Boris Julien

The sphingosine 1-phosphate receptor S1P2 triggers hepatic wound healing

Sphingosine 1‐phosphate (S1P) is a bio‐active sphingolipid produced by sphingosine kinase (SphK1 and 2). We previously showed that S1P receptors (S1P1, S1P2, and S1P3) are expressed in hepatic myofibroblasts (hMF), a population of cells that triggers matrix remodeling during liver injury. Here we investigated the function of these receptors in the wound healing response to acute liver injury elicited by carbon tetrachloride, a process that associates hepato‐cyte proliferation and matrix remodeling. Acute liver injury was associated with the induction of S1P2, S1P3, SphK1, and SphK2 mRNAs and increased SphK activ‐ity, with no change in S1P1 expression. Necrosis, inflammation, and hepatocyte regeneration were similar in S1P2−/− and wild‐type (WT) mice. However, compared with WT mice, S1P2−/− mice displayed reduced accumulation of hMF, as shown by lower induction of smooth muscle α‐actin mRNA and lower induction of TIMP‐1, TGF‐β1, and PDGF‐BB mRNAs, overall reflecting reduced activation of remodeling in response to liver injury. The wound healing response was similar in S1P3−/− and WT mice. In vitrO, S1P enhanced proliferation of cultured WT hMF, and PDGF‐BB further enhanced the mitogenic effect of S1P. In keeping with these findings, PDGF‐BB up‐regulated S1P2 and SphK1 mRNAs, increased SphK activity, and S1P2 induced PDGF‐BB mRNA. These effects were blunted in S1P2−/− cells, and S1P2−/− hMF exhibited reduced mitogenic and comitogenic responses to S1P. These results unravel a novel major role of S1P2 in the wound healing response to acute liver injury by a mechanism involving enhanced proliferation of hMF. –Serriere‐Lanneau, V., Teixeira‐Clerc, F., Li, L., Schippers, M., de Wries, W., Julien, B., Tran‐Van‐Nhieu, J., Manin, S., Poelstra, K., Chun, J., Carpentier, S., Levade, T., Mallat, A., Lotersztajn, S. The sphingosine 1‐phosphate receptor S1P2 triggers hepatic wound healing. FASEB J. 21, 2005–2013 (2007)

ATP release after partial hepatectomy regulates liver regeneration in the rat

BACKGROUND & AIMSnParacrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study.nnnMETHODSnHepatic ATP release after hepatectomy was examined in the rat and in human living donors for liver transplantation. Quinacrine was used for in vivo staining of ATP-enriched compartments in rat liver sections and isolated hepatocytes. Rats were treated with an antagonist for purinergic receptors (Phosphate-6-azo(benzene-2,4-disulfonic acid), PPADS), and liver regeneration after hepatectomy was analyzed.nnnRESULTSnA robust and transient ATP release due to acute portal hyperpressure was observed immediately after hepatectomy in rats and humans. Clodronate liposomal pre-treatment partly inhibited ATP release in rats. Quinacrine-stained vesicles, co-labeled with a lysosomal marker in liver sections and isolated hepatocytes, were predominantly detected in periportal areas. These vesicles significantly disappeared after hepatectomy, in parallel with a decrease in liver ATP content. PPADS treatment inhibited hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in bromodeoxyuridine incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early gene induction.nnnCONCLUSIONnExtracellular ATP is released immediately after hepatectomy from hepatocytes and Kupffer cells under mechanical stress and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP is released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration.

Heme oxygenase-1 is an antifibrogenic protein in human hepatic myofibroblasts☆

BACKGROUND & AIMSnHepatic myofibroblasts play a key role in the development of liver fibrosis associated with chronic liver diseases. We have shown that oxidative stress is a messenger of 15-deoxy-delta-12,14-prostaglandin J2 (15-d-PGJ2) in human hepatic myofibroblasts. The aim of the present study was to investigate the role of a stress-inducible protein, heme oxygenase-1 (HO-1), in the action of 15-d-PGJ2.nnnMETHODSnExpression of HO-1 was characterized in biopsy specimens of normal human liver and active cirrhosis by immunohistochemistry, and in cultured human hepatic myofibroblasts by Northern and Western blot analysis. Functional studies also were performed in cultured human hepatic myofibroblasts.nnnRESULTSnImmunohistochemistry showed that in biopsy specimens from normal livers, HO-1 protein expression was restricted to Kupffer cells. Biopsy specimens from cirrhotic patients displayed HO-1 protein both in macrophages and in myofibroblasts within fibrotic septa. HO-1 messenger RNA (mRNA) and protein also were detected in cultured human hepatic myofibroblasts and increased in response to 15-d-PGJ2 in a time- and dose-dependent manner. Induction of HO-1 in human hepatic myofibroblasts mediated 2 major antifibrogenic properties of 15-d-PGJ2, namely, inhibition of proliferation and of procollagen I mRNA expression. These effects were ascribed to bilirubin, one of the products of HO-1-mediated heme degradation.nnnCONCLUSIONSnThis study shows that HO-1 is expressed in human hepatic myofibroblasts and induced during chronic liver injury. Moreover, these data unravel HO-1 as a major antifibrogenic pathway.

