Fibroblast growth factor (FGF) 19 is a member of the FGF15/19 subfamily of FGFs that includes FGF15/19, FGF21, and FGF23. FGF19 has been shown to have profound effects on liver metabolism and regeneration. FGF19 binds to FGFR4 and its coreceptor β-Klotho to activate intracellular kinases, including Erk1/2. Studies have shown that proinflammatory cytokines such as TNFα impair FGF21 signaling in adipose cells by repressing β-Klotho expression. However, little is known about the effects of inflammation on the FGF19 pathway in the liver. In the present study, we found that lipopolysaccharide (LPS) inhibited β-Klotho and Fgfr4 expression in livers in mice, whereas LPS had no effects on the two FGF19 receptors in Huh-7 and HepG2 cells. Of the three inflammatory cytokines TNFα, IL-1β, and IL-6, IL-1β drastically inhibited β-Klotho expression, whereas TNFα and IL-6 had no or minor effects. None of the three cytokines had any effects on FGFR4 expression. IL-1β directly inhibited β-Klotho transcription, and this inhibition required both the JNK and NF-κB pathways. In addition, IL-1β inhibited FGF19-induced Erk1/2 activation and cell proliferation. These results suggest that inflammation and IL-1β play an important role in regulating FGF19 signaling and function in the liver.
- fibroblast growth factor 19
the mammalian fibroblast growth factor (FGF) family consists of 22 members. Most FGFs function in an autocrine or paracrine fashion to regulate cell growth, differentiation, and morphogenesis. The FGF19 subfamily comprises FGF19/FGF15, FGF21, and FGF23. FGF15 is a mouse ortholog of human FGF19, but they show different tissue distributions, with FGF19 being expressed in the human liver and intestine and FGF15 being expressed only in the mouse intestine (15). FGF19 family members have weak affinity toward heparan sulfate, thus allowing them to escape from the extracellular matrix into circulation. In addition, the intramolecular disulfide bonds formed in the FGF19 subfamily members may increase their stability in plasma and allow them to function as endocrine hormones (12, 13, 19).
Unlike the canonical paracrine-acting FGFs, which use heparin or heparan sulfate to achieve high-affinity receptor binding and signal transduction, the FGF19 subfamily members require Klotho proteins (Klotho and β-Klotho) as coreceptors to facilitate their interactions with FGFRs, activate FGF receptor (FGFR) substrate-2α, and induce Erk1/2 phosphorylation. Klotho is the coreceptor for FGF23 (22, 34), and β-Klotho is the coreceptor for FGF19 and FGF21 (21, 23, 38). Both FGF19 and FGF21 can signal through FGFR1c, -2c, and -3c in the presence of β-Klotho, but only FGF19 effectively signals through FGFR4 bound by β-Klotho, whereas FGF21 does not activate FGFR4. FGFR4 is widely distributed in mouse tissues, with the highest expression found in liver. β-Klotho is most highly expressed in adipose, liver, and pancreas. Liver is the only organ where both β-Klotho and FGFR4 are highly expressed. This unique expression pattern allows FGF19 to act primarily on the liver (21, 23, 32, 39).
FGF19 is expressed abundantly in the distal small intestine (5, 15, 43). It is now well established that FGF19 is an important player in postprandial gut-liver communications. After a meal, bile acids are released into the intestine, where bile acids activate farnesoid X receptor and thereby induce FGF19 expression (14, 15, 25). FGF19 exerts negative feedback on hepatocytes by inhibiting cholesterol 7α-hydroxylase (CYP7A1) transcription and bile acid biosynthesis (14, 15). FGF19 also suppresses insulin-induced fatty acid synthesis in hepatocytes (3), increases metabolic rate and decreases glucose and triglycerides in diabetic mice (10, 33), and stimulates protein and glycogen synthesis in liver cells (18, 19).
