblotting using an IκBα antibody. Actin is shown as a loading control (results representative of 2 independent experiments). (H) Long-term inhibition of endocytosis in dynasore-treated HeLa cells. Transferrin uptake in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (I) Long-term inhibition of endocytosis fails to enhance the basal degradation of IκBα. Basal degradation of IκBα in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours. As positive control of the degradation of IκBα, cells were stimulated for 20 minutes with TNFα (results representative of 2 independent experiments). (J) CLCa knockdown fails to enhance IκBα degradation. Cell lysates from control, CLCa or CHC siRNA transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as loading control. (K) Densitometric quantification of IκBα levels shown in Figure 4F (Graph representative of 2 independent experiments). (L) CLCa knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, CLCa or CHC siRNAs for 72 hours and supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).
Mentions: Through its activity in CME, CHC is involved in the internalization of nutrients, pathogens, antigens, growth factors and receptors [13], [15], [16]. To test whether CHC regulated indirectly the NF-κB pathway via its function in CME, we measured p65 nuclear translocation and IL-8 secretion in cells where CME was disrupted by RNAi-mediated depletion of the μ2-subunit of the main CME adaptor protein AP-2 (AP2M1). The recruitment of AP-2 at the plasma membrane is critical for the initiation of CME [23]. AP-2 interacts with sorting signals present in the cytoplasmic domains of membrane proteins destined to become cargo in the coated vesicles. In addition, AP-2 recruits clathrin onto the membrane, where it functions as a scaffold for vesicle budding. First, in order to demonstrate that CME was impaired in AP2M1 and CHC-depleted cells, the CME-dependent mechanism of transferrin uptake was monitored in HeLa cells. As previously reported [23], depletion of both CHC and AP2M1 impaired the uptake of fluorescently labeled transferrin (Figures 4A and 4B). However, although the depletion of AP2M1 blocked transferrin uptake to the same extent as CHC knockdown, it failed to increase basal IκBα degradation (Figures 4C and 4D) and IL-8 secretion (Figure 4E), suggesting that CHC controls basal NF-κB activation and gene expression independently of its activity in CME. In order to further validate this result, we tested whether chemical inhibition of endocytosis by the drugs phenylarsine oxide (PAO) and dynasore had an effect on NF-κB signaling. PAO is a chemical compound that, at low micromolar concentrations, blocks CME [24]. Dynasore is a cell-permeable small molecule that inhibits the GTPase activity of dynamin and blocks the formation of clathrin-coated vesicles [25]. Whereas a short term incubation with these drugs effectively blocked transferrin uptake (Figure 4F), neither of them had an effect on the degradation of IκBα (Figure 4G). HeLa cells stimulated with the inflammatory cytokine tumor necrosis factor α (TNFα) that rapidly activates NF-κB, were used as positive control for the degradation of IκBα. To better mimic the long-lasting effect of CHC knockdown on endocytosis, endocytosis and IκBα degradation were examined after 48 hours of dynasore treatment. Although endocytosis was still effectively blocked (Figure 4H), the degradation of IκBα was unchanged compared to untreated cells (Figure 4I), showing that inhibition of endocytosis had no effect on the activation of NF-κB. Taken together, these results strongly indicated that CHC regulates basal NF-κB activation independently of its function in endocytosis.

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