From phenotype to mechanism: Prenatal spectrum of NKAP mutation-related disorder and its pathogenesis inducing congenital heart disease.

NKAP mutations are associated with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD, MIM: #301039). Here, we elucidate the potential prenatal manifestation of NKAP mutation-associated disorder for the first time, alongside revealing the relationship between NKAP mutations and congenital heart defect (CHD) in the Chinese population. An NKAP mutation (NM_024528.4: c.988C>T, p.Arg330Cys) was identified in two foetuses presenting with CHD. Subsequent mechanistic exploration revealed a marked downregulation of NKAP transcription within HEK293T cells transfected with NKAP p.R330C. However, no significant change was observed at the protein level. Moreover, the mutation led to a dysregulation in the transcription of genes associated with cardiac morphogenesis, such as DHRS3, DNAH11 and JAG1. Additionally, our research determined that NKAP p.R330C affected Nkap protein intra-nuclear distribution, and binding with Hdac3. Summarily, our study strengthens NKAP mutations as a cause of CHD and prompts the reclassification of NKAP p.R330C as likely pathogenic, thereby establishing a prospective prenatal phenotypic spectrum that provides new insight into the prenatal diagnosis of CHD. Our findings also provide evidence of NKAP p.R330C pathogenicity and demonstrate the potential mechanism by which p.R330C dysregulates cardiac developmental gene transcription by altering Nkap intra-nuclear distribution and obstructing the interaction between Nkap and Hdac3, thereby leading to CHD.

SCI1, a flower regulator of cell proliferation, and its partners NtCDKG2 and NtRH35 interact with the splicing machinery.

Successful plant reproduction depends on the adequate development of floral organs controlled by cell proliferation and other processes. The Stigma/style cell-cycle inhibitor 1 (SCI1) gene regulates cell proliferation and affects the final size of the female reproductive organ. To unravel the molecular mechanism exerted by Nicotiana tabacum SCI1 in cell proliferation control, we searched for its interaction partners through semi-in vivo pull-down experiments, uncovering a cyclin-dependent kinase, NtCDKG;2. Bimolecular fluorescence complementation and co-localization experiments showed that SCI1 interacts with NtCDKG;2 and its cognate NtCyclin L in nucleoli and splicing speckles. The screening of a yeast two-hybrid cDNA library using SCI1 as bait revealed a novel DEAD-box RNA helicase (NtRH35). Interaction between the NtCDKG;2-NtCyclin L complex and NtRH35 is also shown. Subcellular localization experiments showed that SCI1, NtRH35, and the NtCDKG;2-NtCyclin L complex associate with each other within splicing speckles. The yeast two-hybrid screening of NtCDKG;2 and NtRH35 identified the conserved spliceosome components U2a', NF-κB activating protein (NKAP), and CACTIN. This work presents SCI1 and its interactors, the NtCDKG;2-NtCyclin L complex and NtRH35, as new spliceosome-associated proteins. Our findings reveal a network of interactions and indicate that SCI1 may regulate cell proliferation through the splicing process, providing new insights into the intricate molecular pathways governing plant development.

Knockdown of NF-κB activating protein promotes pancreatic cancer growth and metastasis through mTOR signaling pathway.

BACKGROUND: NF-κB activating protein (NKAP) acts as a transcriptional suppressor in the Notch signaling pathway, It plays a role in hematopoiesis maintenance, immune cell development, maturation, and functional competency acquisition. NKAP has been found to act as an oncogene in many tumors, but it has not been reported in PAAD.The purpose of this study was to investigate the effect of NKAP on the growth and metastasis of pancreatic adenocarcinoma(PAAD). METHODS AND RESULTS: In this study, western blot and qRT-PCR showed that highly expressed NKAP was found in PAAD cell lines, and small interfering RNA (siRNA) was employed to reduce the expression of NKAP in PAAD cell lines. The results of CCK-8, clony formation, Transwell and flow cytometry showed that knockdown of NKAP significantly inhibited biological function of PAAD cells, and increased cell apoptosis. Study also observed that knockdown of NKAP inhibited the expression levels of apoptosis proteins and cyclin in PAAD cells. In addition, mTOR's degree of phosphorylation and the expression of its downstream target p70S6K can both be activated by NKAP. This effect was also confirmed in salvage experiments performed with Rapamycin(RaPa), an inhibitor of mTOR. At the end of the experiment, It was investigated how NKAP affected the drug sensitivity of gemcitabine used to treat PAAD. The results showed that knocking down NKAP could increase the drug sensitivity of gemcitabine. CONCLUSIONS: NKAP as an oncogene regulates the development of PAAD cells. The research found that the mTOR signaling pathway is engaged in the oncogenic role of NKAP in PAAD for the first time.