In addition to its role in liver metabolism, FGF19 was found to induce hepatocyte proliferation (26, 35, 39), an effect not seen with FGF21 (39). In line with its mitogenic activity, FGF19 was shown to be a mediator of liver regeneration (20, 35) and an associated factor with hepatocellular carcinoma (1, 26, 28, 30, 31).
Despite the functional importance of hepatic FGF19 signaling in metabolic homeostasis and hepatocyte proliferation, little is known as to how the FGF19 receptor FGFR4 and its coreceptor β-Klotho are regulated in the liver. A recent study demonstrated that β-Klotho expression and FGF19 signaling in the liver are inhibited by miR-34a, a microRNA whose expression in the liver is elevated in human patients with fatty liver and in obese mice (11). β-Klotho expression is also suppressed by TNFα in adipose cells, leading to reduction of FGF21-mediated glucose transporter 1 expression and glucose uptake (7). In the present study, we show that IL-1β inhibited β-Klotho expression in Huh-7 and HepG2 hepatoma cells and in mouse livers. IL-1β inhibited Erk1/2 phosphorylation and cell proliferation induced by FGF19. Our results are consistent with previous findings that IL-1β inhibited liver regeneration (4, 6, 27, 36) and may provide a novel mechanism underlying IL-1β's inhibitory effects on liver regeneration.
MATERIALS AND METHODS
Male C57BL/6 mice at 6–7 wk of age were used for LPS or IL-1β administration. Mice at similar weights received an intraperitoneal (ip) injection of LPS (E. coli O127:B8; Sigma-Aldrich) at a dose of 2.5 μg/g body wt or recombinant mouse IL-1β (R & D Systems) at a dose of 5 ng/g body wt. Control mice were injected with the same amounts of PBS. Mice were euthanized by cervical dislocation at different time points as indicated. Livers were dissected, snap-frozen in liquid nitrogen, and stored at −80°C for preparation of proteins and total RNA.
Mice were housed in standard specific pathogen-free mouse rooms in The Chinese University of Hong Kong Laboratory Animal Services Center. China poplar sawdust was used for bedding. Food (18% protein extruded diet, 2018SX; Harlan) and water were supplied ad libitum. A light-dark cycle of 6 AM to 6 PM was used. All of the procedures were performed in accordance with The Chinese University of Hong Kong animal care regulations.
Human hepatoma cell lines Huh-7 and HepG2 were cultured in DMEM and MEM α-medium (Invitrogen), respectively, supplemented with 10% FBS. Cells were starved in serum-free medium for 12 h before the cells were treated with lipopolysaccharides (LPS; E. Coli O127:B8; Sigma-Aldrich), IL-6 (R & D Systems), TNFα (R & D Systems), IL-1β (R & D Systems), actinomycin D (Sigma-Aldrich), cycloheximide (Sigma-Aldrich), the NF-κB inhibitors 481406 (Calbiochem) and RO 106-9920 (Santa Cruz Biotechnology), the JNK inhibitors SP-600125 (Calbiochem) and CEP-1347 (Tocris Biosciences), FGF19 (R & D Systems), and FGF21 (R & D Systems) as indicated.
Cell proliferation and MTT assay.
HepG2 cells were seeded into 96-well plates at a density of 1 × 104 cells/well. Twenty-four hours later, cells were starved in serum-free medium for 12 h, followed by incubation with or without IL-1β (1 ng/ml) for 8 h. Cells were then treated with FGF19 (50 ng/ml) in the presence or absence of IL-1β for 24 h before the cells were subjected to CyQUANT Cell proliferation assay according to the manufacturer's instructions (Invitrogen) or MTT assay, as described previously (24).
RNA isolation and real-time PCR analysis.
