Relative Selectivity of Covalent Inhibitors Requires Assessment of Inactivation Kinetics and Cellular Occupancy: A Case Study of Ibrutinib and Acalabrutinib.

Kinases form an attractive class of targets for small molecule inhibitors, but similarity among their adenosine triphosphate binding sites presents difficulties for developing selective drugs. Standard methods of evaluating selectivity of most reversibly bound drugs account for binding affinity but not the two-step process, affinity and inactivation, occurring during covalent inhibition. To illustrate this concept, we assessed the selectivity of Bruton's tyrosine kinase (BTK) over TEC kinases by two novel therapeutics: ibrutinib and acalabrutinib. The two-step process and time-dependent inhibition unique to covalent inhibitors were evaluated with two biochemical assays measuring enzymatic function and inhibition kinetics. The selectivity for BTK over TEC found in these biochemical analyses was 1-1.5 for ibrutinib and 3.0-4.2 for acalabrutinib. To further assess drug selectivity in a more physiologically relevant context, we developed cell-based occupancy assays that quantify the percentage of drug-inactivated kinases. Cellular selectivity of BTK over TEC was determined after MWCL-1 cells, and samples from patients with chronic lymphocytic leukemia (CLL) were treated for durations and concentrations based on human pharmacokinetics of each drug. In MWCL-1 cells, BTK/TEC selectivities measured at 0.5, 1, and 3 hours were 2.53, 1.05, and 1.51 for ibrutinib and 0.97, 1.13, and 2.56 for acalabrutinib, respectively. The equivalent selectivity measured in samples from patients with CLL were 1.31 ± 0.27 and 1.09 ± 0.11 for ibrutinib and acalabrutinib, respectively. Collectively, our data show that when properly accounting for time-dependent factors and relevant cellular context, ibrutinib and acalabrutinib demonstrate similar selectivity for BTK over TEC. SIGNIFICANCE STATEMENT: This study shows relative selectivity of covalent inhibitors toward different kinase targets should be assessed with both affinity and inactivation kinetics to accurately account for time-dependent effects of covalent binding and assessed in a cellular matrix to reproduce the physiologic context of target inhibition. This is illustrated with a case study of ibrutinib and acalabrutinib for which selectivity assessment with appropriate assays, as opposed to measuring binding affinity with KINOMEscan alone, corroborate emerging clinical data demonstrating similar safety profiles between the therapies.

The effect of Bruton's tyrosine kinase (BTK) inhibitors on collagen-induced platelet aggregation, BTK, and tyrosine kinase expressed in hepatocellular carcinoma (TEC).

OBJECTIVES: Bruton's tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) are expressed by human platelets. These kinases participate in platelet activation through the collagen receptor glycoprotein VI and may perform overlapping functions. In clinical studies, BTK inhibitors (ibrutinib, acalabrutinib, tirabrutinib, zanubrutinib) have been associated with increased bleeding risk, which may result from inhibition of BTK alone or of both BTK and TEC, although the role of TEC in bleeding risk remains unclear. METHODS: Here, in vitro catalytic and binding activities of ibrutinib and acalabrutinib were determined with four assay systems. Platelet aggregation assays determined inhibitor potency and its relationship to selectivity between BTK and TEC. RESULTS: Neither inhibitor was substantially more selective for BTK over TEC. The potencies at which BTK inhibitors suppressed platelet aggregation correlated with the potencies in on-target BTK assays, including those in cells. At clinically relevant plasma concentration, ibrutinib, acalabrutinib, and tirabrutinib inhibited collagen-induced platelet aggregation to a similar extent, despite differing in vitro IC50 s. CONCLUSIONS: Our results suggest BTK inhibition is the primary driver for inhibition of platelet aggregation. The subtle differences between these inhibitors suggest only randomized, double-blind, placebo-controlled clinical studies can fully address the bleeding risks of different BTK inhibitors.

Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

Targeting IRAK3 for Degradation to Enhance IL-12 Pro-inflammatory Cytokine Production.

Interleukin-1 receptor-associated kinase 3 (IRAK3) is a pseudokinase mediator in the human inflammatory pathway, and ablation of its function is associated with enhanced antitumor immunity. Traditionally, pseudokinases have eluded "druggability" and have not been considered tractable targets in the pharmaceutical industry. Herein we disclose a CRISPR/Cas9-mediated knockout of IRAK3 in monocyte-derived dendritic cells that results in an increase in IL-12 production upon lipopolysaccharide (LPS) stimulation. Furthermore, we disclose and characterize Degradomer D-1, which displays selective proteasomal degradation of IRAK3 and reproduces the 1L-12p40 increases observed in the CRISPR/Cas9 knockout.

