The ESCRT-III molecules regulate the apical targeting of bile salt export pump.

BACKGROUND: The bile salt export pump (BSEP) is a pivotal apical/canalicular bile salt transporter in hepatocytes that drives the bile flow. Defects in BSEP function and canalicular expression could lead to a spectrum of cholestatic liver diseases. One prominent manifestation of BSEP-associated cholestasis is the defective canalicular localization and cytoplasmic retention of BSEP. However, the etiology of impaired BSEP targeting to the canalicular membrane is not fully understood. Our goal was to discover what molecule could interact with BSEP and affect its post-Golgi sorting. METHODS: The human BSEP amino acids (a.a.) 491-630 was used as bait to screen a human fetal liver cDNA library through yeast two-hybrid system. We identified a BSEP-interacting candidate and showed the interaction and colocalization in the co-immunoprecipitation in hepatoma cell lines and histological staining in human liver samples. Temperature shift assays were used to study the post-Golgi trafficking of BSEP. We further determine the functional impacts of the BSEP-interacting candidate on BSEP in vitro. A hydrodynamically injected mouse model was established for in vivo characterizing the long-term impacts on BSEP. RESULTS: We identified that charged multivesicular body protein 5 (CHMP5), a molecule of the endosomal protein complex required for transport subcomplex-III (ESCRT-III), interacted and co-localized with BSEP in the subapical compartments (SACs) in developing human livers. Cholestatic BSEP mutations in the CHMP5-interaction region have defects in canalicular targeting and aberrant retention at the SACs. Post-Golgi delivery of BSEP and bile acid secretion were impaired in ESCRT-III perturbation or CHMP5-knockdown hepatic cellular and mouse models. This ESCRT-III-mediated BSEP sorting preceded Rab11A-regulated apical cycling of BSEP. CONCLUSIONS: Our results showed the first example that ESCRT-III is essential for canalicular trafficking of apical membrane proteins, and provide new targets for therapeutic approaches in BSEP associated cholestasis.

Cells responsible for liver mass regeneration in rats with 2-acetylaminofluorene/partial hepatectomy injury.

BACKGROUND: Whether hepatic progenitor cells (HPCs)/oval cells regenerate liver mass upon chronic liver injury is controversial in mice and has not been conclusively proven in humans and rats. In this study, we examined which cell type-hepatocytes or oval cells-mediates liver regeneration in the classic rat 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) injury where AAF reversibly blocks hepatocyte proliferation, thereby inducing oval cell expansion after the regenerative stimulus of PH. METHODS: We employed lineage tracing of dipeptidyl peptidase IV (DPPIV, a hepatocyte canalicular enzyme)-positive hepatocytes by subjecting rats with DPPIV-chimeric livers to AAF/PH, AAF/PH/AAF (continuous AAF after AAF/PH to nonselectively inhibit regenerating hepatocytes), or AAF/PH/retrorsine injury (2-dose retrorsine after AAF/PH to specifically and irreversibly block existing hepatocytes); through these methods, we determined hepatocyte contribution to liver regeneration. To determine the oval cell contribution to hepatocyte regeneration, we performed DPPIV(+) oval cell transplantation combined with AAF/PH injury or AAF/PH/retrorsine injury in DPPIV-deficient rats to track the fate of DPPIV(+) oval cells. RESULTS: DPPIV-chimeric livers demonstrated typical oval cell activation upon AAF/PH injury. After cessation of AAF, DPPIV(+) hepatocytes underwent extensive proliferation to regenerate the liver mass, whereas oval cells underwent hepatocyte differentiation. Upon AAF/PH/AAF injury where hepatocyte proliferation was inhibited by continuous AAF treatment following AAF/PH, oval cells extensively expanded in an undifferentiated state but did not produce hepatocytes. By substituting retrorsine for AAF administration following AAF/PH (AAF/PH/retrorsine), oval cells regenerated large-scale hepatocytes. CONCLUSIONS: Hepatocyte self-replication provides the majority of hepatocyte regeneration, with supplementary contribution from oval cells in rats under AAF/PH injury. Oval cells expand and maintain in an undifferentiated state upon continuously nonselective liver injury, whereas they can significantly regenerate hepatocytes in a noncompetitive environment.