Total RNA was isolated from Huh-7 and HepG2 cells and mouse livers using the Pure Link RNA mini kit (Ambion). First-strand cDNA synthesis was performed using the PrimeScript RT reagent Kit (TAKARA) and amplified using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems) with specific primers for human and mouse β-Klotho, FGFR4, hepcidin, and ribosomal protein L19 (RPL19). Primers were as follows: human β-Klotho, 5′-TTCTGGGGTATTGGGACTGGA-3′ (forward) and 5′-CCATTCGTGCTGCTGACATTTT-3′ (reverse); mouse β-Klotho: 5′-TGTTCTGCTGCGAGCTGTTAC-3′ (forward) and 5′-CCGGACTCACGTACTGTTTTT-3′ (reverse); mouse Fgfr4: 5′-TTGGCCCTGTTGAGCATCTTT-3′(forward) and 5′-GCCCTCTTTGTACCAGTGACG-3′ (reverse). Human FGFR4, hepcidin and 78-kDa glucose-regulated protein (GRP78), and human and mouse RPL19 were amplified using the previously described primers (16, 29, 41, 42). RPL19 was used as the internal control.
Huh-7 and HepG2 cells and liver samples were lysed as described previously (42). Briefly, the samples were lysed in TBS (50 mM Tris·HCl, 150 mM NaCl, and 1% Triton X-100, pH 7.4) containing protease inhibitor mixture (Pierce Biotechnology) and phosphatase inhibitor mixture (Pierce Biotechnology) for 30 min on ice. After centrifugation for 10 min at 4°C, the supernatant was assayed for protein concentration by colorimetric assay (BCA kit; Pierce). The lysates were subjected to Western blotting analysis under reducing conditions using previously validated mouse and human β-Klotho antibodies (R & D Systems; also see Ref. 11), anti-phospho-Erk1/2, total Erk1/2, phospho-NF-κB p65, total NF-κB p65, phospho-JNK antibodies (Cell Signaling Technology), and anti-β-actin (Sigma-Aldrich) as indicated.
All data are represented as means ± SD of independent replicates (n ≥ 3). Student's t-test was applied for statistical analysis. A P value of ≤0.05 was considered statistically significant. One-way ANOVA with Newman-Keuls posttest was also performed.
LPS inhibited β-Klotho and Fgfr4 expression in livers in mice.
To investigate the effects of inflammation on hepatic β-Klotho and Fgfr4 expression, we used the LPS-induced inflammation mouse model. Male mice at 6–7 wk of age were injected with or without LPS (2.5 μg/g body wt ip), and liver samples were collected at 3, 6, 9, 12, and 24 h after the LPS injection. As shown in Fig. 1A, β-Klotho mRNA levels were significantly reduced (45% of controls) 3 h after LPS treatment and reached the lowest levels (6% of controls) 9 h after LPS treatment. β-Klotho mRNA levels were still significantly reduced by 21% 24 h after LPS treatment (Fig. 1A). β-Klotho protein expression was inhibited from 6 to 24 h after LPS treatment, with the maximum inhibition seen at 12 h after LPS treatment (Fig. 1B). Fgfr4 mRNA levels were significantly reduced compared with the controls from 3 to 12 h after LPS treatment (Fig. 1C). In addition, β-Klotho and Fgfr4 expression were also inhibited by LPS in young male mice at 11–12 days of age (data not shown).
LPS had no effect on β-Klotho and FGFR4 expression in Huh-7 and HepG2 cells.
To determine whether LPS directly regulates β-Klotho and FGFR4, we incubated Huh-7 cells, a widely used human hepatoma cell line, with increasing doses of LPS for 8 h. LPS at concentrations of ≤50 ng/ml did not alter β-Klotho or FGFR4 expression in Huh-7 cells (Fig. 2, A and B). As a positive control (16), LPS inhibited expression of 78-kDa glucose-regulated protein (GRP78) (Fig. 2C). Consistent with a previous report (8), LPS had no effect on expression of hepcidin (Fig. 2D), a key iron regulatory hormone. In HepG2 cells, another widely used human hepatoma cell line, LPS did not alter β-Klotho or FGFR4 expression either (data not shown). These results suggest that LPS does not directly regulate β-Klotho or FGFR4 in hepatocytes.
IL-1β inhibited β-Klotho but not FGFR4 expression in Huh-7 and HepG2 cells.