Treg-specific deletion of NKAP results in severe, systemic autoimmunity due to peripheral loss of Tregs.

Regulatory T cells are critical for the generation and maintenance of peripheral tolerance. Conditional deletion of the transcriptional repressor NKAP in Tregs using Foxp3-YFP-cre NKAP conditional knockout mice causes aggressive autoimmunity characterized by thymic atrophy, lymphadenopathy, peripheral T cell activation, generation of autoantibodies, immune infiltration into several organs, and crusty skin at 3 weeks of age, similar to that of "scurfy" Foxp3-mutant mice. While Treg development in the thymus proceeds normally in the absence of NKAP, there is a severe loss of thymically-derived Tregs in the periphery. NKAP-deficient Tregs have a recent thymic emigrant phenotype, and are attacked by complement in a cell-intrinsic manner in the periphery. Previously, we demonstrated that NKAP is required for conventional T cell maturation as it prevents complement-mediated attack in the periphery. We now show that Tregs undergo a similar maturation process as conventional T cells, requiring NKAP to acquire complement resistance after thymic egress.

Capn3b-deficient zebrafish model reveals a key role of autoimmune response in LGMDR1.

Mutations in calcium-dependent papain-like protease CALPAIN3 (CAPN3) cause Limb-Girdle Muscular Dystrophy Recessive Type 1 (LGMDR1), the most common limb-girdle muscular dystrophy in humans. In addition to progressive muscle weakness, persistent inflammatory infiltration is also a feature of LGMDR1. Despite the underlying mechanism remaining poorly understood, we consider that it may relate to the newly defined role of CAPN3/Capn3b in the nucleolus. Here, we report that the loss-of-function of zebrafish capn3b, the counterpart of human CAPN3, induces autoimmune response akin to that in LGMDR1 patients. Mutant capn3b larvae are more susceptible to Listeria monocytogenes injection, characterized by recruiting more macrophages. Under germ-free conditions, transcriptome analysis of the capn3b mutant muscle reveals a significant upregulation of the chemokine-production-related genes. Coincidently, more neutrophils are recruited to the injury site imposed by either muscle stabbing or tail fin amputation. Nucleolar proteomic analysis and enzymatic assays reveal NKAP, an activating factor of the NF-κB pathway, to be a target of CAPN3. We conclude that the accumulation of Nkap and other factors in the capn3b mutant may be involved in the over-activation of innate immunity. Our studies indicate that the zebrafish capn3b mutant is a powerful model for studying the immunity-related progression of human LGMDR1.

LncRNA LINC00707 serves as a sponge of miR-382-5p to alleviate lipopolysaccharide (LPS)-induced WI-38 cell injury through upregulating NKAP in infantile pneumonia.

Infantile pneumonia (IP) is an acute lower respiratory infection that imposes a heavy burden on children's health. Increasing evidence has demonstrated that long non-coding RNA (lncRNA) LINC00707 participates in the regulation of the pneumonia process. Cell proliferative ability and apoptosis were measured using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Bcl-2 related X protein (Bax), NF-kB activating protein (NKAP), p-P65, P65, p-IκBα, and IκBα protein levels were detected using western blot assay. The binding between miR-382-5p and LINC00707 or NKAP was predicted by starBase v2.0 and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. LINC00707 and NKAP levels were increased, and miR-382-5p was decreased in LPS-stimulated WI-38 cells. Furthermore, the silencing of LINC00707 could abrogate LPS-engendered WI-38 cell proliferation, apoptosis, and oxidative stress. LINC00707 deficiency could relieve LPS-triggered WI-38 cell damage by regulating the miR-382-5p/NKAP axis, providing a new therapeutic strategy for IP treatment.

IL-1ß promotes hypoxic vascular endothelial cell proliferation through the miR-24-3p/NKAP/NF-κB axis.