RBMY, a novel inhibitor of glycogen synthase kinase 3ß, increases tumor stemness and predicts poor prognosis of hepatocellular carcinoma.

UNLABELLED: Male predominance of hepatocellular carcinoma (HCC) occurs particularly among young children aged 6-9 years, indicative of a possible role of the Y chromosome-encoded oncogene in addition to an androgenic effect. The discovery of oncogenic activation of RBMY (RNA-binding motif on Y chromosome), which is absent in normal hepatocytes but present in male HCC tissues, sheds light on this issue. Herein, we report on a critical hepatocarcinogenic role of RBMY and its ontogenic origin. During liver development, the Ser/Thr phosphorylated RBMY is expressed in the cytoplasm of human and rodent fetal livers. It is then silenced in mature hepatocytes and restricted to scarce expression in the bile ductular cells. Upon hepatocarcinogenesis, a noteworthy increase of cytoplasmic and nuclear RBMY is observed in HCC tissues; however, only the former is expressed dominantly in hepatic cancer stem cells and correlates significantly to a poor prognosis and decreased survival rate in HCC patients. Cytoplasmic expression of RBMY, which is mediated by binding to nuclear exporter chromosome region maintenance 1 and further enriched upon Wnt-3a stimulation, confers upon tumor cells the traits of cancer stem cell by augmenting self-renewal, chemoresistance, cell-cycle progression, proliferation, and xenograft tumor growth. This is achieved mechanistically through increasing Ser9 phosphorylation-inactivation of glycogen synthase kinase 3ß by RBMY, thereby impeding the glycogen synthase kinase 3ß-dependent degradation of ß-catenin and eventually inducing the nuclear entry of ß-catenin for the transcription of downstream oncogenes. CONCLUSION: RBMY is a novel oncofetal protein that plays a key role in attenuating glycogen synthase kinase 3ß activity, leading to aberrant activation of Wnt/ß-catenin signaling, which facilitates malignant hepatic stemness; because of its absence from normal human tissues except the testis, RBMY represents a feasible therapeutic target for the selective eradication of HCC cells in male patients.

Transplantation speed offers early hepatocyte engraftment in acute liver injured rats: A translational study with clinical implications.

The impact of the rate of intraportal hepatocyte transplantation on early engraftment and repopulation is unclear. The aim of this study was to address this and to improve the engraftment and repopulation efficiencies of hepatocyte transplantation for the treatment of a rat model of acute liver failure in a clinically useful way without preconditioning. Acute hepatic injury was induced into Sprague-Dawley rats with D-galactosamine. Hepatocytes were infused intraportally over a period of 30, 70, or 100 seconds to study early engraftment (2 days) and repopulation (7 days). Three groups had significant differences in hepatocyte engraftment (P = 0.018) and repopulation efficiencies (P = 0.037), and an infusion over a period of 70 seconds produced superior outcomes. After the 70-second infusion, the transplanted cells immediately transmigrated the sinusoidal endothelial layer and rarely accumulated in the portal venules, with liver function improving significantly. The mean first peak pressures, without significant differences, were 14.8 ± 6.5, 17.7 ± 3.7, and 13.6 ± 3.0 mm Hg in the 30-, 70-, and 100-second groups, respectively. Differential hepatocyte transfusion rates contributed to accelerated early engraftment and repopulation in rats with acute liver injury. These proof-of-concept findings are of clinical significance because they are easy to translate into practice.

Contribution of mature hepatocytes to small hepatocyte-like progenitor cells in retrorsine-exposed rats with chimeric livers.

