In this study, the macro domain protein LRP16 was identified as a novel interactor of NF-κB component p65. By a series of independent approaches, we demonstrated that LRP16 participates in the NF-κB enhanceosome and is crucial for the recruitment of NF-κB for essential coactivators such as p300 and CBP. Knockdown of LRP16 led to impaired NF-κB activity and signaling output. The regulatory role of LRP16 on NF-κB activity was further corroborated by a positive relationship between the staining intensity of LRP16 in the nuclei of tumor cells and the level of NF-κB activity in human gastric carcinomas. These findings highlight an important nuclear function of LRP16 in the regulation of NF-κB transcriptional activity and suggest that LRP16 might be an important contributor to elevated NF-κB activity in tumors.
The biochemical feature of binding PAR is required for macro domain proteins including LRP16 to participate into PARP-1-based complex in response to DNA damage 
. In this study, the luciferase results as shown in did not demonstrate the link between the LRP16 coactivation of TNF-α-induced NF-κB transcriptional activity and its biochemical feature of binding ADP-ribose metabolites. These results suggest that the biochemical activity of binding ADP-ribose metabolites is dispensable for macro domain proteins when they serve as transcriptional cofactors.
By knockdown assays, we demonstrated that LRP16 is essential for NF-κB activation. In addition, this interference also sensitizes TNF-α-induced cell apoptosis. Although we also demonstrated that the ectopic expression of LRP16 can significantly increase TNF-α-induced κB-luc activity, this ectopic expression in 293T cells did not further enhance TNF-α-induced NF-κB-dependent gene expression, and its DNA binding ability. These results indicate that the endogenous amount of LRP16 inside the nuclei of some cell types is in a saturate status for maximally activating the endogenous NF-κB activity. However, exogenous LRP16 is required for maximally activate the exogenous κB-luc activity.
Coactivators are frequently observed to be shared by different transcription factors, but the differential requirement for shared coactivators may occur in these cases 
. In addition to NF-κB, LRP16 can interact with some steroid receptors such as ERα and AR, and is essential for their transactivation 
. The macro domain alone of LRP16 is enough to mediate its interaction with both p65 and AR. The macro domain alone can augment AR-dependent gene transcription in the same way as the wild type protein 
; however, the macro domain and the non-macro domain regions of LRP16 are both required to activate NF-κB-dependent transcription activity.
Although regulatory events in the nucleus all shape the strength and duration of the NF-κB transcriptional response, the different factors recruited by NF-κB posses differential action mechanisms. p300/CBP was found to associate directly with NF-κB and form a bridge between NF-κB and the basal transcriptional machinery 
. CARM-1 synergistically coactivates NF-κB-mediated transcription in a promoter-dependent manner, in concert with p300/CBP and p160 
. In this study, we demonstrated that the nuclear translocation of NF-κB in the presence of TNF-α stimulation was not affected by LRP16 knockdown. However, the formation or stability of NF-κB transcription complex (NF-κB/p300/CBP/CARM1) was markedly impaired in nuclei of LRP16-deficient cells. Our findings support the notion that LRP16 is essential for the assembly or stability of the functional NF-κB transcription complex in the nucleus. The precise mechanism regarding how LRP16 affects the formation of functional NF-κB complex still remains to be studied and is an interesting and important subject of further investigation.
NF-κB-associated pathways have been widely implicated in oncogenesis and tumor progression by stimulating cell proliferation, inhibiting apoptosis, and promoting metastasis and angiogenesis 
. Aberrant activation of NF-κB is frequently observed in several tumor types including gastric carcinomas 
. In this study, a positive link between the prominent staining of LRP16 inside the nucleus and the elevated NF-κB activity was established in gastric carcinoma specimens. In view of the fact that the clinicopathological data we obtained previously from a large number of samples, which included breast cancer, gastric cancer and colorectal carcinoma, indicated that predominant nuclear staining of LRP16 in tumor cells is significantly linked to high-risk prognostic indices and short survival term 
, we proposed that the aberrant nuclear accumulation of LRP16 would be a common mechanism employed by tumor cells by which NF-κB was excessively activated. The predominant nuclear staining of LRP16 in tumor cells may be a valuable indicator for the evaluation of excessive NF-κB activity.
Aberrant stimulation of estrogen and androgen plays a central role in the genesis and progression of breast cancer and prostate cancer, respectively 
. Crosstalk or reciprocal regulation between hormone-dependent pathways, including ERα- and AR-mediated signal transduction, and NF-κB signaling has been extensively established at multiple signaling nodes 
. However, reasonable explanations for excessive NF-κB activity in hormone-dependent cancers remain to be investigated 
. Previously, we demonstrated that estrogen can upregulate LRP16 expression in estrogen-dependent breast cancer, endometrial cancer, and ovary cancer cell lines 
. Androgen can also upregulate LRP16 expression in androgen-sensitive prostate cancer cell lines 
. The expression level of LRP16 in hormone-dependent cancer cell lines is dependent on hormone action. In addition, we also previously revealed the promotion of LRP16 to proliferation or invasive growth of hormone-dependent cells 
. The identification of the hormone-responsive gene LRP16 acting as an crucial coactivator of NF-κB in this study may provide a novel clue for linking hormone-dependent signaling and NF-κB activity in hormone-dependent cancers.
In conclusion, these findings provide definite evidence to support the integration of the macro domain protein LRP16 into the NF-κB transcriptional complex via interaction with p65 and its crucial role for NF-κB activation inside the nucleus. Aberrant nuclear accumulation of LRP16 in tumor cells might represent a therapeutic target for the control of excessive NF-κB activity.