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.
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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)