A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis

Abstract

Hepatic stellate cells (HSCs) are key cellular components of hepatic wound healing and fibrosis. There is emerging evidence that the fibrogenic function of HSCs may be influenced by neurochemical and neurotrophic factors. This study addresses the potential for the serotonin (5-HT) system to influence HSC biology. Rat and human HSCs express the 5-HT1B, 5-HT1F 5-HT2A 5-HT2B, and 5-HT7 receptors, with expression of 5-HT1B 5-HT2A and 5-HT2B being induced on HSC activation. Induction of 5-HT2A and 5-HT2B was 106+/-39- and 52+/-8.5-fold that of quiescent cells, respectively. 5-HT2B was strongly associated with fibrotic tissue in diseased rat liver. Treatment of HSCs with 5-HT2 antagonists suppressed proliferation and elevated their rate of apoptosis; by contrast 5-HT was protective against nerve growth factor-induced apoptosis. 5-HT synergized with platelet-derived growth factor to stimulate increased HSC proliferation. HSCs were shown to express a functional serotonin transporter and to participate in both active uptake and release of 5-HT. We conclude that HSCs express key regulatory components of the 5-HT system enabling them to store and release 5-HT and to respond to the neurotransmitter in a profibrogenic manner. Antagonists that selectively target the 5-HT class of receptors may be exploited as antifibrotic drugs.

Figure 1

Detection of 5-HT receptor subtype expression in rat HSCs. A–E: Real-time RT-PCR analysis was performed on total mRNA isolated from rat HSCs at regular 24-hour time periods after between 1 and 6 days of culture on plastic dishes. Primers specific to the individual subtypes of the rat 5-HT receptors were used to prime PCR reactions (see Table 1 for list). 5-HT receptor expression levels were normalized to the expression levels of BTF3, which was found not to change significantly with HSC activation. Results shown are for all receptor subtypes that were within the assay detection limits; those receptors absent were not detected. All PCR reactions had efficiencies ≥95%, and melt analysis of the amplified DNA demonstrated the generation of a single DNA species in each reaction (results not shown). Western blotting analysis (F) was also performed on 20 μg of whole-cell protein extracted from day-1 and day-10 rat HSCs. Once transferred to nitrocellulose membranes, the proteins were probed with antibodies specific to β-actin, α-SMA, desmin, 5-HT_2A, and 5-HT_2B receptors. Approximate molecular weights of the visible bands were estimated from prestained protein markers run simultaneously with the whole-cell protein. F: Whole-cell protein extracted from the rat stomach fundus used as a positive control for the 5-HT_2B receptor. G: 5-HT₂ receptor/ligand interaction was visualized using a fluorescently labeled spiperone derivative (10 μmol/L NBD-spiperone) (panel 1), and binding specificity was demonstrated when rat HSCs were incubated with both NBD-spiperone and unlabeled 10 μmol/L spiperone (panel 2). The results shown here are representative of three independent experiments from at least three separate cell preparations.

Figure 2

RT-PCR and Western blotting detection of 5-HT₂ receptor expression in human HSCs. A: RT-PCR analysis was performed on total mRNA isolated from passage 3 human HSCs. Primers specific to the human 5-HT_2A (2A) and 5-HT_2B (2B) receptors were used to prime the PCR reaction. PCR reactions were terminated after a predetermined number of cycles (no greater than 40). Controls performed were as follows: water replacing cDNA (lane W) was run simultaneously with test conditions, PCR was performed for 40 cycles using β-actin primers (B) where cDNA was replaced by mRNA (negative control), and the positive control was detection of β-actin gene expression using 20 cycles of PCR analysis. The approximate size of each amplified fragment was estimated from a 1-kb DNA ladder. Western blotting analysis (C) was also performed on 20 μg of whole-cell protein extracted from passage 3 human HSCs. Once transferred to nitrocellulose membranes, the proteins were probed with antibodies specific to α-SMA, desmin, 5-HT_2A, and 5-HT_2B receptors. Approximate molecular weights of the visible bands were estimated from prestained protein markers run simultaneously with the whole-cell protein. The results shown here are representative of three independent experiments from at least three separate cell preparations.

