Functional variations of NFKB1 and NFKB1A in inflammatory disorders and their implication for therapeutic approaches

Being described as immune activation within the arterial wall in the presence of inflammatory mediators, atherosclerosis (AT) is believed to be a chronic immunoinflammatory disease. As NF-κB is accepted as regulating the expression of a wide range of genes cooperating with the distinct aspects of atherosclerotic pathogenesis [17], NFKB1 may be an appropriate candidate for investigating arterial wall inflammation-related disorders such as atherosclerosis and coronary artery disease (CAD) [18]. Özbilüm et al. [19] draw our attention to alveolar epithelial cells as a piece of evidence for the mechanism of the IκBα–NF-κB-dependent anti-inflammatory effect. Our results differed from those of Özbilüm et al.: we reported that rs28362491 polymorphism has no association with AT; however, the AA genotype of the rs696 polymorphism of NFKB1A gene was considerably elevated in atherosclerotic patients in a Turkish population. Moreover, the combined genotype association of rs28362491 and rs696 polymorphisms with atherosclerosis was not statistically significant, suggesting that these two polymorphisms together play no role in the pathogenesis of atherosclerosis [20]. Although the findings represent NFKB1A rs696 polymorphism as a novel marker of susceptibility to atherosclerosis, the results from such analyses should be treated with caution because of the limited sample size.

NFKB1A −826 C/T promoter polymorphism was reported as a risk factor for susceptibility to CAD, but NFKB1A −297C/T and −881A/G polymorphisms had no associations, in a Turkish population [19], whereas Lai et al. [21] found that rs28362491 polymorphism had a critical role and was related to functional interleukin (IL)-6 expression in a Chinese Uygur population. In an Iranian subpopulation, rs28362491 polymorphism, but not NFKB1A −826C/T polymorphism, was found to be significantly associated with the development and severity of CAD as an independent risk factor [22]. In research into an NFKB1 promoter variant in coronary heart diseases (CHDs) of people of European ancestry, Vogel et al. [23] reported that a variant previously found to lead to partial depletion of NF-κB p50 is associated with CHD. The participation of rs28362491 polymorphism in CAD in Korean and Chinese populations was reported more recently [24, 25]. There is general agreement that inflammation occupies a pivotal role in both initiation and progression of coronary atherosclerosis is the necessarily condition for CHD. Jin et al. [25] concluded that the del/del genotype of rs28362491 is associated with the risk and the severity of the disease.

NF-κB is a good illustration of two sides of the same coin. While a majority of authors mention that NF-κB activation is associated with normal conditioning, Peterson et al. [26], using a murine model of Duchenne muscular dystrophy, report the importance of NF-κB blocking in normal cardiac function.

MicroRNAs (miRNAs) are noncoding, single-stranded RNAs around 20–22 nucleotides in length, which organize gene expression through posttranscriptional repression [27] and are members of a large array of remodification processes, such as cellular differentiation, proliferation, development, immunity, apoptosis, and angiogenesis [28,29,30], but these are linked to several chronic and acute diseases, such as heart diseases, cancer, acute organ injury, autoimmunity, and ischemic stroke, when they are dysregulated [31,32,33,34]. In a major advance for understanding inflammatory diseases, Taganov et al. [35] noted that an NF-κB-targeting actor, namely, mir-146a, enhances the inflammatory response by being used as a feedback inhibitor of NF-κB activation. Generally, the expression level of mature miRNA-146a may be regulated by SNPs of pre-miR-146a. An SNP located in the stem region, namely, rs2910164, may modify the transcription of genes involved putatively in the pathogenesis of inflammation-related diseases, such as cardiovascular diseases, reducing the total amount of mature miRNA [36, 37].

More recently, literature has emerged that describes consistent findings for mir-146a SNP. Xiong et al. [38] reported that a G-to-C substitution in the precursor of mir-146a seems to lead to the enhanced expression of mature mir-146a. This is in agreement with findings by Guo et al. [39] that the function of TH-1 cells, which is necessary for the progression of acute coronary syndrome, is stimulated by increased mature mir-146a expression levels.

The following findings also seem to be consistent with this research, which found significant differences within atherosclerotic patients compared with control subjects, concerning pre-miR-146a rs2910164 polymorphism. The G allele and GG genotype were associated with an elevated risk for atherosclerosis thus pre-miR-146a rs2910164 is assumed to play a role as a novel marker for possible atherosclerosis susceptibility [20].

The link between obesity and obesity-related disorders can be explained by inflammation, which is noticed as an elevation in circulating levels of C-reactive protein (CRP) with low-grade inflammation [40, 41]. The debate about the association of NFKB1 with obesity has gained fresh prominence, with many arguing that NF-κB is weakly initiated in a nonobese murine model of type 1 diabetes [42], whereas NF-κB is directly correlated with pancreatic β-cell failure within diabetes [43]. However, we previously concluded that the ins allele of NFKB1 rs28362491 and/or ins/ins genotype is related to morbid obesity in a Turkish population [44]. These findings are in contrast to those of Stegger et al. [45], who found no substantial interaction between rs28362491 and gluteofemoral, abdominal, and general obesity in a Danish population.

