Role of CD14 Promoter Polymorphisms in Helicobacter pylori Infection–Related Gastric Carcinoma

Purpose: Genetic variation in CD14 may affect CD14 expression and susceptibility to Helicobacter pylori infection–related cancers. This study examined functional single nucleotide polymorphisms (SNP) in the CD14 promoter and their associations with risk of developing gastric carcinoma in relation to H. pylori infection.

Experimental Design: Thirty individual DNAs were sequenced to identify variants, and the function of the variants was examined by reporter gene assays. Genotypes and haplotypes were analyzed in 470 patients and 470 controls, and odds ratios (OR) and 95% confidence intervals (95% CI) were estimated by logistic regression. Serologic H. pylori antibody and soluble CD14 (sCD14) levels were measured by ELISA.

Results: Two SNPs (−651C>T and −260C>T) were identified, of which the −260CT and −260TT genotypes were associated with elevated risk of gastric carcinoma (OR, 1.77; 95% CI, 1.09-2.85 and OR, 1.95; 95% CI, 1.20-3.16, respectively). Haplotype analysis suggested a synergistic effect of the two SNPs (OR for the T₋₆₅₁-T₋₂₆₀ haplotype, 3.39 versus OR for the C₋₆₅₁-T₋₂₆₀ haplotype, 1.45; P = 0.02), which is consistent with reporter gene assays. A multiplicative joint effect between H. pylori infection and −260C>T polymorphism was observed (OR for the presence of both −260TT genotype and H. pylori infection, 4.03; 95% CI, 1.80-9.04). Patients had significantly higher sCD14 than controls (1,866 ± 2,535 ng/mL versus 1,343 ± 2,119 ng/mL; P < 0.001), and this difference was associated with the CD14 −260 polymorphism and H. pylori infection.

Conclusions: Functional polymorphism in CD14 is associated with greater risk of H. pylori–related gastric carcinoma, which might be mediated by elevated sCD14.

Helicobacter pylori has been associated with etiology of gastric cancer (1). However, the clinical outcome of H. pylori infection is highly variable, and only a small fraction of exposed individuals develops the malignancy (2), which may be influenced by both microbial and host factors (3). Immunogenetic factors play important roles in the progression and severity of inflammation, thereby influencing the clinical outcome of H. pylori infection (4). The evolutionarily conserved innate immunity system is the first-line defense against microbial invasion (5), and this system may function as major modulator in the chronic inflammation of persistent H. pylori infection (6). As important components of innate immunity, the germ line–encoded pattern-recognition receptors play a central role in pathogen recognition and immune responses. The signaling of Toll-like receptors (TLR), a pattern-recognition receptor family, not only leads to innate immunity activation but also instructs the antigen-specific adaptive immunity (7). It has been shown that variations in innate immunity may influence immune responses and thus contribute to the diversity of infectious disease (8, 9).

CD14 is a pattern-recognition receptor that plays a central role in innate immunity and directs the adaptive immune responses (10). As a coreceptor of TLRs, CD14 acts primarily by transferring lipopolysaccharide (LPS) and other bacterial ligands from circulating LPS-binding protein to the TLR4/MD-2 signaling complex. This results in the activation of innate host-defense mechanisms by release of inflammatory cytokines (11), such as interleukin-12, an obligatory signal for the differentiation of naive T cells to T helper 1 cells (12). Therefore, CD14 is essential to maintain the T helper 1/2 balance and to coordinate adaptive immune responses. CD14 is a single-copy gene mapped to 5q31.1 (13), but CD14 protein exists as two distinct forms: a glycosylphosphatidylinositol-anchored membrane molecule (mCD14; ref. 14) expressed mainly on the surface of monocytes/macrophages and neutrophils and a soluble form (sCD14) lacking the glycosylphosphatidylinositol anchor found in serum and urine (15). sCD14 is thought to be derived from both protease-dependent shedding of the mCD14 on myeloid cells and protease-independent release from intracellular compartments (16). However, accumulating evidence indicates that sCD14 is also produced by hepatocytes (17) and plays a role that is different from mCD14 in LPS signaling by transferring LPS to plasma lipoproteins but not TLR4/MD-2 complex. This effect of sCD14 dramatically limits the amount of LPS to bind to the monocytes and substantially reduces cytokine responses (18, 19). This effect of sCD14 may also mediate the effects of LPS and other microbial products on mCD14-lacking cells, such as epithelial and endothelial cells, leading to the signaling cascades of immune responses (20, 21). Moreover, sCD14 may be an important molecule in modulating LPS-induced apoptosis of epithelial and endothelial cells (22–24) and a regulatory factor capable of modulating cellular and humoral immune responses (25, 26).

