NF-кB increases LPS-mediated procalcitonin production in human hepatocytes

Scientific Reports, Jun 2018

For years, procalcitonin (PCT) has been employed as a diagnostic biomarker for the severity of sepsis and septic shock, as well as for guiding the application of antibiotics. However, the molecular/cellular basis for the regulation of PCT production is not fully understood. In this study, we identified the signalling pathway by which the expression of PCT was induced by lipopolysaccharide in human hepatocytes at the mRNA and protein levels. This expression was dependent on nuclear transcription factor κB (NF-κB), as indicated by a NF-κB binding site (nt −53 to −44) found in the PCT promoter region. We also showed that microRNA-513b (miR-513b) was also able to bind to the 3′-untranslated region (UTR) of the PCT promoter sequence. Meanwhile, the activation of NF-κB down-regulated the expression of miR-513b. In conclusion, we suggest that NF-κB is capable of enhancing the expression of PCT by either directly activating the transcription of the PCT gene or indirectly modulating the expression of its regulatory component, miR-513b. Our results indicate a molecular mechanism responsible for the regulation of PCT production.

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NF-кB increases LPS-mediated procalcitonin production in human hepatocytes

SCientiFiC RepoRts | NF-?B increases LPS-mediated procalcitonin production in human hepatocytes Yongfeng Bai Jun Lu Ying Cheng Feng Zhang Xueyu Fan Yuanyuan Weng Jin Zhu OPEN For years, procalcitonin (PCT) has been employed as a diagnostic biomarker for the severity of sepsis and septic shock, as well as for guiding the application of antibiotics. However, the molecular/cellular basis for the regulation of PCT production is not fully understood. In this study, we identified the signalling pathway by which the expression of PCT was induced by lipopolysaccharide in human hepatocytes at the mRNA and protein levels. This expression was dependent on nuclear transcription factor ?B (NF-?B), as indicated by a NF-?B binding site (nt ?53 to ?44) found in the PCT promoter region. We also showed that microRNA-513b (miR-513b) was also able to bind to the 3?-untranslated region (UTR) of the PCT promoter sequence. Meanwhile, the activation of NF-?B down-regulated the expression of miR-513b. In conclusion, we suggest that NF-?B is capable of enhancing the expression of PCT by either directly activating the transcription of the PCT gene or indirectly modulating the expression of its regulatory component, miR-513b. Our results indicate a molecular mechanism responsible for the regulation of PCT production. - Procalcitonin (PCT), the precursor of the hormone calcitonin, comprises 116 amino acids and is present in minute quantities under healthy conditions1. The level of PCT has been observed to increase significantly with bacterial infection and correlate to the severity of the infection2. Therefore, serum PCT has been considered a powerful biomarker for the diagnosis of bacterial infection. This marker has also been employed successfully to guide the administration of antibiotics3. Since the discovery of its association with sepsis in the 1990s, many studies on PCT and its clinical applications have been conducted4. However, the molecular details of the regulation of PCT production in relation to bacterial infection remain partially understood. Nuclear transcription factor ?B (NF-?B), an important master transcription factor, is involved in the regulation of numerous components of the host immune response5. NF-?B is naturally located in the cytoplasm bound to its inhibitory proteins, known as inhibitors of NF-?B (I?Bs). Upon stimulation by various activators, one of which is lipopolysaccharide (LPS), the I?B complex is degraded to release NF-?B protein6, and the latter is then allowed to translocate to the nucleus and attach to specific binding site(s) on target genes. As a master transcriptional regulator, NF-?B is responsible for modulating the production of many cellular components, including antimicrobial peptides, cytokines, chemokines, stress-response proteins and anti-apoptotic proteins7. MicroRNAs (miRs) are a group of small, non-coding, single-stranded RNAs that regulate gene expression by base pairing with the untranslated regions (UTRs) of their target genes. Those small molecules are considered the tools for fine tuning the stability of mRNA and consequently affecting the translational outcomes8. The miR-513 subfamily belongs to the miR-506?514 cluster. Evidence shows that different members of the miR-513 subfamily (miR-513a/b/c) lead to functional divergences and that miR-513b can affect male sexual maturation by negatively regulating the development stage-related function of DR19. Recent studies have indicated that miR-513b inhibits cell proliferation in testicular embryonal carcinoma10 and gastric cancer11. Furthermore, miR-513b is decreased in cholangiocytes following Cryptosporidium parvum infection or LPS stimulation and is associated with B7-H1 expression12,13. Collectively, these observations suggest that miR-513b plays an important role in regulating the host inflammatory response. In this study, we conclude that NF-?B signalling activation is necessary for PCT expression in human hepatocytes. On the one hand, NF-?B can directly regulate the production of PCT by binding to its promoter. On the other hand, NF-?B is a negative regulator of miR-513b, which is an inhibitor of PCT expression. Taken together, our results not only indicate that NF-?B is crucial for PCT production in bacterial infection but also provide a detailed mechanism of PCT generation in hepatocytes. Results LPS stimulates PCT expression in hepatocytes. To verify the effect of LPS on PCT expression, we initially measured PCT expression upon stimulation with different concentrations of LPS in HepG2 cells. The level of PCT expression was examined by real-time PCR. The results showed that the level of PCT expression increased upon LPS treatment in a dose-dependent manner. LPS treatment triggered a much more dramatic change in PCT expression when increasing the concentration from 1 ?g/mL to 5 ?g/mL than from 5 ?g/mL to 10 ?g/mL (Fig.?1a). We chose 5 ?g/mL as the LPS concentration for further study. The temporal patterns of PCT produ (...truncated)


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Yongfeng Bai, Jun Lu, Ying Cheng, Feng Zhang, Xueyu Fan, Yuanyuan Weng, Jin Zhu. NF-кB increases LPS-mediated procalcitonin production in human hepatocytes, Scientific Reports, 2018, Issue: 8, DOI: 10.1038/s41598-018-27302-7