Morbid obesity is correlated with elevated circulating systemic acute-phase proteins, namely, CRP, and the expression of CRP is regulated by the p50 homodimer of NFKB1. We found that the rs28362491 deletion variant tends to decrease the p50 subunit level and that increased serum CRP levels are correlated with NFKB1 rs28362491 polymorphism [46]. By modulating serum levels of CRP, this polymorphism may be correlated with morbid obesity.

There is good evidence for an association among toll-like receptor (TLR) polymorphisms, hepatic fat accumulation, and chronic inflammation, and the NF-κB gene appears to play a key role. We reported that TLR2 variants, such as rs5743708 (G/G), and high levels of liver enzymes, such as alanine transaminase, alkaline phosphatase, aspartate transaminase, and γ-glutamyl transpeptidase, are associated with high levels of CRP [44]. These results are important for 2 main reasons. First, detecting the ins allele of rs28362491 and Arg753Gln allele of rs5743708, combined with a high level of CRP, may offer better treatments in the future. Second, NFKB1 may be useful to monitor both the course and severity of liver disease, suggesting new trends of therapies for these conditions.

Male infertility accounts for approximately 50% of infertility cases, even though 60% of whole infertility cases are idiopathic [47]. Severe oligozoospermia or azoospermia is responsible for most idiopathic male infertility. The interaction between the egg and the sperm is a key point, but the molecular mechanisms of egg–sperm membrane protein binding and fusion reactions are not fully clarified.

NFKB1 is a candidate gene in spermatogenesis, whose failure may lead to male infertility [48, 49]. The relationship of the variants of the promoter sites of NFKB1 with numerous diseases has been studied widely. Ranganathan et al. [50] have associated decreased NFKB1 expression with poor sperm production.

The E-cadherin short interfering RNA (siRNA) is known to trigger the stimulation of NF-κB transcriptional activity [51]. α-Catenin and E-cadherin are validated to be good indices of infertility [52]. Hernandez Gifford et al. [53] proposed anti-catenin/anti-cadherin antibodies for male contraception, and Purohit et al. [54] reported the absence of E-cadherin on the head domain of spermatozoa from oligospermic individuals; however, as yet, the precise mechanisms of both recognition and fusion processes remain to be elucidated. We found increased risk of development of male infertility associated with the presence of the ins allele of NFKB1. Moreover, in the context of low E-cadherin expression, NFKB1 rs28362491 ins/ins and del/del genotypes are likely to display an essential role in the case of susceptibility to oligospermic male infertility [55]. In other works, we were not able to find any correlation of rs28362491 within NFKB1 or the rs696 polymorphisms within NFKB1A in cases of elevated apoptosis in oligospermia. However, we concluded that the rs28362491 heterozygosity is likely to play a protective role by altering oligospermia susceptibility [56]. Consistent with these findings, in the context of E-cadherin and fibronectin levels, we found that the insertion allele within the promoter region of NFKB1 is presumably a key function in the susceptibility to normospermic male infertility [57].

There is other evidence to suggest that the NF-κB also contributes to female infertility. In a murine model, Wang et al. confirmed the idea that intrauterine adhesion results in impaired pregnancy and concluded that NF-κB activation is notably increased within the endometrial tissues of patients with Asherman syndrome [58].

Hashimoto disease is a common chronic inflammation-based disorder present in the thyroid gland influenced by the interplay between various cytokines, and the potential risk factors for Hashimoto disease have been studied widely for decades [59].

As NF-κB is at the center of all autoimmune diseases, and since inflammation is inevitable, we have previously analyzed the associations of polymorphisms of rs28362491 within NFKB1 and rs696 within NFKBIA with susceptibility to Hashimoto disease. There was no significant association between the frequency of the alleles of these two variants and the genotypes. In a combined genotype analysis of NFKB1 and NFKBIA polymorphisms, instead, the ins/ins/AG combined genotype was negatively associated with Hashimoto disease, which predominantly depends on the G allele of rs696 in the Turkish population. In the presence of at least one G allele, the IL-6 levels were also higher in patients with the del/del genotype [60]. Elevated serum IL-6 levels were correlated with the del allele of rs2836249. Therefore, polymorphism of the functional NFKB1 rs28362491 promoter was significantly associated with modulated IL-6 levels in the population with Hashimoto disease [61].