UNLABELLED: The potential lineage relationship between hepatic oval cells, small hepatocyte-like progenitor cells (SHPCs), and hepatocytes in liver regeneration is debated. To test whether mature hepatocytes can give rise to SHPCs, rats with dipeptidyl peptidase IV (DPPIV) chimeric livers, which harbored endogenous DPPIV-deficient hepatocytes and transplanted DPPIV-positive hepatocytes, were subjected to retrorsine treatment followed by partial hepatectomy (PH). DPPIV-positive hepatocytes comprised about half of the DPPIV chimeric liver mass. Tissues from DPPIV chimeric livers after retrorsine/PH treatment showed large numbers of SHPC clusters. None of the SHPC clusters were stained positive for DPPIV in any analyzed samples. Furthermore, serial sections stained for gamma-glutamyl-transpeptidase (GGT, a marker of fetal hepatoblasts) and glucose-6-phosphatase (G6Pase, a marker of mature hepatocytes) showed inverse expression of the two enzymes and a staining pattern consistent with a lineage that begins with GGT(+)/G6Pase(-) to GGT(-)/G6Pase(+) within a single SHPC cluster. Using double immunofluorescence staining for markers specific for hepatic oval cells and hepatocytes in serial sections, oval cell proliferations with CK-19(+)/laminin(+) and OV-6(+)/C/EBP-α(-) were shown to extend from periportal areas into the SPHC clusters, differentiating into hepatic lineage by progressive loss of CK-19/laminin expression and appearance of C/EBP-α expression towards the cluster side. Cells in the epithelial cell adhesion molecule (EpCAM(+)) SHPC clusters showed membranous EpCAM(+)/HNF-4α(+) (hepatocyte nuclear factor-4α) staining and were contiguous to the surrounding cytoplasmic EpCAM(+)/HNF-4α(-) ductular oval cells. Extensive elimination of oval cell response by repeated administration of 4,4'-methylenedianiline (DAPM) to retrorsine-exposed rats impaired the emergence of SHPC clusters. CONCLUSION: These findings highly suggest the hepatic oval cells but not mature hepatocytes as the origin of SHPC clusters in retrorsine-exposed rats.

Hepatocyte transplantation in bile salt export pump-deficient mice: selective growth advantage of donor hepatocytes under bile acid stress.

The bile salt export pump (Bsep) mediates the hepatic excretion of bile acids, and its deficiency causes progressive familial intrahepatic cholestasis. The current study aimed to induce bile acid stress in Bsep(-/-) mice and to test the efficacy of hepatocyte transplantation in this disease model. We fed Bsep(-/-) and wild-type mice cholic acid (CA) or ursodeoxycholic acid (UDCA). Both CA and UDCA caused cholestasis and apoptosis in the Bsep(-/-) mouse liver. Wild-type mice had minimal liver injury and apoptosis when fed CA or UDCA, yet had increased proliferative activity. On the basis of the differential cytotoxicity of bile acids on the livers of wild-type and Bsep(-/-) mice, we transplanted wild-type hepatocytes into the liver of Bsep(-/-) mice fed CA or CA + UDCA. After 1-6 weeks, the donor cell repopulation and canalicular Bsep distribution were documented. An improved repopulation efficiency in the CA + UDCA-supplemented group was found at 2 weeks (4.76 ± 5.93% vs. 1.32 ± 1.48%, P = 0.0026) and at 4-6 weeks (12.09 ± 14.67% vs. 1.55 ± 1.28%, P < 0.001) compared with the CA-supplemented group. Normal-appearing hepatocytes with prominent nuclear staining for FXR were noted in the repopulated donor nodules. After hepatocyte transplantation, biliary total bile acids increased from 24% to 82% of the wild-type levels, among which trihydroxylated bile acids increased from 41% to 79% in the Bsep(-/-) mice. We conclude that bile acid stress triggers differential injury responses in the Bsep(-/-) and wild-type hepatocytes. This strategy changed the balance of the donor-recipient growth capacities and was critical for successful donor repopulation.

Hepatocyte and mesenchymal stem cell co-transplantation in rats with acute liver failure.