Effects of ibrutinib on in vitro platelet aggregation in blood samples from healthy donors and donors with platelet dysfunction.

Background: Ibrutinib, a first-in-class, once-daily inhibitor of Bruton's tyrosine kinase (BTK), is approved in the US and EU for the treatment of various B-cell malignancies. In clinical studies, BTK inhibitors have been associated with increased bleeding risk, which may result from BTK inhibition in platelets.Methods: To better understand the mechanism of ibrutinib in bleeding events, we isolated platelet-rich plasma from healthy donors (n = 8) and donors with conditions associated with impaired platelet function or with potentially increased bleeding risk (on hemodialysis, taking aspirin, or taking warfarin; n = 8 each cohort) and used light transmission aggregometry to assess platelet aggregation in vitro after exposure to escalating concentrations of ibrutinib, spanning and exceeding the pharmacologic range of clinical exposure.Results: Platelet aggregation was induced by agonists of 5 major platelet receptors: adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP6), ristocetin, collagen, or arachidonic acid (AA). Platelet aggregation induced by ADP, TRAP6, ristocetin, and AA was not meaningfully inhibited by the maximal concentrations of ibrutinib (10 µM). In contrast, collagen-induced platelet aggregation was dose-dependently inhibited by ibrutinib in all donor cohorts (maximum aggregation % with 10 µM ibrutinib, -64% to -83% of agonist activity compared to control agonist samples but without ibrutinib).Conclusion: These results confirm prior reports and support a mechanistic role for the inhibition of collagen-induced platelet aggregation in bleeding events among susceptible individuals receiving ibrutinib therapy.

A class of small molecules that inhibit TNFalpha-induced survival and death pathways via prevention of interactions between TNFalphaRI, TRADD, and RIP1.

Small-molecule library screening to find compounds that inhibit TNFalpha-induced, but not interleukin 1beta (IL-1beta)-induced, intercellular adhesion molecule 1 (ICAM-1) expression in lung epithelial cells identified a class of triazoloquinoxalines. These compounds not only inhibited the TNFalpha-induced nuclear factor kappaB (NFkappaB) survival pathway but also blocked death-pathway activation. Such dual activity makes them unique against other known NFkappaB-pathway inhibitors that inhibit only a subset of TNFalpha signals leading to increased TNFalpha-induced cytotoxicity. Interestingly, these compounds inhibited association of TNFalpha receptor (TNFalphaR) I with TNFalphaR-associated death domain protein (TRADD) and receptor interacting protein 1 (RIP1), the initial intracellular signaling event following TNFalpha stimulation. Further study showed that they blocked ligand-dependent internalization of the TNFalpha-TNFalphaR complex, thereby inhibiting most of the TNFalpha-induced cellular responses. Thus, compounds with a triazoloquinoxaline scaffold could be a valuable tool to investigate small molecule-based anti-TNFalpha therapies.

R-253 disrupts microtubule networks in multiple tumor cell lines.

PURPOSE: The design and development of synthetic small molecules to disrupt microtubule dynamics is an attractive therapeutic strategy for anticancer drug discovery research. Loss of clinical efficacy of many useful drugs due to drug resistance in tumor cells seems to be a major hurdle in this endeavor. Thus, a search for new chemical entities that bind tubulin, but neither are a substrate of efflux pump, P-glycoprotein 170/MDR1, nor cause undesired side effects, would potentially increase the therapeutic index in certain cancer treatments. EXPERIMENTAL DESIGN: A high-content cell-based screen of a compound library led to the identification of a new class of compounds belonging to a thienopyrimidine series, which exhibited significant antitumor activities. On structure-activity relationship analysis, R-253 [N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent antiproliferative agent (average EC(50), 20 nmol/L) when examined in a spectrum of tumor cell lines. RESULTS: R-253 is structurally unique and destabilizes microtubules both in vivo and in vitro. Standard fluorescence-activated cell sorting and Western analyses revealed that the effect of R-253 on cell growth was associated with cell cycle arrest in mitosis, increased select G(2)-M checkpoint proteins, and apoptosis. On-target activity of R-253 on microtubules was further substantiated by immunofluorescence studies and selected counter assays. R-253 competed with fluorescent-labeled colchicine for binding to tubulin, indicating that its binding site on tubulin could be similar to that of colchicine. R-253 neither is a substrate of P-glycoprotein 170/MDR1 nor is cytotoxic to nondividing human hepatocytes. CONCLUSION: Both biochemical and cellular mechanistic studies indicate that R-253 could become a promising new tubulin-binding drug candidate for treating various malignancies.

Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth.

Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (<100 nmol/L); in addition, the IC50s were lower than that of lapatinib and dacomitinib. Inhibition of cell growth by ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2+ MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinib's unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835-44. ©2016 AACR.

