LRP16 integrates into NF-κB transcriptional complex and is required for its functional activation

Abstract

Background: Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.

Methodology: GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.

Results: We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.

Conclusions: Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.

Figure 1. Physical interaction of LRP16 and p65.

(A) GST and GST-LRP16 stained with Coomassie Blue are shown in the left panel. [³⁵S]methionine-labeled UXT, p65, p50 or c-Rel was incubated with GST or GST-LRP16 as indicated in the middle and right panels. (B) The left panel shows a schematic illustration of p65 and its mutants. GST pull-down assays were performed with various [³⁵S]-labeled mutants of p65 and GST-LRP16 or GST alone (right panel). (C) The left panel shows a schematic illustration of LRP16 and its mutants. The middle panel shows GST, GST-LRP16 and its mutants stained with Coomassie Blue. GST pull-down assays were performed with [³⁵S]-labeled p65 and GST-LRP16 mutants or GST alone (right panel). (D) 293T cells were treated with TNF-α (10 ng/ml) for the indicated times. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (E) 293T cells were cotransfected with p65 and Flag-tagged constructs. Forty-eight hours after transfection, cells were lysed and the lysates subjected to CoIP using the indicated antibodies. The asterisk indicates a non-specific band. NsIgG: non-specific IgG.

Figure 2. Ectopic expression of LRP16 markedly upregulates cytokine-induced κB-luc activity.

(A–D) 293T cells were cotransfected with 3×κB-luc and the indicated vectors. Forty-two hours after transfection, cells were simulated with 10 ng/ml TNF-α or 20 ng/ml IL-1βfor 7 h before luciferase assays were performed. The relative levels of luciferase activity were normalized to the activity obtained after cotransfection of 3×κB-luc and the empty expression vector, which was arbitrarily assigned a value of 1. All experiments were performed in triplicate and were repeated at least three times, and the results are expressed as means±SD. ^* P<0.05.

Figure 3. Ectopic expression of LRP16 does not significantly augment TNF-α-induced expression of the endogenous NF-κB target genes and NF-κB DNA binding capacity.

(A) 293T cells, which were stably transfected with the LRP16 expression construct or empty vector, were treated with TNF-α (10 ng/ml) for the indicated times. Endogenous expression of IκBα and A20 mRNA was measured by qPCR. Data represent means±SD (error bars) of at least three independent experiments. (B) 293T cells, which were stably transfected with the LRP16 expression or empty vector, were treated with TNF-α (10 ng/ml) for the indicated times, lysed and the nuclear extracts subjected to EMSA assays. Sp1 was used as a loading control.

Figure 4. Knockdown of LRP16 markedly attenuates NF-κB transcriptional activity.

(A) 293T cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, the endogenous expression of LRP16 mRNA was monitored by RT-PCR, and the protein levels of LRP16, p65, and p50 were determined by immunoblotting. β-actin was used as a loading control. (B–D) 293T cells were cotransfected with 3×κB-luc, siRNA and vectors as indicated. Forty-two hours after transfection, cells were stimulated with 10 ng/ml TNF-α for 7 h and then luciferase assays were performed. The relative levels of luciferase activity were normalized to the activity obtained after cotransfection of 3×κB-luc and the empty expression vector, which was arbitrarily assigned a value of 1. All experiments were performed in triplicate and were repeated at least three times, and the results are expressed as means±SD. (E) 293T cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were stimulated with 10 ng/ml TNF-α for the indicated times. Endogenous expression of the indicated genes was measured by qPCR. Data represent means±SD (error bars) of at least three independent experiments. (F) 293T cells were treated as in (E), cells were stimulated with 10 ng/ml TNF-α for 90 min. Endogenous expression of the indicated genes was measured by qPCR. Data represent means±SD (error bars) of at least three independent experiments. *P<0.05, **P <0.01, ^N P>0.05.

Figure 5. Knockdown of LRP16 sharply attenuates the DNA binding capacity of NF-κB.

(A) 293T or HeLa cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were stimulated with 10 ng/ml TNF-α for the indicated times, lysed and the nuclear extracts subjected to EMSA assays. Sp1 was used as a loading control. (B) 293T cells were treated as in (A). The ChIP assays were performed for the A20, IκBα and GAPDH promoters using the indicated antibodies and corresponding primers described in Materials and Methods.

Figure 6. Knockdown of LRP16 impairs the formation or stability of NF-κB/p300/CBP/CARM1 complex inside the nucleus.

(A) 293T cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were treated with 10 ng/ml TNF-α for the indicated times and the nuclear fraction was purified. Equal amounts of nuclear lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (B) 293T cells were treated as in (A), ChIP assays were performed for the A20, IκBα and GAPDH promoters using the indicated antibodies and corresponding primers described in Materials and Methods.

Figure 7. Knockdown of LRP16 sensitizes TNF-α-induced cell apoptosis.

(A) 293T cells were transfected with the indicated siRNAs. Forty-eight hours later, cells were treated with TNF-α plus CHX or left untreated for 22 h. Representative microscopic images are shown (right panel for DAPI staining). Bar = 20 µm. (B) 293T cells were treated as in (A), the percentage of apoptotic cells was monitored by annexin V staining followed by FACS analysis. Experiments to analyze apoptosis were performed in triplicate and were repeated at least three times, and the results are expressed as means±SD. (C) 293T cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were stimulated with 10 ng/ml TNF-α for an additional 12 h. Total protein was prepared and immunoblotting was performed with the indicated antibodies.

Figure 8. The expression level of LRP16 in nuclei is positively associated with the elevated NF-κB activity in human gastric carcinoma specimens.

(A) Immunostaining of LRP16 in three representative gastric carcinoma samples. LRP16 is stained dark brown; nuclei were counterstained with hematoxylin (light blue). The left panel shows no staining. Predominant cytoplasmic staining of LRP16 is shown in the middle panel. The right panel shows predominant nuclear staining of LRP16. Bar = 20 µm. (B) The DNA binding activity of NF-κB in the LRP16 negative and positive groups was measured by using an ELISA-based chemiluminescent kit as described in Materials and Methods.