Background: Cell therapy is considered a potential alternative to liver transplantation in acute liver failure (ALF). We aimed to evaluate the add-on therapeutic benefit of hepatocyte and mesenchymal stem cell (MSC) cotransplantation over hepatocyte-only transplantations in a rat model of ALF. Methods: ALF was induced by D-galactosamine in Sprague-Dawley rats. Freshly isolated donor hepatocytes were derived from Tg (UBC-emGFP) rats and MSCs were collected from the bone marrow cells of DsRed rats. Donor hepatocytes (1×107/mL) were intraportally transplanted 24 hours after treatment with D-galactosamine over a 70-second interval, and donor MSCs (0.5, 1, or 2×106/0.5 mL) were intraportally transplanted 1 hour after the hepatocyte transplantation was complete. Animals were sacrificed after 7 and 14 days and subjected to donor cell identification, liver histology, serologic testing, and immunohistopathological examination. Results: MSCs were observed in the periportal area, 1 and 2 weeks after transplantation. Transplanted hepatocytes did not actively proliferate when compared to hepatocyte-only transplantation. Morphologically, transplanted MSCs did not appear to differentiate into hepatocytes even 2 weeks after transplantation. Cotransplantation of MSCs was associated with lower macrophage infiltration, and reduced type I collagen, hepatocyte growth factor, tumor necrosis factor-α, and interleukin 10 expression, with similar gene expression profiles for epidermal growth factor and interleukin 6, when compared to hepatocyte-only transplantation. Conclusions: Hepatocyte and MSC cotransplantation is feasible and safe in rat models of ALF. MSCs were found to survive the process and could be located within the periportal niches 2 weeks after treatment, without enhancing transplanted hepatocyte proliferation or differentiating into hepatocytes, while ameliorating the inflammatory response.

Coupling Dichloroacetate Treatment with Curcumin Significantly Enhances Anticancer Potential.

BACKGROUND/AIM: Dichloroacetate (DCA) and curcumin have been shown to be potent drug candidates in cancer therapy. Our study aimed to investigate the combined effects of DCA and essential oil-blended curcumin (ECUR) using the hepatoma Huh-7 cell model. MATERIALS AND METHODS: Muse™ Cell Cycle assay, Muse™ Annexin V & Dead Cell assay, Muse™ Oxidative Stress assay, and western blot analysis were applied to explore the underlying mechanisms. RESULTS: DCA combined with ECUR dramatically augmented inhibition of cell survival and enhanced apoptotic induction. The enhanced apoptosis was accompanied by mitochondria-dependent apoptotic signaling activation and corroborated with significant cellular morphological alternations. CONCLUSION: Apoptosis was the major event contributing to the synergistically boosted antiproliferative effect. Coupling DCA treatment with curcumin may rationally be expected to lower the DCA dose needed and relatively reduce accompanying toxicity and oxidative damage while enhancing anticancer potential. This novel 'add-on' approach is, thus, of enormous value to the current DCA therapy.

Histopathological evidence for the existence of primary liver progenitor cell cancer: insight from cancer stem cell pathobiology.

BACKGROUND: Primary liver progenitor cell cancer is a rare disease entity. Current nomenclature of primary liver cancer with prominent progenitor features is not comprehensive. This study was aimed to investigate the existence of this type of primary liver cancer and characterize it immunohistopathologically based on the emerging understanding of cancer stem cell pathobiology. METHODS: Surgical specimens from a primary liver cancer were stained with antibodies against well-defined markers of progenitor cells, stemness, and differentiation toward hepatocytes or cholangiocytes. Comparative interpretation of images was processed considering the histological morphology and characteristic markers. RESULTS: The primary liver cancer consisted of CD24+ cancer progenitor cells and CD90+ mesenchymal stromal cells, which were intimately mixed. CD24+ cancer cells demonstrated bi-directional trends of differentiation: bile ductule transformation and trabecular or nested cell clusters toward hepatic lineage. Moderate number of CD4+ and CD8+ T cells infiltrated the CD90+ cancer-associated stroma. CONCLUSIONS: We provided the corroboration that liver progenitor cells can form primary liver cancer, not just presented as a few side populations of cancer stem cells. Its existence might have significance for future stem cell therapeutic intervention targeting liver diseases.

Role of matrix metalloproteinase-9 in ex vivo expansion of human limbal epithelial cells cultured on human amniotic membrane.