Cytokine regulation of liver development.

Liver development is a sequential array of distinct biological events. Each step of differentiation is regulated by intrinsically programmed mechanisms as well as by extracellular signals. The establishment of cell culture systems that recapitulate each stage of liver development has led to the identification of several extracellular signals that affect hepatocytic differentiation. Furthermore, studies on genetically engineered animals, especially knockout and transgenic mice, have highlighted a number of molecules essential for liver development. By applying primary culture techniques to analyses of mutant mice, it is now possible to link extracellular signals to intracellular pathways that provoke cellular responses of differentiation. Improvement in gene transfer technology utilizing viral vectors has further expanded the molecular analysis of liver development. In this review article, we summarize recent advances and attempt to describe the molecular basis of liver development from beginning to end as a sequential event.

Cultivation of fetal liver cells in a three-dimensional poly-L-lactic acid scaffold in the presence of oncostatin M.

To investigate the feasibility of fetal liver cells for liver tissue engineering, the supporting function of poly-L-lactic acid (PLLA) for fetal liver cells and the effects of oncostatin M (OSM) on hepatic differentiation were studied. After preparing three-dimensional biodegradable PLLA scaffold having a well-developed open-pore structure by a gas-forming method with ammonium chloride particles as a porogen and a gas-forming reagent, fetal liver cells separated from E14.5-C57BL/6CrSlc murine embryos were inoculated in the PLLA scaffolds. Cells were cultured in Williams' E medium with or without OSM (10 ng/ml) for 30 days with a medium change every 2 days. Results showed that there were significant increases in the number of cells and in albumin secretion in PLLA culture compared with in monolayer culture on day 15. In addition, a significant increase in albumin secretion was observed in OSM-added PLLA culture compared with OSM-free culture, and there was only a slightly enhanced albumin secretion in monolayer cultures with OSM. These results suggest that PLLA may enhance the biological activity of OSM for inducing maturation of fetal liver cells. Interestingly, the number of cells in PLLA culture with OSM decreased compared with OSM-free PLLA culture at day 15. This may be because promotion of hepatic development by OSM simultaneously suppressed in vitro hematopoiesis (i.e., blood cell production). In summary, our results indicate that the three-dimensional PLLA scaffold is a good support material for the cultivation of fetal liver cells and that OSM is capable of not only terminating hematopoiesis of the fetal liver but also stimulating the maturation of hepatic parenchymal cells in vitro.

Noninvasive imaging of in vivo MuRF1 expression during muscle atrophy.

Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3' untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.

AMPK activation through mitochondrial regulation results in increased substrate oxidation and improved metabolic parameters in models of diabetes.

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.

Preclinical characterization of Aurora kinase inhibitor R763/AS703569 identified through an image-based phenotypic screen.

PURPOSE: Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. METHODS: Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. RESULTS: R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. CONCLUSIONS: R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

Maturation of fetal hepatocytes in vitro by extracellular matrices and oncostatin M: induction of tryptophan oxygenase.

Previously, we described that embryonic day 14.5 (E14.5) mouse fetal hepatocytes differentiate to express tyrosine amino transferase (TAT) and glucose-6-phosphatase, which are expressed in the perinatal liver, in response to oncostatin M (OSM) or in high-cell-density culture. However, under such conditions, fetal hepatic cells failed to express genes for adult liver-specific enzymes, such as tryptophan oxygenase (TO). Although phenobarbital (PB) and dimethylsulfoxide (DMSO) have been known to maintain the functions of adult hepatocytes in vitro, they failed to induce TO expression in fetal hepatic cells. Thus far, no system has been developed that reproduces terminal differentiation of fetal hepatocytes in vitro. Here, we describe that extracellular matrices derived from Engelbreth-Holm-Swarm sarcoma (EHS) in combination with OSM or high-cell-density culture induced expression of TO as well as cytochrome P450 genes that are involved in detoxification. However, EHS alone was insufficient to induce expression of TO, although it induced TAT expression in fetal hepatocytes. In addition, high-density culture further augmented differentiation. In conclusion, the combination of signals by cytokines, cell-cell contact, and cell-matrix interaction is required for induction of adult liver functions in fetal hepatocytes in vitro. This primary culture system will be useful for studying the mechanism of liver development.

STAT3 down-regulates the expression of cyclin D during liver development.