It has been shown that serum sCD14 levels are associated with both genetic variants in the promoter of CD14 and H. pylori infection and consequently associated with certain diseases or disease outcome (27–29). Based on these observations, we hypothesized that the functional genetic variant in the CD14 promoter may affect CD14 expression, which shapes the mCD14/sCD14–mediated innate and adaptive immune responses to persistent H. pylori infection and thus contributes to the development of gastric cancer. In this study, we sought to identify the functional polymorphisms in the 5-flanking region of the CD14 gene and examined their effects on serum sCD14 levels among individuals. We also conducted a large case-control study to investigate the association between CD14 promoter genotypes, alone and in combination with H. pylori infection, and the risk for developing gastric cancer.

Single nucleotide polymorphism identification. Thirty DNA samples derived from peripheral blood of randomly selected healthy subjects (all were Han Chinese) were used to search for single nucleotide polymorphisms (SNP) within the promoter region of CD14 (1,047 bp). These samples included 60 chromosomes, providing at least a 95% confidence level to detect SNPs with a minor allele frequency above 5%. In reference to the human CD14 gene sequences,⁴

PCR primers 5′-AATGGATAGTGTAAGTGACCCAGAG-3′ and 5′-GTGCTTTAGCTTCTTTCCTACACAG-3′ were designed for amplifying the CD14 promoter. SNPs within the fragment from each subject were identified by directly bidirectional sequencing the PCR products with an ABI PRISM 3700 automatic sequencer (Applied Biosystems, Foster City, CA). SNP candidates were identified using CodonCode Aligner program (CodonCode Co., Dedham, MA) and further verified and typed by PCR-based RFLP methods.

Subjects for the case-control study. The case-control study consisted of 470 patients with histopathologically verified primary gastric adenocarcinoma and 470 controls. All subjects were unrelated Han Chinese. Patients were recruited between January 2000 and January 2003 at the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing). All eligible patients diagnosed at the hospital during the study period were recruited, with a response rate of 90%. Patients were from Beijing city and its surrounding regions, and there were no age, sex, and disease stage restrictions. The exclusion criteria included previous cancer and previous chemotherapy or radiotherapy. The pathologic stage of gastric carcinoma was evaluated according to the International Union against Cancer Tumor-Node-Metastasis classification at diagnosis based on postoperative pathologic examination of specimens. Population controls were accrued from a community nutritional survey conducted in the same region during the period of patient collection. The controls were randomly selected from a database consisting of 2,500 individuals based on a physical examination. The selection criteria included no history of cancer and frequency matching to the cases on sex and age (±5 years). At recruitment, informed consent was obtained from each subject, and this study was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.