PURPOSE: To investigate the expression and pivotal role of matrix metalloproteinase (MMP)-9 in the ex vivo expansion of human limbal explants with or without amniotic membrane (AM). METHODS: Corneoscleral buttons were cultured on intact, denuded AM or plastic dishes for 3 weeks. To determine the role of MMP-9 in cell migration, either the MMP inhibitor GM6001 or an MMP-9 antibody was used. Expression of MMP-9 was determined by gelatin zymography, reverse transcription-polymerase chain reaction, and immunohistochemical staining. RESULTS: The expression of MMP-9 in all culture conditions increased in a time-dependent manner. However, the active form of MMP-9 emerged only in cultures on both intact and denuded AM from the second week. The averaged corrected ratio of MMP-9 expression in cultures on intact AM versus those on denuded AM or plastic dishes was 2.76 +/- 0.69- or 4.25 +/- 0.30-fold, respectively, when total RNA was used as an internal control. MMP-9 transcripts were upregulated in cultures on intact AM compared with the other two culture conditions. Immunohistochemical staining demonstrated that the MMP-9 protein was located on the limbal epithelial cells. Upregulation of MMP-9 associated with cell migration was significantly attenuated by both GM6001 and MMP-9 antibody, consistent with the inhibition of MMP-9 activity, as determined by gelatin zymography. In contrast, the sizes of limbal outgrowth were not different between the control and MMP-9 antibody-treated plastic dishes. CONCLUSIONS: These results demonstrated that MMP-9 not only was upregulated, it was also involved in the outgrowth of limbal epithelial cells. These results suggest that cell-cell matrix interaction is involved in the expansion of limbal epithelial cells on intact AM, and MMP-9 may be a key element.

Induction of cyclooxygenase-2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB pathways.

Lipopolysaccharide (LPS) was found to induce inflammatory responses in the airways and exerted as a potent stimulus for PG synthesis. This study was to determine the mechanisms of LPS-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). LPS markedly increased the expression of COX-2 and release of PGE(2) in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Both the expression of COX-2 and the generation of PGE(2) in response to LPS were attenuated by a tyrosine kinase inhibitor genistein, a phosphatidylcholine-phospholipase C inhibitor D609, a phosphatidylinositol-phospholipase C inhibitor U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. Furthermore, LPS-induced NF-kappaB activation correlated with the degradation of IkappaB-alpha, COX-2 expression, and PGE(2) synthesis, was inhibited by transfection with dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. LPS-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK inhibitor), but these two inhibitors had no effect on LPS-induced NF-kappaB activation, indicating that NF-kappaB is activated by LPS independently of activation of p42/p44 MAPK and p38 MAPK pathways in TSMCs. Taken together, these findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from LPS-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways. LPS-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.

Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS, and NF-kappaB pathways in canine tracheal smooth muscle cells.

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB.

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.

Substance P-induced activation of p42/44 mitogen-activated protein kinase associated with cell proliferation in human tracheal smooth muscle cells.

Substance P (SP) released from sensory nerve endings in the airways induces several responses including cell proliferation. However, the mechanisms were not completely understood in tracheal smooth muscle cells (TSMCs). We therefore investigated the effect of SP on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. SP stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Both DNA synthesis and phosphorylation of MAPK in response to SP were attenuated by pretreatment with pertussis toxin, genistein, D609, U73122, staurosporine, removal of Ca(2+) by BAPTA/AM plus EGTA, PD98059, and SB202190. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by SP and PDGF-BB. These results conclude that the mitogenic effect of SP was mediated through the activation of Ras/Raf/MEK/MAPK pathway, which was modulated by PC-PLC, PI-PLC, Ca(2+), and PKC in cultured human TSMCs.

Thrombin-stimulated cell proliferation mediated through activation of Ras/Raf/MEK/MAPK pathway in canine cultured tracheal smooth muscle cells.