LPS induces a massive release of inflammatory cytokines, including IL-6, TNFα, and IL-1β in the infected host (17). To identify the factors that mediate LPS action on hepatic β-Klotho and FGFR4 expression, we examined whether IL-6, TNFα, and IL-1β regulate β-Klotho and FGFR4 expression in Huh-7 cells. We treated Huh-7 cells with increasing concentrations of TNFα, IL-1β, or IL-6 for 8 h. We found that IL-1β suppressed β-Klotho mRNA expression in a dose-dependent manner. IL-6 and TNFα had no (IL-6) or little (TNFα) effect on β-Klotho expression (Fig. 3A), whereas the same IL-6 and TNFα preparations dramatically stimulated suppressor of cytokine signaling 3 (SOCS3) and inhibited growth hormone receptor (GHR), respectively (Fig. 3C) (44). All the three cytokines with concentrations of ≤7.5 nM had no effect on FGFR4 mRNA expression (Fig. 3B).
We next performed a time course of 0.5, 1, 2, 3, 4, 8, and 24 h for IL-1β treatments (1 ng/ml) in Huh-7 cells. β-Klotho mRNA remained unchanged at 0.5 and 1 h, but it started to decrease at 2 h and reached the lowest level with 65% reduction compared with the control at 8 h after the IL-1β treatment (Fig. 3D). We further used HepG2 cells to confirm the inhibitory effects of IL-1β on β-Klotho expression. As seen in Huh-7 cells, IL-1β inhibited β-Klotho mRNA expression in a time-dependent manner in HepG2 cells (Fig. 3E).
To examine the effect of IL-1β on β-Klotho protein expression in liver cells, we incubated Huh-7 and HepG2 cells with IL-1β for 1, 4, and 8 h and then analyzed β-Klotho protein expression. IL-1β treatment inhibited β-Klotho protein expression at 4 and 8 h after IL-1β treatment in both Huh-7 (Fig. 3F) and HepG2 cells (Fig. 3G).
IL-1β inhibited β-Klotho in livers in mice.
To investigate the effects of IL-1β on hepatic β-Klotho expression in vivo, we injected male mice at 6–7 wk of age with IL-1β (5 ng/g body wt ip). Liver samples were collected at 1, 4, and 8 h after the IL-1β injections. β-Klotho mRNA in livers was decreased compared with the controls at all the three time points (Fig. 4A). β-Klotho protein did not change at 1 h but was significantly decreased at 4 and 8 h after IL-1β treatments (Fig. 4B). As seen in Huh-7 and HepG2 cells, IL-1β treatment did not alter Fgfr4 mRNA expression in livers in mice (Fig. 4C). Taken together, our results suggest that IL-1β inhibits hepatic β-Klotho expression but does not regulate hepatic FGFR4 expression either in vitro or in vivo.
IL-1β signaling directly inhibited β-Klotho mRNA expression.
To investigate whether IL-1β decreases β-Klotho mRNA levels by inhibiting β-Klotho at the transcriptional level, Huh-7 and HepG2 cells were pretreated with actinomycin D (5 μg/ml), an inhibitor of gene transcription, before the cells were incubated with IL-1β. Pretreatment with actinomycin D abolished IL-1β's ability to inhibit β-Klotho mRNA expression in both cell lines (Fig. 5, A and B). These results suggest that IL-1β inhibits β-Klotho expression at the transcriptional level.
We then examined whether the action of IL-1β on β-Klotho mRNA expression is a direct regulation or is mediated by other factors whose expression is regulated by IL-1β. Huh-7 and HepG2 cells were pretreated with cycloheximide (10 μg/ml), an inhibitor of new protein synthesis, before the cells were incubated with IL-1β. As shown in Fig. 5, C and D, IL-1β still suppressed β-Klotho mRNA expression after pretreatment with cycloheximide. These results suggest that the inhibitory effect of IL-1β on β-Klotho mRNA expression is independent of de novo protein synthesis.