CD14 genotyping. Genotypes of the CD14 promoter were determined by PCR-RFLP methods. Genomic DNA isolated from peripheral blood lymphocytes of the study subjects were used as templates to amplify CD14 promoter region containing −651T>C or −260T>C site using the PCR primer pairs 651F5′-AGGAAGGGGGAATTTTTCTTTAGGC-3′/651R5′-CCACCAAATCCAAGTCCCTGAA-3′ and 260F5′-TGAGGATCATCCTTTTCCCAAAC-3′/260R5′-CAGGCTTCACACTTGTGAACTCTTC-3′, respectively. PCR products were digested with HaeIII (New England BioLabs, Inc., Beverly, MA) and separated on 1.5% (for the −260T>C site) or 3.0% (for the −651T>C site) agarose gel. Genotypes revealed by PCR-RFLP were further confirmed by directly DNA sequencing of the PCR products. Genotyping was done without knowledge of subjects' case/control status. A 10% random sample of cases and controls was tested twice by different investigators to ensure quality control, and the results were identical.

Serologic determinations of anti–H. pylori antibody and sCD14. Serologic assays for anti–H. pylori antibody were done in Dr. W. You's laboratory (Beijing Institute for Cancer Research, Beijing) as described (30). Briefly, H. pylori strains cultured from gastric biopsies of five patients in the previous study were used to provide a local antigen preparation for serology. Serum levels of anti–H. pylori IgG and IgA were measured separately in duplicate with ELISA procedures. Quality control samples were assayed using the same method in Dr. M.J. Blaser's laboratory at Vanderbilt University, Nashville, TN. An individual was determined to be positive for H. pylori infection when the mean absorbance for either the IgG or the IgA was >1.0, a cutoff value from the examination of a group of H. pylori–negative persons and reference sera. Serum sCD14 levels were measured using ELISA kits (Quantikin, R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. The concentration of each sample was determined by extrapolation from a standard curve estimated from a panel of sCD14 standards of known concentrations. Absorbance values were measured at 450 nm with a multilabel counter (Wallac Victor² 1420, Perkin-Elmer Life Sciences, Boston, MA). Serum levels of sCD14 were expressed as ng/mL.

Construction of reporter plasmids. To verify whether the identified SNPs and their haplotypes influence the transcriptional activity of CD14 and response to H. pylori infection, reporter plasmids encompassing −360 to +29 bp (containing the −260C allele) or −1090 to +20 bp (containing both −260T and −651C alleles) of human CD14 promoter were constructed, respectively. The primers used for amplifying the two DNA fragments were 5′-GACCGCTAGCGAGRAAACAGGGCATTCAC-3′/5′-CGTCAAGCTTGTTCGACCCCAAGACCCTAC-3′ (−360 to +29 bp) and 5′-GACCGCTAGCGGATAGTGTAAGTGACCCAGAG-3′/5′-CGTCAAGCTTCAAGACCCTACACTCACCATG-3′ (−1090 to +20 bp), which contain 5′-NheI and 3′-HindIII cloning sites (underlined sequences), respectively. PCR products were digested with NheI and HindIII and then directionally cloned into pGL3-Basic vector (Promega, Madison, WI). The resulting constructs were named as p260C and p651C-260T according to sequence analysis. Site-specific mutagenesis using p260C and p651C-260T as templates was subsequently manipulated to generate other constructs containing other allele and all possible haplotypes (i.e., p260T, p651C-260C, p651T-260T, and p651T-260C). All constructs used in this study were restriction mapped and sequenced to confirm their authenticity.

Transient transfection and luciferase assays. Because human hepatocytes were shown to express CD14, and because the liver may serve as an important source of sCD14 in plasma (17), human hepatocellular carcinoma cell lines HepG2 and SMMC-7721, cultured in RPMI 1640 with 10% fetal bovine serum, were used for reporter gene expression. Cells were seeded in 48-multiwell plates at a density of 1 × 10⁴ per well and placed in a humidified incubator with 5% CO₂. After grown overnight to 80% confluence, cells were cotransfected with 500 ng p260T, p260C, p651C-260T, p651C-260C, p651T-260T, p651T-260C, or pGL3-Basic and 1.0 ng Renilla luciferase reporter plasmid pRL-SV40 (Luciferase Assay System, Promega). After transfection for 12 h, H. pylori TN2, kindly provided by Dr. W. You and prepared as described (30), were added into the medium of cultured cells at a 1:10 multiplicity of infection. Following another 12-h incubation, luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega) on a TD-20/20ⁿ luminometer (Turner Designs, Promega). Results were normalized for Renilla activity and were expressed as relative luciferase activity of the CD14 reporter constructs compared with the empty vector pGL3-Basic. Three independent transfection experiments were done, and each was done in triplicate.