As the expression of cyclin D1 is induced during liver regeneration and also in hepatic tumor cells, cyclin D1 is likely to play an important role in the proliferation and transformation of hepatocytes. However, the role of cyclin D1 in liver development remains unknown. Here we show that the expression of D-type cyclins including cyclin D1, D2, and D3 is down-regulated along with liver development. In addition, oncostatin M (OSM), an interleukin-6 family cytokine, down-regulated the expression of cyclin D1 and D2 in a primary culture of fetal hepatocytes in which OSM induces hepatic differentiation. Ectopic expression of receptor mutants defective in the activation of either STAT3 or SHP-2/Ras indicated that the down-regulation of D1 and D2 cyclins by OSM was mediated by STAT3 but not by SHP-2/Ras. Consistently, expression of dominant negative STAT3 but not Ras relieved OSM-induced suppression of cyclin D expression. Activation of STAT3 in fetal hepatocytes of transgenic mice expressing the STAT3-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in the suppression of cyclin D1 and D2 expression. These results indicate that STAT3 activation is necessary and sufficient for down-regulation of D1 and D2 cyclins in fetal hepatocytes. Furthermore, STAT3-C, a constitutively active form of STAT3, suppressed transcription of the cyclin D1 promoter in fetal hepatocytes, whereas it activated the transcription in hepatic tumor cells, huH7 and HepG2. Thus, STAT3-mediated down-regulation of cyclin D expression is rather specific to fetal hepatocytes that are undergoing maturation processes including a reduction of their proliferation potential.

Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow.

During mammalian development, definitive hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) region and colonize the fetal liver (FL) before hematopoiesis occurs in the bone marrow. The FL is a unique hematopoietic organ where both HSCs and mature blood cells are actively generated along with functional maturation of hepatic cells as a metabolic organ. To characterize HSCs and FL microenvironments during development, this study establishes a coculture system composed of AGM-originated HSCs and FL nonhematopoietic cells. The results demonstrate that FL cells support significant expansion of lineage-committed hematopoietic cells as well as immature progenitors. More important, long-term repopulating activity was amplified from AGM-originated HSCs in this coculture system. Engraftment of HSCs to the bone marrow was strongly enhanced by coculture. In addition, AGM HSCs produced significantly more hematopoietic cells than E14.5 and E18.5 FL HSCs in vitro. These results suggest that the FL microenvironment not only stimulates expansion of the hematopoietic system, but also possibly modifies the characteristics of AGM HSCs. Thus, this coculture system recapitulates the developmental process of HSCs and the FL microenvironment and provides a novel means to study the development of hematopoiesis.

K-Ras mediates cytokine-induced formation of E-cadherin-based adherens junctions during liver development.

The E-cadherin-based adherens junction (AJ) is essential for organogenesis of epithelial tissues including the liver, although the regulatory mechanism of AJ formation during development remains unknown. Using a primary culture system of fetal hepatocytes in which oncostatin M (OSM) induces differentiation, we show here that OSM induces AJ formation by altering the subcellular localization of AJ components including E-cadherin and catenins. By retroviral expression of dominant-negative forms of signaling molecules, Ras was shown to be required for the OSM-induced AJ formation. Fetal hepatocytes derived from K-Ras knockout (K-Ras-/-) mice failed to form AJs in response to OSM, whereas AJ formation was induced normally by OSM in mutant hepatocytes lacking both H-Ras and N-Ras. Moreover, the defective phenotype of K-Ras-/- hepatocytes was restored by expression of K-Ras, but not by H-Ras and N-Ras. Finally, pull-down assays using the Ras-binding domain of Raf1 demonstrated that OSM directly activates K-Ras in fetal hepatocytes. These results indicate that K-Ras specifically mediates cytokine signaling for formation of AJs during liver development.

Hepatoma-derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation.

Hepatoma-derived growth factor (HDGF) is a heparin-binding protein, which has been purified from the conditioned media of HuH-7 hepatoma cells. Recent studies have suggested the involvement of HDGF in development of the kidney and cardiovascular systems. In the present study, we investigated the possibility that HDGF was also involved in liver development. Northern blot and immunostaining revealed unique expression patterns of HDGF in liver development. HDGF expression was strongly detected in the fetal liver of the midgestation stage and was markedly decreased near birth. Its expression was mainly detected in stromal cells, including immature hepatocytes. Expression in hepatocytes decreased with differentiation. Administration of recombinant HDGF enhanced the growth of primary cultured fetal hepatocytes significantly, although the effect was small. The effect of exogenous HDGF on the proliferation of neonatal hepatocytes was also small and significant only at one point, despite the lower expression of endogenous HDGF, suggesting that the differences exist between fetal and neonatal hepatocytes. However, adenoviral introduction of HDGF antisense cDNA into the fetal hepatocytes significantly suppressed their proliferation, and the inhibitory effect of HDGF antisense virus was reversed by exogenous HDGF. In conclusion, HDGF helps regulate the hepatocyte proliferation in liver development.