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients and shown to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). However, the implication of thrombin in the cell proliferation was not completely understood. In this study, thrombin stimulated [3H]thymidine incorporation and p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C inhibitor GF109203X, removal of Ca2+ by addition of BAPTA/AM plus EGTA, PI 3-kinase inhibitors wortmannin and LY294002, and inhibitor of MEK1/2 PD98059. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca2+, PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in canine cultured TSMCs.

Contribution of Mature Hepatocytes to Biliary Regeneration in Rats with Acute and Chronic Biliary Injury.

Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4'-methylene-dianiline (DAPM) and D-galactosamine (DAPM+D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM+D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1ß (HNF-1ß) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation.

Hepatocyte transplantation and the differentiation fate of host oval cells in acute severe hepatic injury.

Oval cells and hepatocytes rarely proliferate simultaneously. This study aimed to determine the impacts of hepatocyte transplantation on the response and fate of oval cells that are activated to proliferate in acute severe hepatic injury. Retrorsine + D-galactosamine (R+D-gal) treatment was used to induce acute hepatic injury and to elicit extensive activation of oval cells in male dipeptidyl peptidase IV-deficient F344 rats. These rats were then randomized to receive wild-type hepatocyte transplantation or vehicle intraportally. The kinetics of oval cell response and their differentiation fate were analyzed. Results showed that oval cells were activated early and differentiated into hepatocytes in R+D-gal-treated rats without hepatocyte transplantation. With hepatocyte transplantation, the oval cells were recruited later and continued to proliferate in parallel with the massive proliferation of transplanted hepatocytes. They formed ductules and differentiated into biliary cells. When hepatocytes were transplanted at the day when oval cells were at their peak response, the numerous activated oval cells ceased to differentiate into hepatocytes and remained in ductular form. The ductular oval cells were capable of differentiating into hepatocytes again when the donor hepatocytes were inhibited to proliferate. We conclude that hepatocyte transplantation changes the mechanism of liver reconstitution and affects the differentiation fate of host oval cells in acute severe hepatic injury.

Impaired hepatocyte regeneration in acute severe hepatic injury enhances effective repopulation by transplanted hepatocytes.

Efficient repopulation by transplanted hepatocytes in the severely injured liver is essential for their clinical application in the treatment of acute hepatic failure. We studied here whether and how the transplanted hepatocytes are able to efficiently repopulate the toxin-induced acute injured liver. Male dipeptidyl peptidase IV-deficient F344 rats were randomized to receive retrorsine plus D-galactosamine (R+D-gal) treatment or D-galactosamine-alone (D-gal) to induce acute hepatic injury, and retrorsine-alone. In these models, retrorsine was used to inhibit the proliferation of endogenous hepatocytes while D-galactosamine induced acute hepatocyte damage. Wild-type hepatocytes (1 x 10(7)/ml) were transplanted intraportally 24 h after D-galactosamine or saline injection. The kinetics of proliferation and repopulation of transplanted cells and the kinetics of cytokine response, hepatic stellate cell (HSC) activation, and matrix metalloproteinase (MMP2) expression were analyzed. We observed that early entry of transplanted hepatocytes into the hepatic plates and massive repopulation of the liver by transplanted hepatocytes occurred in acute hepatic injury induced by R+D-gal treatment but not by D-gal-alone or retrorsine-alone. The expressions of transforming growth factor-alpha and hepatocyte growth factor genes in the R+D-gal injured liver were significantly upregulated and prolonged up to 4 weeks after hepatocyte transplantation. The expression kinetics were parallel with the efficient proliferation and repopulation of transplanted hepatocytes. HSC was activated rapidly, markedly, and prolongedly up to 4 weeks after hepatocyte transplantation, when the expression of HGF gene and repopulation of transplanted hepatocytes were reduced afterward. Furthermore, the expression kinetics of MMP2 and its specific distribution in the host areas surrounding the expanding clusters of transplanted hepatocytes are consistent with those of activated HSC. Impaired hepatocyte regeneration after acute severe hepatic injury may initiate serial compensatory repair mechanisms that facilitate the extensive repopulation by transplanted hepatocytes that enter early the hepatic plates.

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells.

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.