Haplotype construction and statistical analysis. Logistic regression was used to assess the association between genotypes or H. pylori infection and risk of gastric carcinoma using the Statistical Analysis System software (SAS Institute, Cary, NC). Odds ratios (OR) and their 95% confidence intervals (95% CI) were adjusted for age, sex, genotype, or H. pylori infection, where it was appropriate. We tested the null hypothesis of multiplicative joint effect between genotype and H. pylori infection and evaluated the departures from multiplicative joint effect models. A multiplicative joint effect (31) was suggested when OR₁₁ > OR₁₀ × OR₀₁. The relationship among serum sCD14 levels, CD14 genotypes, H. pylori infection, and disease status were examined by one-way ANOVA with comparison between two groups using Bonferroni's t test. Haploview 3.2 software (32) was used to construct the haplotypes, evaluate the linkage disequilibrium of two SNPs, and test the association of single markers and haplotypes with gastric carcinoma, correcting for multiple testing bias by permutation tests. Haplo.stats software package (33, 34) developed using the R language was used to estimate adjusted ORs and 95% CIs for each haplotype. Simulations were run for 1,000 times for empirical P values. The differences in the luciferase reporter activity between each allele and haplotype with or without H. pylori stimulation were analyzed by paired t test. A two-tailed P < 0.05 was considered statistically significant.

The baseline clinical characteristics of patients and controls are shown in Table 1. The distributions of age and sex among patients and controls were not significantly different, indicating that the matching of controls to cases was adequate. However, H. pylori infection significantly differed between the patients and controls; 73.2% of patients were anti–H. pylori antibody positive, whereas this value was 54.3% in controls (P < 0.0001). Subjects with H. pylori infection were at >2-fold increased risk of developing gastric carcinoma (adjusted OR, 2.31; 95% CI, 1.76-3.04). Of the 470 patients, 383 (81.5%) underwent gastrectomy and had detailed tumor stage data, whereas the rest (18.5%) had not undergone gastrectomy and had unknown tumor stage (Table 1).

Two C>T polymorphisms, located at −651 bp and −260 bp upstream of the translational start site, were identified by resequencing the promoter region of CD14 from 30 healthy subjects. The latter one (−260C>T) is the same as −159C>T designated according to the transcriptional start site in the literature. The allele frequencies for the −651C and −260C were 0.55 and 0.12, respectively, among 470 controls. These two CD14 polymorphisms are in linkage disequilibrium (with D′ = 0.909, LOD = 149.43, and r² = 0.558) in our study population.

CD14 genotyping data are summarized in Table 2. The genotype distributions of the −651C>T and −260C>T polymorphisms in controls fitted the Hardy-Weinberg equilibrium. The frequencies of CD14 −260CC, −CT, and −TT genotypes were 7.0%, 47.9%, and 45.1% in patients compared with 11.9%, 48.3%, and 39.8% in controls (P = 0.023), respectively. Logistic regression analysis showed that an elevated risk for developing gastric carcinoma was associated with the −260CT (adjusted OR, 1.77; 95% CI, 1.09-2.85; P = 0.020) and −260TT (adjusted OR, 1.95; 95% CI, 1.20-3.16; P = 0.007) genotypes. For the −651C>T polymorphism, however, the difference in genotype frequencies between patients and controls was not significant (P = 0.496). No association between CD14 genotypes and H. pylori infection status or tumor stage was found (data not shown).