Cytosolic calcium regulates liver regeneration in the rat

Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium‐binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two‐thirds PH 4 days after Ad‐PV‐NES‐DsR or Ad‐DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura‐2‐acetoxymethyl ester in hepatocytes isolated from Ad‐infected rats and in Ad‐infected Hela cells. Also, isolated hepatocytes were infected with Ad‐DsR or Ad‐PV‐NES‐DsR and assayed for bromodeoxyuridine incorporation. Ad‐PV‐NES‐DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist‐induced cytosolic calcium oscillations were attenuated in both PV‐NES–expressing Hela cells and hepatocytes, as compared to DsR‐expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV‐NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV‐NES rats exhibited reduced c‐fos induction and delayed extracellular signal‐regulated kinase 1/2 phosphorylation after PH. Finally, primary PV‐NES–expressing hepatocytes exhibited less proliferation and agonist‐induced cyclic adenosine monophosphate responsive element binding and extracellular signal‐regulated kinase 1/2 phosphorylation, as compared with control cells. Conclusion: Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle. (HEPATOLOGY 2010;)

The P2X4 purinergic receptor regulates hepatic myofibroblast activation during liver fibrogenesis

BACKGROUND & AIMSnLiver fibrosis is characterized by the accumulation of extracellular matrix produced by hepatic myofibroblasts (hMF), the activation of which is critical to the fibrogenic process. Extracellular ATP, released by dying or stressed cells, and its purinergic receptors, constitute a powerful signaling network after injury. Although the purinergic receptor P2X4 (P2RX4) is highly expressed in the liver, its functions in hMF had never been investigated during liver fibrogenesis.nnnMETHODSnIn vivo, bile duct ligation was performed and methionine- and choline-deficient diet administered in wild-type and P2x4 knock-out (P2x4-KO) mice. In vitro, hMF were isolated from mouse (wild-type and P2x4-KO) and human liver. P2X4 pharmacological inhibition (in vitro and in vivo) and P2X4 siRNAs (in vitro) were used. Histological, biochemical and cell culture analysis allowed us to study P2X4 expression and its involvement in the regulation of fibrogenic and fibrolytic factors, as well as of hMF activation markers and properties.nnnRESULTSnP2X4 genetic invalidation or pharmacological inhibition protected mice from liver fibrosis and hMF accumulation after bile duct ligation or methionine- and choline-deficient diet. Human and mouse hMFs expressed P2X4, mainly in lysosomes. Invalidation of P2X4 in human and mouse hMFs blunted their activation marker expression and their fibrogenic properties. Finally, we showed that P2X4 regulates calcium entry and lysosomal exocytosis in hMF, impacting on ATP release, profibrogenic secretory profile, and transcription factor activation.nnnCONCLUSIONnP2X4 expression and activation is critical for hMF to sustain their activated and fibrogenic phenotype. Therefore, the inactivation of P2X4 may be of therapeutic interest during liver fibrotic diseases.nnnLAY SUMMARYnDuring chronic injury, the liver often repairs with fibrotic tissue, which impairs liver function, and for which there is currently no treatment. We found that a previously unexplored pathway involving the purinergic receptor P2X4, can modulate fibrotic liver repair. Therefore, this receptor could be of interest in the development of novel therapies for fibrotic liver diseases.

102 Sphingosine-1-phosphate triggers wound healing via the S1P2 receptor following acute liver injury

Sphingosine-l-phosphate (SIP) is a bioactive sphingolipid metabolite of ceramide produced by phosphorylation of sphingosine by sphingosine kinases (SphK) type 1 and 2. We previously identified SIP receptors (S1P1, S1P2 and S1P3) in human hepatic myofibroblasts. In the present study, we investigated the role of SIP receptors in the wound healing response elicited by acute toxic liver injury. Acute liver injury was induced by a single i.p. injection of carbon tetrachloride (CC14) in wild type (WT), S1P2 / and S1P3 / mice. Matrix remodelling was evaluated 72 hrs after CC14 injection by quantification of liver 0~-smooth muscle actin (0~SMA), TGF-[3, PDGE TIMP-1, collagen I and III mRNAs. Culture experiments were conducted in hepatic myofibroblasts isolated from WT and S1P2 / mice. The expression of S1P2, S1P3, SphK1 and SphK2 mRNAs was strongly increased following CC14 treatment, suggesting an involvement of the SIP system in the wound healing response; expression of S1P1 was unaffected by CC14 treatment. Induction of c~SMA mRNA and protein were markedly decreased in S1P2 / mice. In addition, induction of TGF-[3, PDGF-BB and TIMP-1 mRNAs were significantly lower in S1P2 / mice, whereas up-regulation of collagen I and III mRNAs was similar in both groups of animals. In contrast, 0~SMA and TGF-[3 mRNAs were similarly induced in WT and S1P3 / mice. S 1P dose-dependently stimulated DNA synthesis in hepatic myofibroblasts from WT mice and PDGF-BB enhanced the mitogenic effect of SIR In keeping with these findings, PDGF-BB strongly induced the expression of S1P2. In contrast, in hepatic myofibroblasts isolated from S1P2 / mice, there was a marked reduction of the mitogenic and comitogenic effects of SIR These results demonstrate that PDGF-BB potentiates the mitogenic effect of SIP, by a mechanism involving up-regulation of S1P2 expression. In conclusion, S1P2 knock-out mice show a reduced wound healing response, by a mechanism involving reduced proliferation of hepatic myofibroblasts. These results point out a novel role of S1P2 in matrix remodelling and suggest that S1P2 may promote liver fibrogenesis during chronic liver injury.