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Nov 28, 2011

Liver Regeneration Boosted by Blocking Cell-Specific Serotonin Receptor

  • Blocking activity of the 5-HT2B  serotonin receptor on fibrogenic hepatic stellate cells (HSCs) in the liver may provide a new approach to boosting liver regeneration in injury and disease, scientists suggest. They report on research demonstrating that scar-causing hepatic stellate cells (HSCs) in the liver are negative regulators of hepatocyte regeneration, and that this negative regulatory activity requires stimulation of the 5-hydroxytryptamine 2B receptor (5-HT2B) on HSCs by serotonin.

    The studies, which were led by the Newcastle University’s Institute of Cellular Medicine, showed that selective antagonism of 5-HT2B on HSCs enhanced hepatocyte growth in rodent models of acute and chronic liver injury. Similar effects were also seen in mice lacking 5-HT2B. The findings are reported in Nature Medicine in a paper titled “Stimulating healthy tissue regeneration by targeting the 5-HT2B receptor in chronic liver disease.”

    Liver disease is characterized by reduced hepatocyte regeneration, which is accompanied by fibrogenesis and the development of liver cirrhosis and cancer, the authors explain. Unfortunately, the complexity of pathways that regulate hepatocyte proliferation, including the contribution of fibrogenic HSCs, is not well understood.

    What has been shown, however, is that in the diseased liver HSCs transdifferentiate into activated myofibroblasts that drive fibrogenesis and secrete soluble factors such as hepatocyte growth factor, TGF-β1, and interleukin-6 (IL-6), which might impact on hepatocyte proliferation.

    To investigate the role of HSCs in hepatocyte regeneration the team first evaluated the effects of triggering selective apoptosis-mediated  depletion of HSCs on hepatocyte proliferation in bile duct-ligated  (BDL) mice, a well-established rodent model of extrahepatic cholestasis. To effect selective apoptosis, HSCs were targeted using a single-chain antibody, C1-3, conjugated to gliotoxin. This mycotoxin is specific to synaptophysin, an antigen expressed on myofibroblasts positive for α-smooth muscle actin (α-SMA+ myofibroblasts), which are specifically derived from the transdifferentation of HSCs.

    Treatment of BDL mice using C1–3 gliotoxin resulted in marked but not complete, deletion of hepatic α-SMA+ cells in mice ( α-SMA+ myofibroblasts derived from other cell types weren’t affected) and the stimulation of hepatocyte proliferation. Importantly, there was no accompanying change in the expression of the hedgehog target gene Gli2, which indicated that stimulation of hepatocyte growth after HSC depletion wasn’t due to activation of the hedgehog pathway, the authors note.

    The team’s previous work had identified functional 5-HT2B serotonin receptors on activated HSCs in liver disease. Hence, they next looked at whether paracrine signaling between HSCs and hepatocytes might explain the antiregenerative properties of HSCs. To investigate the influence of 5-HT2B on hepatocyte regeneration during liver injury the researchers used a drug called SB-204741, which is a highly specific 5-HT2B antagonist but has negligible effects on the 5-HT2A and 5-HT2C receptor subtypes.

    Studies in the experimental mice showed that administration of SB-204741 stimulated hepatocyte proliferation in progressive BDL-induced liver injury and in liver damage induced by acute carbon tetrachloride (CCl4) administration. These results indicated a specific antiregenerative role for 5-HT2B signal, a notion supported by studies in 5-HT2B knockout mice (Htr2b-/-).

    Partial hepatectomy (PHX) in these knockout animals led to elevated hepatocyte proliferation.  Levels of IL-6 and TNF-α, which are primers of hepatocyte regeneration and expressed transiently shortly after surgery, were modestly increased  in 5-HT2B knockout mice at four hours of PHX. Crucially, though, the production of TGF-β1, which is induced in the end stage of liver regeneration and acts to repress hepatocyte proliferation, was evident in the livers of wild-type mice at 36 hours after PHX but not in the livers of 5-HT2B knockout animals.

    Interestingly, the investigators report, the liver-to-bodyweight ratios of wild-type mice treated using SB-204741 increased after PHX, indicating that selective antagonism of 5-HT2B results in sustained stimulation of liver regeneration.

    5-HT2B is expressed on HSCs in diseased liver but at lower levels on cholangiocytes and Kupffer cells, the researchers continue. Studies indicated that 5-HT2B was induced in HSCs after PHX, but its expression was reduced in hepatocytes. Treatment with C1-3-gliotoxin increased hepatocyte proliferation after PHX, and this was associated with reduced hepatic expression of TGF-β1.

    Thus far, it appeared that HSC depletion and 5-HT2B antagonism had similar effects on hepatocyte proliferation in models of liver damage, but what wasn’t known was whether they acted through independent mechanisms. If so, then additive effects should be observed when combining HSC depletion with 5-HT2B antagonism. However, while the combination of HSC depletion and SB-204741 treatment in CCl4-injured mice enhanced hepatocyte proliferation and inhibited TGF-β1 expression, there were no additive effects.    

    The investigators next designed chromatin immunoprecipitation studies to identify the intracellular signaling pathways through which serotonin and 5-HT2B exert their antiregenerative effects. They found that either antagonism of 5-HT2B or treatment using the ERK inhibitor PD98059 suppressed serotonin-induced recruitment of JunD, which is one half of the heterodimer making up the AP-1 transcription factor that controls transcription of TGFβ1. Likewise serotonin-induced phosphorylation of JunD was also suppressed on administration of PD98059 or SB-204741.

    “On the basis of these data, we propose that phosphorylation and activation of JunD by ERK mediates the stimulation of TGF-β1 transcription by serotonin and 5-HT2B in HSCs,” the authors conclude. “If this pathway operates in the context of the injured liver, then JunD would be predicted to function as a transcriptional repressor of hepatocyte proliferation.”

    In confirmation of this, the team found that compared with wild-type mice, JunD-knockout mice recovering from CCl4 injury demonstrated higher numbers of mitotic hepatocytes, which was associated with reduced TGF-β1 expression.

    The researchers finally moved on to evaluate whether 5-HT2B antagonism would have an antifibrogenic effect in mouse models of progressive liver disease in which both fibrogenesis and regeneration result in remodeling of the hepatic architecture. Experimental animals were given CCl4 injections over three weeks to establish fibrotic disease, and then CCl4 treatment was continued either with or without treatment with SB-204741.

    “Treatment with SB-204741 significantly reduced the number of hepatic α-SMA+ fibrogenic cells, fibrotic matrix, hepatic expression of TGF-β1, and expression of the fibrogenic genes encoding TIMP-1 and pro-collagen I, confirming an antifibrogenic effect at the molecular level,” they write.

    Moreover, SB-20741 administration was linked with a higher rate of cellular apoptosis in the fibrotic matrix. Administration of SB-20471 in the model of progressive BDL-induced liver disease also resulted in a protective antifibrotic effect as well as improvements in liver function.

    The overall results demonstrate that the negative regulation of hepatocyte regeneration by HSCs in the liver requires stimulation of the 5-HT2B receptor on HSCs by serotonin, which activates expression of TGF-β1 through signaling by ERK1 and the transcription factor JunD, the authors conclude.

    “5-HT2B is selectively expressed by activated human HSCs and the signaling pathway we describe here is conserved in human HSCs. Potent and selective antagonists of 5-HT2B are already available and have been reported as being safe for clinical use in humans. This class of drug may therefore have therapeutic potential in liver disease, both as stimulants of hepatocyte regeneration and as anti-fibrotic agents.”


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