Because −651C>T and −260C>T polymorphisms are in linkage disequilibrium, we constructed two-marker haplotypes (Table 3). A significant difference in haplotype frequencies was observed between controls and patients (χ² = 11.84; P = 0.008; degree of freedom, 3). Compared with the C₋₆₅₁-C₋₂₆₀ haplotype, all the −260T allele–containing haplotypes were associated with increased risk of developing gastric carcinoma, with the adjusted ORs being 1.45 (95% CI, 1.05-2.00; P = 0.026) and 3.39 (95% CI, 1.38-8.31; P = 0.003) for the C₋₆₅₁-T₋₂₆₀ and T₋₆₅₁-T₋₂₆₀ haplotypes, respectively. Although the T₋₆₅₁-C₋₂₆₀ haplotype was not associated with increased risk (adjusted OR, 1.23; 95% CI, 0.87-1.74; P = 0.243), the adjusted OR for the T₋₆₅₁-T₋₂₆₀ haplotype was significantly higher than that for the C₋₆₅₁-T₋₂₆₀ haplotype (P = 0.020, test for homogeneity), suggesting a synergistic effect between −651T and −260T alleles in the context of haplotype.

We next examined joint effects of CD14 polymorphisms and H. pylori infection. Because the −651C>T polymorphism alone was not associated with the risk, only −260C/T genotypes were analyzed (Table 4). Logistic regression analysis showed that the −260C>T polymorphism was not associated with increased risk of gastric carcinoma among subjects without H. pylori infection. However, among subjects infected with H. pylori, the −260CT or −260TT genotype carriers were at significantly elevated risk for developing the cancer, with adjusted ORs being 3.70 (95% CI, 1.65-8.28; P = 0.002) and 4.03 (95% CI, 1.80-9.04; P < 0.001), respectively. Because the OR (4.03; 95% CI, 1.8-9.04) for subjects with the −260TT genotype and H. pylori infection versus subjects with the −260CC genotype and without H. pylori infection is more than the product of the OR for subjects with the −260TT genotype but without H. pylori infection and the OR for subjects with the −260CC genotype and H. pylori infection (i.e., 1.71 × 1.85 = 3.16), a multiplicative joint effect between H. pylori infection and the CD 14 −260TT genotype in intensifying the risk for developing gastric carcinoma is suggested (31).

We also analyzed the relationship among CD14 genotypes, H. pylori infection, disease status, and serum sCD14 levels (Table 5). Although serum sCD14 levels (ng/mL) varied greatly among individuals, the mean value ± SEM in patients was significantly higher than that in controls (1,866 ± 117 versus 1,343 ± 97, P < 0.001), and this significantly higher sCD14 levels in patients than in controls was related to H. pylori infection (1,909 ± 139 versus 1,174 ± 125, P < 0.001) and CD14 −260TT genotypes (1,997 ± 190 versus 1,310 ± 153, P = 0.006). Analyses with combined CD14 genotypes and H. pylori infection also showed that patients with the CD14 −260CT or TT genotype and H. pylori infection had significantly higher sCD14 levels compared with controls with the same genotype and infection (Table 5). However, there were no significant differences in serum sCD14 levels for CD14 −260 genotypes or H. pylori infection either among patients or among controls. No significant differences in sCD14 levels were found when analyses were stratified by sex, age, or tumor stages (data not shown).