PURPOSE: Our previous data indicated that miR-24-3p is involved in the regulation of vascular endothelial cell (EC) proliferation and migration/invasion. However, whether IL-1ß affects hypoxic HUVECs by miR-24-3p is still unclear. Therefore, the present study aimed to investigate the role and underlying mechanism of interleukin 1ß (IL-1ß) in hypoxic HUVECs. METHODS: We assessed the mRNA expression levels of miR-24-3p, hypoxia-inducible factor-1α (HIF1A) and NF-κB-activating protein (NKAP) by quantitative real-time polymerase chain reaction (RT-qPCR). ELISA measured the expression level of IL-1ß. Cell counting kit-8 (CCK-8) assays evaluated the effect of miR-24-3p or si-NKAP+miR-24 on cell proliferation (with or without IL-1ß). Transwell migration and invasion assays were used to examine the effects of miR-24-3p or si-NKAP+miR-24-3p on cell migration and invasion (with or without IL-1ß). Luciferase reporter assays were used to identify the target of miR-24-3p. RESULTS: We demonstrated that in acute myocardial infarction (AMI) patient blood samples, the expression of miR-24-3p is down-regulated, the expression of IL-1ß or NKAP is up-regulated, and IL-1ß or NKAP is negatively correlated with miR-24-3p. Furthermore, IL-1ß promotes hypoxic HUVECs proliferation by down-regulating miR-24-3p. In addition, IL-1ß also significantly promotes the migration and invasion of hypoxic HUVECs; overexpression of miR-24-3p can partially rescue hypoxic HUVECs migration and invasion. Furthermore, we discovered that NKAP is a novel target of miR-24-3p in hypoxic HUVECs. Moreover, both the overexpression of miR-24-3p and the suppression of NKAP can inhibit the NF-κB/pro-IL-1ß signaling pathway. However, IL-1ß mediates suppression of miR-24-3p activity, leading to activation of the NKAP/NF-κB pathway. In conclusion, our results reveal a new function of IL-1ß in suppressing miR-24-3p up-regulation of the NKAP/NF-κB pathway.

MARCKS cooperates with NKAP to activate NF-kB signaling in smoke-related lung cancer.

Rationale: Cigarette smoking is a major risk factor for lung cancer development and progression; however, the mechanism of how cigarette smoke activates signaling pathways in promoting cancer malignancy remains to be established. Herein, we aimed to determine the contribution of a signaling protein, myristoylated alanine-rich C kinase substrate (MARCKS), in smoke-mediated lung cancer. Methods: We firstly examined the levels of phosphorylated MARCKS (phospho-MARCKS) in smoke-exposed human lung cancer cells and specimens as well as non-human primate airway epithelium. Next, the MARCKS-interactome and its gene networks were identified. We also used genetic and pharmacological approaches to verify the functionality and molecular mechanism of smoke-induced phospho-MARCKS. Results: We observed that MARCKS becomes activated in airway epithelium and lung cancer cells in response to cigarette smoke. Functional proteomics revealed MARCKS protein directly binds to NF-κB-activating protein (NKAP). Following MARCKS phosphorylation at ser159 and ser163, the MARCKS-NKAP interaction was inhibited, leading to the activation of NF-κB signaling. In a screen of two cohorts of lung cancer patients, we confirmed that phospho-MARCKS is positively correlated with phospho-NF-κB (phospho-p65), and poor survival. Surprisingly, smoke-induced phospho-MARCKS upregulated the expression of pro-inflammatory cytokines, epithelial-mesenchymal transition, and stem-like properties. Conversely, targeting of MARCKS phosphorylation with MPS peptide, a specific MARCKS phosphorylation inhibitor, suppressed smoke-mediated NF-κB signaling activity, pro-inflammatory cytokines expression, aggressiveness and stemness of lung cancer cells. Conclusion: Our results suggest that phospho-MARCKS is a novel NF-kB activator in smoke-mediated lung cancer progression and provide a promising molecular model for developing new anticancer strategies.

Silencing of lncRNA MIAT alleviates LPS-induced pneumonia via regulating miR-147a/NKAP/NF-κB axis.