Both the NF-κB and JNK pathways were required for the inhibition of β-Klotho expression by IL-1β.
By interacting with IL-1 receptor, IL-1β activates the NF-κB pathway and JNK pathway to regulate expression of target genes (37). We first evaluated whether the NF-κB pathway participates in IL-1β-induced downregulation of β-Klotho. We used the NF-κB inhibitor 481406, which inhibits NF-κB transcriptional activity. Huh-7 cells were pretreated with 1 μM 481406 before the cells were treated with IL-1β. As shown in Fig. 6A, the inhibitory effect of IL-1β on β-Klotho expression was slightly attenuated by the NF-κB inhibitor 481406 in Huh-7 cells. As a control, inhibition of NF-κB activity dramatically reduced the stimulation of hepcidin expression by IL-1β (Fig. 6B). We also used another NF-κB inhibitor, RO 106-9920, which blocks IκBα degradation, thus inhibiting the NF-κB pathway. As with 481406, RO 106-9920 (5 μM) did not affect the inhibitory effect of IL-1β on β-Klotho expression (Fig. 6C), whereas it inhibited NF-κB p65 phosphorylation (Fig. 6D) and attenuated the stimulation of hepcidin expression by IL-1β (Fig. 6E).
In HepG2 cells, inhibition of NF-κB activity had no effect on the inhibition of β-Klotho expression by IL-1β either (Fig. 7A). This suggests the NF-κB pathway alone has no major effect on IL-1β-induced inhibition of β-Klotho.
We then examined whether the JNK pathway mediates IL-1β-induced suppression on β-Klotho expression. Huh-7 cells were pretreated with the JNK inhibitors SP-600125 (10 μM) or CEP-1347 (500 nM) before the cells were treated with IL-1β. As shown in Fig. 6, F and H, IL-1β inhibited β-Klotho mRNA expression to similar extents in the presence or absence of SP-600125 or CEP-1347, although the two inhibitors dramatically inhibited IL-1β-induced JNK phosphorylation (Fig. 6, G and I). Similar results were seen in HepG2 cells (Fig. 7B). These results suggest that the JNK pathway alone has no effect on IL-1β's action on β-Klotho.
To determine the effects of inhibition of both NF-κB and JNK pathways on IL-1β activity, Huh-7 cells were pretreated with a combination of 481406 (1 μM) and SP-600125 (10 μM) before the cells were treated with IL-1β. Simultaneous inhibition of both the NF-κB and JNK pathways abolished the ability of IL-1β to inhibit β-Klotho expression in Huh-7 cells (Fig. 6J). In HepG2 cells, the inhibition of β-Klotho by IL-1β was much less in the presence of the two inhibitors than in the absence of the two inhibitors (Fig. 7C). These results suggest that the NF-κB and JNK pathways play a permissive role for each other in mediating IL-1β's action on β-Klotho.
IL-1β inhibited FGF19 signaling.
To examine whether the inhibitory effect of IL-1β on β-Klotho affects FGF19 and FGF21 signaling, we first examined whether HepG2 cells respond to FGF19 and FGF21 stimulation. As shown in Fig. 8, A and B, FGF19 robustly stimulated Erk1/2 phosphorylation in a dose- and time-dependent manner. Consistent with previous observations (21, 23, 32, 38), FGF21 had much smaller effects on Erk1/2 phosphorylation.
We then examined the effects of IL-1β on FGF19 signaling. We incubated HepG2 cells with or without IL-1β for 8 h before the cells were stimulated with or without FGF19 at 50 ng/ml for 30 min. IL-1β at 1 ng/ml slightly increased Erk1/2 phosphorylation (Fig. 8, C and D), but it dramatically inhibited β-Klotho protein expression (Fig. 8, C, D, and F). In the absence of IL-1β, FGF19 induced a 4.7-fold increase in Erk1/2 phosphorylation levels. Cotreatment with IL-1β significantly attenuated Erk1/2 phosphorylation induced by FGF19, with the induction by FGF19 being decreased to 2.1-fold (Fig. 8, D and E). These results suggest that IL-1β inhibits FGF19 signaling.