Figure 3

Induction of rat HSC apoptosis by 5-HT₂ receptor-specific antagonists. A: After treatment with 10 μmol/L spiperone for 24 hours, nuclear condensation and fragmentation was visualized with acridine orange (1 μg/ml) (as indicated by white arrows). B: Cells were treated with indicated compounds (all known to bind members of the 5-HT₂ receptor family) at indicated concentrations for 24 hours. Condensed and fragmented nuclei were then counted, and results were expressed as a percentage of total number of nuclei visible. The concentration of the indicated compound that caused optimal nuclear condensation in part (B) was then incubated with rat HSCs for various time periods as indicated (C). Again, condensed and fragment nuclei were counted and expressed as a percentage of the total number nuclei visible. The specific activity of caspase-3 (D) was then assessed after aHSC treatment with spiperone (100 μmol/L for 24 hours) in the absence or presence of 10 μmol/L 5-HT, Z-VAD-FMK caspase 3 inhibitor (50 μmol/L), and gliotoxin (1.5 μmol/L for 3 hours) in the absence or presence of Z-VAD-FMK (50 μmol/L). The ability of 5-HT to inhibit apoptosis in HSCs initiated by NGF was also investigated (E). Cells were preincubated with various 5-HT concentrations for 30 minutes before the application of 100 ng/ml NGF for 6 hours. Nuclear condensation and fragmentation (as identified by acridine orange) were then assessed and expressed as a percentage of total cell nuclei visible. The results shown here are representative of three independent experiments from three separate cell preparations, and in the case of nuclear condensation and fragmentation, data were generated from three separate fields of view per treatment per data point. ★/★★ indicates SD values (P < 0.05/0.01).

Figure 4

Effects of 5-HT₂ antagonists on activated rat HSC proliferation. Culture-activated rat HSCs (day 10 or greater) were cultured in DMEM plus 0.1% fetal calf serum and 50 mmol/L HEPES for 24 hours before incubation with various 5-HT receptor antagonists methiothepin maleate (A), spiperone (B), ritanserin (C), LY53,857 (D), ketanserin (E), and WAY100635 (F) at the indicated concentrations. Cells were treated for 2 hours before the addition of BrdU to the cell culture medium, and the extent of the BrdU incorporation was determined 16 hours later. Data are expressed relative to control values and are presented as mean values ± SEM for three separate experiments done in triplicate.

Figure 5

Effects of 5-HT proliferation and CTGF gene expression in rat-activated HSCs. Culture-activated rat HSCs (day 10 or greater) were cultured in DMEM plus 0.1% fetal calf serum and 50 mmol/L HEPES for 24 hours before incubation with 5-HT (A) or PDGF-BB (B) (±10 μmol/L 5-HT) at the indicated concentrations. Cells were treated for 2 hours before the addition of BrdU to the cell culture medium, and the extent of the BrdU incorporation was determined 16 hours later. C: Using a single submaximal concentration of PDGF-BB (20 ng/ml), the dose dependency of the effects of 5-HT on PDGF BB-stimulated proliferation was also tested. Cells were deprived of serum for 16 hours before cells were treated with 5-HT (at the indicated concentrations) for 30 minutes. PDGF-BB (10 ng/ml) was then introduced into the culture medium for a further 23.5 hours. After 24 hours, 20 μl of an MTS tetrazolium reagent were added to each well, and the absorbance was read after 2 hours. Results here were corrected for the change in absorbance at 490 nm induced by PDGF-BB alone. Serum-deprived, activated HSCs were also incubated with various concentrations of 5-HT for 3 hours before RNA extraction and first-strand cDNA synthesis. Changes in CTGF RNA expression were monitored after 5-HT incubation (D) using real-time PCR SYBR green technology. Data for all experiments are expressed relative to control values and are presented as mean values ± SEM for three separate experiments done in triplicate. ★ indicates SD values (P < 0.05).