PURPOSE: Pneumonia is a respiratory disease with an increasing incidence in recent years. More and more studies have revealed that lncRNAs can regulate the transcriptional expression of target genes at different stage. Herein, we aimed to explore the effect of lncRNA MIAT in LPS-induced pneumonia, and further illuminate the possible underlying mechanisms. METHOD AND RESULTS: Mice were intraperitoneally injected with LPS, and the lung inflammation was evaluated. Microarray showed lncRNA MIAT was up-regulated in LPS-induced pulmonary inflammation. And qRT-PCR and FISH assay indicated that MIAT was increased in mice with LPS injection. Functional analysis showed sh-MIAT inhibited LPS-induced inflammation response, inhibited apoptosis level and protected lung function. As well, si-MIAT removed the injury of LPS on mouse lung epithelial TC-1 cells, and inhibited the activation of NF-κB signaling. Furthermore, MIAT acted as a sponge of miR-147a, and miR-147a directly targeted NKAP. Functionally, AMO-147a or NKAP remitted the beneficial effects of si-MIAT on LPS-induced inflammation response of TC-1 cells. CONCLUSION: Deletion of MIAT protected against LPS-induced lung inflammation via regulating miR-147a/NKAP, which might provide new insight for pneumonia treatment.

NKAP alters tumor immune microenvironment and promotes glioma growth via Notch1 signaling.

BACKGROUND: Glioma is one of the most aggressive malignant brain tumors which is characterized with highly infiltrative growth and poor prognosis. NKAP (NF-κB activating protein) is a widely expressed 415-amino acid nuclear protein that is overexpressed by gliomas, but its function in glioma was still unknown. METHODS: CCK8 and EDU assay was used to examine the cell viability in vitro, and the xenograft models in nude mice were established to explore the roles of NAKP in vivo. The expressions of NKAP, Notch1 and SDF-1 were analyzed by immunofluorescence analysis. The expression of NKAP and Notch1 in glioma and normal human brain samples were analyzed by immunohistochemical analysis. In addition, CHIP, Gene chip, western blot, flow cytometry, immunofluorescence, ELISA and luciferase assay were used to investigate the internal connection between NKAP and Notch1. RESULTS: Here we showed that overexpression of NKAP in gliomas could promote tumor growth by contributing to a Notch1-dependent immune-suppressive tumor microenvironment. Downregulation of NKAP in gliomas had abrogated tumor growth and invasion in vitro and in vivo. Interestingly, compared to the control group, inhibiting NKAP set up obstacles to tumor-associated macrophage (TAM) polarization and recruitment by decreasing the secretion of SDF-1 and M-CSF. To identify the potential mechanisms involved, we performed RNA sequencing analysis and found that Notch1 appeared to positively correlate with the expression of NKAP. Furthermore, we proved that NKAP performed its function via directly binding to Notch1 promoter and trans-activating it. Notch1 inhibition could alleviate NKAP's gliomagenesis effects. CONCLUSION: these observations suggest that NKAP promotes glioma growth by TAM chemoattraction through upregulation of Notch1 and this finding introduces the potential utility of NKAP inhibitors for glioma therapy.

MiR-4766-5p Inhibits The Development And Progression Of Gastric Cancer By Targeting NKAP.

PURPOSE: It is widely known that some specific microRNAs can regulate the expressions of genes in gastric cancer cells at the post-transcriptional level. Previous studies have identified that miRNA-4766-5p was involved in tumor cell proliferation and can be an independent prognostic indicator for malignant pleural mesothelioma. However, the mechanism underlying gastric cancer via the miRNA-4766-5p pathway remains to be blank. METHODS: We investigated the expression of miR-4766-5p in gastric cancer tissues and cells through qRT-PCR. We used RNAi to change the expressions of miR-4766-5p in gastric cancer cell lines, AGS and MKN45. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the mRNA expression of miR-4766-5p. We identified cell proliferation by CCK8 and clone formation assays. We analyzed the cell apoptosis and cycle through flow cytometry. At last, we used a dual-luciferase reporter assay to illustrate the interaction between miR-4766-5p and NKAP and used Western blot to determine the protein expression of signaling pathways. RESULTS: We found that 1) miR-4766-5p was down-regulated in gastric cancer tissues and cells lines; 2) miR-4766-5p inhibited cell proliferation of gastric cancer cell lines significantly; 3) miR-4766-5p significantly inhibited cell migration and invasion of gastric cancer cells; 4) miR-4766-5p induced gastric cancer cell apoptosis. 5) NKAP was a direct target gene of miR-4766-5p; and 6) miR-4766-5p induced inactivation of AKT/mTOR pathway. CONCLUSION: The above results indicate that miR-4766-5p suppressed the proliferation and metastasis of gastric cancer cells through targeting NKAP. Our findings could probably contribute to the diagnostics and prognostics of gastric cancer through new methodologies.