IL-1β inhibited FGF19-indued cell proliferation.
We determined whether the effects of IL-1β on β-Klotho expression and FGF19 signaling are translated into changes in FGF19 functions. HepG2 cells were pretreated with IL-β before they were treated with and without FGF19. Similar to its role in primary hepatocytes (35), FGF19 promoted cell proliferation in HepG2 cells as determined by CyQUANT Cell proliferation assay and MTT assay. IL-β had no effects on cell proliferation by itself, but it significantly attenuated the stimulation of cell proliferation by FGF19 (Fig. 9A).
We measured several cell cycle-related genes. FGF19 stimulated cyclin A2 and cyclin B1 expression, and these stimulatory effects were blocked by IL-β. FGF19 did not significantly affect the expression of cyclin D1, cyclin E1, or P21 (Fig. 9B).
In the present study, we observed that LPS inhibited expression of β-Klotho and FGFR4, the two key receptors for FGF19 signaling, in livers in both young and adult mice. However, LPS did not have any effects on β-Klotho and FGFR4 expression in Huh-7 and HepG2 cells. Therefore, the downregulation of β-Klotho and FGFR4 in livers is not likely to be a direct action of LPS on liver cells. Since LPS induces the release of inflammatory cytokines into the circulation in infected hosts, we investigated the roles of TNFα, IL-1β, and IL-6 in the expression of β-Klotho and FGFR4 in liver cells in vitro. We found that IL-1β, but not TNFα or IL-6, drastically inhibited expression of β-Klotho in Huh-7 cells. IL-1β also inhibited β-Klotho expression in HepG2 cells and in mouse livers. These results suggest that IL-1β may be at least one of the factors that mediates LPS's action on β-Klotho in vivo. Whether there are any other inflammatory cytokines and chemokines induced by LPS that also inhibit β-Klotho expression in the liver remains unknown. Nevertheless, our results demonstrate for the first time that IL-1β and inflammation inhibit β-Klotho expression in the liver. Interestingly, none of the three cytokines had any effect on FGFR4 expression in Huh-7 cells, and IL-1β did not affect hepatic FGFR4 expression in mice. Therefore, how LPS regulates FGFR4 in livers remained to be addressed by screening more inflammatory factors.
LPS induces expression of proinflammatory factors in many cell types, including macrophages and monocytes (2). One would expect that LPS should inhibit β-Klotho expression in Huh-7 cells by inducing the expression of inflammatory factors, including IL-1β. However, results by others (8) and us demonstrate that LPS does not induce hepcidin expression in Huh-7 cells. IL-1β, IL-6 and TNFα are potent inducers of hepcidin expression in hepatocytes (9). Failure of LPS to induce hepcidin expression suggests that Huh-7 cells do not express the major proinflammatory factors or increase expression of these factors in response to LPS stimulation.
We also studied the mechanisms involved in IL-1β-induced suppression of β-Klotho expression. We found that, in the presence of actinomycin D, IL-1β was no longer able to inhibit β-Klotho mRNA in Huh-7 and HepG2 cells, whereas in the presence of cycloheximide, IL-1β still inhibited β-Klotho expression. Therefore, it is clear that IL-1β signaling directly inhibits β-Klotho transcription.
Studies have shown that the NF-κB pathway and JNK pathway are the two main pathways that transduce IL-1β signaling (37). It has been shown that the JNK pathway is involved in the inhibition of β-Klotho expression and FGF21 signaling by TNFα in 3T3-L1 adipocytes (7). Unexpectedly, TNFα did not significantly inhibit β-Klotho expression in Huh-7 cells. Moreover, inhibition of the JNK pathway alone did not affect the inhibitory effect of IL-1β on β-Klotho expression in Huh-7 or HepG2 cells. These results suggest that β-Klotho may be regulated by different mechanisms in adipocytes and hepatocytes.