Figure 6

The expression and function of the serotonin transporter in rat HSCs. A: RT-PCR analysis was performed on total mRNA isolated from day-1 (d1) and day-10 (d10) culture-activated rat HSCs. Primers specific to the rat SERT were used to prime the PCR reaction. PCR reactions were terminated after a predetermined number of cycles (no greater than 40). Controls performed were as follows: water replacing cDNA (lane W) was run simultaneously with test conditions, PCR was performed for 40 cycles using β-actin primers (B) where cDNA was replaced by mRNA (negative control), and the positive control was detection of β-actin gene expression using 20 cycles of PCR analysis. The approximate size of each amplified was estimated from a 1-kb DNA ladder. C: Western blotting analysis was also performed on 20 μg of whole-cell protein extracted from d1 and d10 rat HSCs. Once transferred to nitrocellulose membranes, the proteins were probed with rabbit polyclonal antibodies raised against the rat SERT. Approximate molecular weights of the visible bands were estimated from prestained protein markers run simultaneously with the whole-cell protein. The results shown here are representative of three independent experiments from at least three separate cell preparations.

Figure 7

Quantitative determination of 5-HT in rat HSC growth medium by ELISA. Culture-activated rat HSCs were washed twice with DMEM containing 0% FCS and incubated overnight in DMEM containing 0% FCS. Growth medium was again changed, this time to DMEM containing zimelidine (10 μmol/L), which was used to block the uptake of serotonin by rat HSCs. Medium was then harvested at indicated times where 5-HT concentration was determined by ELISA. Data are representative of two experiments done in duplicate.

Figure 8

The expression of 5-HT_2A and 5-HT_2B in 8-week CCl₄-induced fibrotic rat liver. Livers were harvested from rats injected twice weekly with CCl₄ for 8 weeks. Isolated livers were then fixed using paraformaldehyde in PBS for 24 hours. Sections from at least three rat control livers (olive oil alone) and three fibrotic livers were then subjected to immunohistological analysis using antibodies specific to 5-HT_2A (A) and 5-HT_2B (B) receptors. In both cases, panel 1 represents staining observed in fibrotic rat liver, and panel 2 represents staining observed in fibrotic rat liver when antibodies specific to 5-HT_2A or 5-HT_2B receptors were absent from experimental protocol. Panel 3 represents the staining observed in control liver sections (no fibrosis), and panel 4 represents positive control tissue (rat stomach) for 5-HT_2A or 5-HT_2B receptors. Panel 5 (5-HT_2B receptor only) represents a high-power image of the 5-HT_2B receptor staining observed in fibrotic rat liver. Red arrows denote positively stained HSCs; yellow arrows denote stained hepatocytes.

Figure 8

The expression of 5-HT_2A and 5-HT_2B in 8-week CCl₄-induced fibrotic rat liver. Livers were harvested from rats injected twice weekly with CCl₄ for 8 weeks. Isolated livers were then fixed using paraformaldehyde in PBS for 24 hours. Sections from at least three rat control livers (olive oil alone) and three fibrotic livers were then subjected to immunohistological analysis using antibodies specific to 5-HT_2A (A) and 5-HT_2B (B) receptors. In both cases, panel 1 represents staining observed in fibrotic rat liver, and panel 2 represents staining observed in fibrotic rat liver when antibodies specific to 5-HT_2A or 5-HT_2B receptors were absent from experimental protocol. Panel 3 represents the staining observed in control liver sections (no fibrosis), and panel 4 represents positive control tissue (rat stomach) for 5-HT_2A or 5-HT_2B receptors. Panel 5 (5-HT_2B receptor only) represents a high-power image of the 5-HT_2B receptor staining observed in fibrotic rat liver. Red arrows denote positively stained HSCs; yellow arrows denote stained hepatocytes.