The transcriptional activity of the CD14 −260C and T variants and the four CD14 −651/−260 haplotypes were compared by reporter gene assays in SMMC-7721 and HepG2 cells. Because H. pylori infection–induced sCD14 levels were reported to be associated with CD14 genotypes (29), we also compared the reporter gene expressions in the presence of H. pylori stimulation. As shown in Fig. 1, the −260T-containing CD14 promoter (p260T) drove a >2-fold increased reporter expression compared with the −260C-containing counterpart (p260C) in SMMC-7721 cells (0.61 ± 0.15 versus 2.08 ± 0.58, P = 0.003). Although H. pylori stimulation greatly enhanced the reporter expression, the significant difference between the −260T and −260C alleles remained apparent (2.44 ± 0.24 versus 5.75 ± 0.93, P = 0.004). The basal and H. pylori–stimulated transcriptional activity of p651C-260C, p651T-260C, p651C-260T, and p651T-260T were also significantly different (P = 0.0001), with the −260T-containing haplotypes having heightened promoter activity (Fig. 1). Similar results were also observed in assays with HepG2 cells (data not shown).

In this study, we identified two SNPs in the CD14 promoter region and investigated their effects on CD14 transcriptional activity, circulating sCD14 levels, and risk for developing gastric carcinoma in Han Chinese population. We found that subjects carrying the −260CT or −260TT genotype had significantly increased risk for developing the cancer compared with those carrying the −260CC genotype. Consistent results were obtained when the association between −260C>T polymorphism and risk of the cancer was analyzed in the context of haplotype with −651C>T polymorphism, showing that the −260T-containing haplotypes had higher risk, which is in agreement with the results of functional assays. We also found that although CD14 polymorphisms were not associated with H. pylori infection, there was a multiplicative joint effect between the CD14 −260T allele and H. pylori infection in intensifying the risk. In addition, patients with H. pylori infection or carrying the CD14 −260TT genotype had higher sCD14 levels compared with the controls, indicating that CD14 −260C>T polymorphism might play a role in the outcome of H. pylori infection, especially the development of gastric carcinoma. These findings suggest that inherited variations in genes encoding components of the innate immune system may confer predisposition to gastric cancer.

Overexpression of CD14 and TLRs in gastric mucosa with H. pylori infection, especially in gastric tumor tissues, has been observed (35, 36). It has been well recognized that CD14 plays a crucial role not only in the innate immune responses to bacterial infection but also in the initiation of the cytokine cascades. Besides as a component of the TLR4/MD-2 complex, CD14 is also involved in the signaling mediated by the TLR2 and TLR6 receptors (37), thereby leading to the induction of various transcription factors such as IFN regulatory factors, activating protein-1, and nuclear factor-κB upon bacterial infection, which results in the production of proinflammatory cytokines and chemokines (38). CD14-dependent activation of macrophages/monocytes leads to the release of T helper 1 cytokines, such as interleukin-12, and establishes the chronic inflammation stimulated by H. pylori infection (39). The activated monocytes/macrophages also up-regulate the expressions of many bioactive molecules such as matrix metalloproteinases, vascular endothelial growth factor, cyclooxygenase-2, and inducible NO synthase (40–42), which have been strongly linked to gastric carcinogenesis and cancer progression. In this scenario, it would be expected that genetic variants in CD14 that enhance the gene expression might contribute to the formation of H. pylori–associated cancer.

The association between the CD14 −260T allele and serum sCD14 levels has not been clearly elucidated, and inconsistent reports exist in the literature (27–29, 43). It was reported in a previous study that elevated sCD14 levels were associated with H. pylori infection, especially in subjects with the CD14 CC genotype (29). In the present study, we did not observe any significant elevation of sCD14 in subjects with H. pylori versus those without H. pylori, or in subjects with the CD14 −260CT or TT genotype versus those with the CC genotype either in cases and controls, although a nonsignificantly higher mean value of sCD14 was observed in subjects with the CC genotype and H. pylori infection. Nevertheless, we did observe that the mean level of serum sCD14 in patients was significantly higher than that in controls and patients with the CD14 −260TT genotype and H. pylori infection had higher serum sCD14 levels than controls with the CD14 −260TT genotype and H. pylori infection. Furthermore, combined analyses also showed that significant differences in sCD14 levels associated with CD14 genotypes and H. pylori infection were only seen among patients compared with controls. These findings suggest that the serum sCD14 level might also serve as a disease marker of gastric carcinoma. It has been shown that sCD14 may have several functions in cancer formation. sCD14 could mediate the effects of LPS and other microbial products on epithelial cells that lack mCD14, leading to the signaling cascades of immune responses (22–25). Because sCD14 is involved in LPS-induced apoptosis, it may facilitate the inflammation of gastric epithelia in local site of H. pylori infection and thus play important roles in apoptosis and atrophy of gastric mucosa, which is a crucial event in gastric carcinogenesis (44). On the other hand, circulating sCD14 may also play a role in immunosuppressive responses of cancer because previous study has shown that sCD14 is a negative regulator of human T-cell activation and function (26). Genetic polymorphisms in the CD14 promoter region might play a role in balancing mCD14 and sCD14 levels, thereby modulating the downstream signaling events of immune responses, which lead to different clinical outcome of H. pylori infection.