NKAP Regulates Senescence and Cell Death Pathways in Hematopoietic Progenitors.

NKAP is a multi-functional nuclear protein that has been shown to be essential for hematopoiesis. Deletion of NKAP in hematopoietic stem cells (HSCs) was previously found to result in rapid lethality and hematopoietic failure. NKAP deficient cells also exhibited diminished proliferation and increased expression of the cyclin dependent kinase inhibitors (CDKIs) p19 Ink4d and p21 Cip1. To determine how dysregulation of CDKI expression contributes to the effects of NKAP deficiency, NKAP was deleted in mice also deficient in p19 Ink4d or p21 Cip1 using poly-IC treatment to induce Mx1-cre. Hematopoietic failure and lethality were not prevented by deficiency in either CDKI when NKAP was deleted. Inducible deletion of NKAP in cultured hematopoietic progenitors ex vivo resulted in a senescent phenotype and altered expression of numerous cell cycle regulators including the CDKI p16 INK4a. Interestingly, while combined deficiency in p16 INK4a and p21 Cip1 did not reverse the effect of NKAP deficiency on hematopoiesis in vivo, it did shift the consequence of NKAP deficiency from senescence to apoptosis in ex vivo cultures. These results suggest that NKAP may limit cellular stress that can trigger cell cycle withdrawal or cell death, a role critical for the maintenance of a viable pool of hematopoietic progenitors.

Synthetic Essentiality of Metabolic Regulator PDHK1 in PTEN-Deficient Cells and Cancers.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers.

NKAP functions as an oncogene and its expression is induced by CoCl2 treatment in breast cancer via AKT/mTOR signaling pathway.

PURPOSE: NKAP plays an important role in transcriptional repression, T-cell development, maturation and function acquisition, maintenance and survival of hematopoietic stem cells, and RNA splicing. In this study, we tried to explore the physiological role of NKAP in breast cancer. METHODS: We investigated NKAP expression in breast cancer patients and normal controls and its correlation with survival in breast cancer patients by searching on GEPIA. We knocked down the expression of NKAP in MCF-7 cells by RNAi technique and studied its effect on cell proliferation, migration, invasion, and apoptosis. And we revealed the effect of NKAP on MCF-7 cells under hypoxic conditions in vitro. RESULTS: NKAP was differentially expressed in breast cancer and normal tissues and is a potential prognostic indicator of breast cancer. Subsequently, NKAP knockdown significantly inhibited the proliferation and clonality of MCF-7 cells and induced its apoptosis through caspase 3-dependent pathway. In addition, knockdown of NKAP could strongly inhibit the migration and invasion of MCF-7 cells. In MCF-7 cells, NKAP affected the AKT/mTOR signaling pathway and markedly reduced the phosphorylation of AKT and mTOR, as well as the downstream protein. What's interesting is CoCl2 was found to induce NKAP expression in MCF-7 cells. Downregulation of NKAP hindered the impact of CoCl2 on the MCF-7 cells, including cell proliferation and invasion, by adjusting AKT/mTOR signaling. CONCLUSION: NKAP functioned as an oncogene, and its expression was induced by hypoxia in breast cancer via AKT/mTOR signaling pathway.

The Notch co-repressor protein NKAP is highly expressed in adult mouse subventricular zone neural progenitor cells.

In the adult mammalian brain niches for neural stem cells are maintained, which enable a steady-state neurogenesis. This process is tightly regulated by multiple niche factors, including Notch and NF-κB signaling. The NF-κB-activating-protein (NKAP) has previously been shown to act as Notch co-repressor component by binding CIR and recruiting HDAC3 in T-cell development and furthermore to regulate NF-κB-dependent transcription. Here, we provide first evidence for the expression of NKAP in neurogenic cells of the adult mammalian brain. NKAP is highly expressed in Mash1(+) transit amplifying cells and PSA-NCAM(+) migrating neuroblasts throughout the subventricular zone (SVZ) and the rostral migratory stream (RMS), as well as in the hippocampus. We further show that NKAP expression levels are downregulated during the course of the RMS. Eventually, most differentiated cells in the olfactory bulb (OB) and the corpus callosum only display low levels of NKAP expression. Finally, large subsets of mature neurons in the OB, the hippocampus and the thalamus express NKAP at high levels, suggesting an additional role of NKAP outside of SVZ progenitor cells.