Graves disease is classified as a distinctive organ-specific inflammatory and autoimmunity-based disease of the thyroid, defined by dermopathy, hyperthyroidism, an ocular disorder, and especially diffuse goiter. At least 79% of genetic factors are involved in this disorder; cooperation between several inflammation-related genes, such as NFKB1 and NFKBIA, and cytokines, may have an impact on the development of Graves disease. An association study within a Turkish population found that the rs28362491 del/ins genotype is a possible candidate variation for the development of Graves disease, whereas the ins/ins genotype is likely to play a protective role in this disorder. By contrast, in the case of rs696, there were no statistical differences between the groups [62]. The levels of cytokines IL-1β, IL-6, and tumor necrosis factor (TNF)-α showed remarkable associations with variations in NFKB1 rs28362491. Kurylowicz et al. [63] found that susceptibility to Graves disease is associated with NFKB1 rs28362491 polymorphism within a Polish population.

Behçet syndrome is both a systemic autoimmune disorder and a chronic inflammatory disease defined by ocular inflammation, recurrent vasculitis, oral and genital ulcers, and skin lesions [64]. Although there is some evidence for the involvement of both environmental and genetic factors, Behçet syndrome remains classified as a disease of unknown origin. NFKB1 and its inhibitor, NFKBIA, are important actors in the inflammatory cascade of Behçet syndrome.

The promoter region polymorphisms of NFKB1 in Behçet syndrome could lead to modified NFKB1 expression, which then promotes altered inflammatory cytokine transcription and might clarify the elevated expression and serum concentrations of these cytokines. In an association study in a Turkish population, the association of NFKB1 rs28362491 polymorphism with the ins allele and ins/ins genotype of rs28362491 is found to increase susceptibility to Behçet syndrome by 1.8- and 2.5-fold, respectively [65] (Tables 1 and 2). There appears to be no doubt that NF-κB inhibition is caused by binding of an inhibitor (IKBα) that is encoded by NFKBIA, and any instability of this binding may lead to progression of inflammatory diseases. Hung et al. [67], in their study of Behçet syndrome, found an association with NFKBIA promoter polymorphisms, including −826 T/T, −826 C/T, 519C/T, and −881A/G, and −550A/T, and reported a correlation with the −826 T/T genotype and characteristic skin lesions. Consistent with these studies, we observed an rs696 AA genotype within NFKBIA as a factor that leads to a 2.5-times-increased risk for development of Behçet syndrome [65]. By contrast, assuming them as variations, Kaustio et al. [68] associated NFKB1 mutations, such as I553M and H67R, with Behçet syndrome-like phenotypes.

Elevated NF-κB activation has also been related to cancer progression [69]. In cancer, the process that controls gene expression in response to inflammatory stimuli combines its survival with both its phenotype and function with the rest of the tissue [70]. This is generally apparent in strictly compromised regulation of NF-κB activity, which enables abnormal cohorts of the NF-κB target gene expression in cancer cells [71]. The conclusion is not only that the cells of surrounding tissue change their function and fail to support the organism exclusively, but also that the cancer cells function abnormally. Instability of NF-κB and IκB interactions have been observed generally in several diseases; however, the mechanisms behind the association between certain variations within different genes and cancer development remain elusive.

Glioblastoma (glioblastoma multiforme), accounting for less than 2% of all human cancers, is the most malignant primary brain tumor in the adult nervous system [72, 73]. Although the origin of glioblastoma development is still unknown, some genetic modifications that lead to aberrant activity of pathways, such as proliferation, apoptosis, and cell cycle regulation, are considered to contribute to their pathogenesis [74, 75].

Deregulated NF-κB activity has become a central issue in the development of most human cancers [76, 77]. NF-κB organizes cancer aggressiveness and development by increasing angiogenesis, antiapoptosis, and tumor proliferation and by reducing the immune response and thus managing pathogenetic regulation [78].

Numerous studies advocate the idea that several malignant tumor types, including glioblastomas, have strong connections with NF-κB cascades [5, 79]. We have evaluated 120 glioma samples and 225 controls. There are significant associations between insertion allele carriers and elevated risk of gliomas [66] (Tables 1 and 2). Previous studies reported conflicting results. Meta-analyses such as those conducted by Sun et al. [80] and Yu et al. [81], for instance, concluded that the deletion allele is both a risk and a protection against cancer susceptibility in populations with European or Asian ancestry. The rs28362491 del/del genotype appears related to bladder, prostate, oral, and ovarian cancers but not with hepatocellular carcinoma [82]. Concetti and Wilson reviewed the status of this polymorphism and concluded that rs28362491 polymorphism is disadvantageous for colorectal, gastric, liver, bladder, and thyroid cancers but advantageous for prostate, ovarian, and cervical cancers [83]. Similarly, rs28362491 polymorphism was found to be associated with decreased cancer risk [84], as well as the development of head and neck cancer [85], and rs696 polymorphism was found to be associated with colorectal cancer risk [86].

Various NFKB1 variations in specific pathways are associated with cancer progression, but blocking a signaling pathway in multifactorial diseases such as cancer may not be appropriate because the polymorphisms can be advantageous for the individual.