Consistent with previous studies (28, 37), reporter gene assays showed that the CD14 −260T promoter drove significantly heightened luciferase gene expression compared with the CD14 −260C promoter, and the notable difference between the two alleles was still apparent after stimulation with H. pylori. Similarly, the basal and H. pylori–induced transcriptional activities of the −260T-containing haplotypes were significantly greater than those of the −260C-containing haplotypes. These findings were in line with the population results, indicating that the −260T allele is the risk allele. Interestingly, we found that the p651T-260T construct had higher reporter gene expression than other haplotype constructs, suggesting that there may be a joint effect between the −651C>T and −260TC>T polymorphisms in controlling the CD14 gene transcription, although the mechanism remains unknown. This functional feature was also in agreement with the population data showing that the highest risk of the cancer was associated with the T₋₆₅₁-T₋₂₆₀ haplotype. However, although the serum sCD14 level associated with the CD14 −260T allele was higher in patients than in controls, it was generally not associated with the CD14 genotypes. These results indicate that factors other than genetic polymorphisms may also be involved in the production of serum sCD14. It has been shown, for example, that CD14 can be proteolytically cleaved from the cell surface to form sCD14 by matrix metalloproteinases (45), which are frequently up-regulated by H. pylori in gastric carcinoma (46–49).

The promoter motifs and transcriptional factors involved in the regulation of CD14 expression and how does it response to H. pylori stimulation are not fully understood or elucidated yet. A 227-bp region upstream of the major transcriptional start site was found to drive higher reporter activity compared with the 128-bp, 449-bp, 2.3-kb, and 4.2-kb ones in monocytes (47). Stimulatory protein 1 and CCAAT/enhancer-binding protein binding sites, located at nucleotides −110 and −135, were shown to be critical for the CD14 expression (50, 51). In silico analysis indicated neither gain nor loss of binding site of −651C>T change for transcription factors. However, our reporter constructs encompassing −1090 to +20 bp of CD14 regulation region containing both −651 and −260 polymorphic sites showed much more powerful transcription activity than the constructs encompassing the region from −360 to +29 bp, despite the influences of genotypes and haplotypes. These results indicate that there must be other cis-acting elements located within the region between −1090 and −360 bp that are critical for the CD14 expression in hepatocytes because it has been reported that a 0.7-kb enhancer located ∼6 kb 5′ of the CD14 transcription start site is essential for the CD14 expression in hepatocytes (17). Therefore, it would be interesting to uncover the potential cis-acting elements located in the region between −1090 and −360 bp upstream CD14 and their mechanisms in regulating this gene expression.

In conclusion, we have shown an association between increased risk of gastric carcinoma and genetic polymorphism in the CD14 promoter alone and in combination with H. pylori infection, and this might be mediated by elevated sCD14. These results provide further evidence supporting H. pylori infection as a major risk factor for developing gastric carcinoma and highlight important roles of naturally occurring genetic polymorphisms of innate immunity in susceptibility to H. pylori infection–related cancers.