In the 2.5-kb sequence upstream of the transcription start site of the β-Klotho gene, we found multiple potential AP-1 and NF-κB binding sites (data not shown). Interestingly, our results showed that inhibition of the JNK and NF-κB pathways abolished IL-1β's activity. However, single pathway inhibitions did not significantly affect IL-1β's activity. These results indicate that the NF-κB pathway and the JNK pathway can compensate each other in mediating IL-1β's action on β-Klotho. TNFα also activates the NF-κB pathway and JNK pathway, but in the present study it did not have any significant effect on β-Klotho expression in Huh-7 cells. The different roles of IL-1β and TNFα in regulation of β-Klotho expression cannot be explained by the present study, but it is possible that some unknown factors regulated specifically by IL-1β may play a permissive role in allowing the NF-κB and JNK pathways to inhibit β-Klotho expression in hepatocytes.
FGF19 activates the Erk1/2 pathway (21, 23, 38, 39), which mediates the regulation of hepatic bile acid synthesis and cell proliferation by FGF19 (11, 18, 40). Our present study demonstrated that IL-1β inhibited FGF19-induced cell viability in HepG2 cells. IL-1β did not alter cell viability in HepG2 cells in the absence of FGF19. In addition, IL-1β did not induce caspase-3 cleavage (data now shown). These results indicate that IL-1β did not induce apoptosis/cell death in HepG2 cells. Therefore, our data suggest that IL-1β inhibits FGF19-induced cell proliferation. Interestingly, it has been demonstrated that IL-1β inhibits liver regeneration in mice and rats (4, 6, 27, 36). Therefore, whether IL-1β inhibits FGF19's activity in liver regeneration warrants further investigation.
FGF19 plays multiple metabolic roles, including inhibiting bile acid biosynthesis (14, 15) and gluconeogenesis (19) in liver cells. We examined the effects of IL-1β on the inhibition of Cyp7a1, the key gene for bile acid synthesis, and glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, genes required for gluconeogenesis, by FGF19 in Huh-7 and HepG2 cells. We found that IL-1β was much more potent than FGF19 in suppressing these genes, and FGF19's effect was completely overwhelmed by IL-1β (data not shown). Therefore, the role of the suppression of β-Klotho by IL-1β in FGF19's activity in bile acid synthesis and gluconeogenesis cannot be determined. Nevertheless, our results support the notion that inflammation changes the landscape of metabolic signaling pathways.
Taken together, our results demonstrate that inflammation inhibits β-Klotho and FGFR4 expression in livers. IL-1β inhibits β-Klotho expression and FGF19 signaling and function in hepatocytes. IL-1β directly inhibits β-Klotho transcription by activating the NF-κB pathway and JNK pathway.
J. -F. Zhang was supported by the National Natural Science Foundation of China (81201699 and 81372450). H. Zhao was supported by a grant from the Research Grants Council (RGC) of the Hong Kong Special Administrative Region, China (CUHK; 480709). B. Feng was supported by an RGC grant (CUHK; 464613). P. S. Leung was supported by an RGC grant (CUHK; 470413). Y. Xia was supported in part by the startup fund offered by The Chinese University of Hong Kong, an RGC grant (CUHK; 477311), and a CUHK direct grant (4054217).
The authors declare that they have no conflicts of interest, financial or otherwise.
Y.Z. and Y.X. conception and design of research; Y.Z., C.M., Y.W., H.H., and W.L. performed experiments; Y.Z., C.M., Y.W., H.H., W.L., and Y.X. analyzed data; Y.Z. and Y.X. interpreted results of experiments; Y.Z., C.M., and Y.X. prepared figures; Y.Z., J.-F.Z., H.Z., B.F., P.S.L., and Y.X. drafted manuscript; Y.Z., J.-F.Z., H.Z., B.F., P.S.L., and Y.X. edited and revised manuscript; Y.Z., C.M., Y.W., H.H., W.L., J.-F.Z., H.Z., B.F., P.S.L., and Y.X. approved final version of manuscript.
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